Pattern Formation in the Cerebellum (Colloquium the Developing Brain) 9781615044566, 9781615044573, 1615044566

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ISSN 2159-5194

The Developing Brain

Series Editor: Margaret M. McCarthy, University of Maryland

Pattern Formation in the Cerebellum Carol Armstrong and Richard Hawkes, University

life sciences

M OR G A N & C L AY P O OL LI FE SCI ENCES w w w. m o r g a n c l a y p o o l . c o m

ISBN: 978-1-61504-512-9

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9 781615 045129

M OR GA N & C L AY PO OL

This volume is a printed version of a work that appears in the Colloquium Digital Library of Life Sciences. Colloquium titles cover all of cell and molecular biology and biomedicine, including the neurosciences, from the advanced undergraduate and graduate level up to the post-graduate and practicing researcher level. They offer concise, original presentations of important research and development topics, published quickly, in digital and print formats. For more information, visit www.morganclaypool.com

PATTERN FORMATION IN THE CEREBELLUM

The involvement of key factors operating independently or in cooperation with others contributes to physical and physiological mechanisms to help engineer a vertebrate hypothalamus. The actions of these key factors influence developmental mechanisms including neurogenesis, cell migration,cell differentiation, cell death, axon guidance, and synaptogenesis. On a molecular level, there are several ways to categorize the actions of factors that drive brain development. These range from the actions of transcription factors in cell nuclei that regulate the expression of developmental genes,to external factors in the cellular environment that mediate interactions and cell placements, and to effector molecules that contribute to signaling from one cell to another. Sexual dimorphism is a hallmark of the vertebrate hypothalamus that may arise as a direct consequence of hormone actions or gene actions. These actions may work through any of the mechanisms outlined above. Given the arrangement of cells in groups within the hypothalamus, cell migration may be one particularly important target for early molecular actions that help build the bases for appropriate functions.

ARMSTRONG • HAWKES

Colloquium series in

Colloquium series in

The Developing Brain Series Editor: Margaret M. McCarthy

Pattern Formation in the Cerebellum

Carol Armstrong Richard Hawkes

MOR G A N life sciences

& CL AYP OOL

LIFE SCIENCES

Pattern Formation in the Cerebellum

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Colloquium Digital Library of Life Sciences This e-book is an original work commissioned for the Colloquium Digital Library of Life Sciences, a curated collection of time-saving pedagogical resources for researchers and students who want to quickly get up to speed in a new area of life science/biomedical research. Each e-book available in Colloquium is an in-depth overview of a fast-moving or fundamental area of research, authored by a prominent contributor to the field. We call these resources ‘Lectures’ because authors are asked to provide an authoritative, state-of-the-art overview of their area of expertise, in a manner that is accessible to a broad, diverse audience of scientists (similar to a plenary or keynote lecture at a symposium/meeting/colloquium). Readers are invited to keep current with advances in various disciplines, gain insight into fields other than their own, and refresh their understanding of core concepts in cell & molecular biology. For the full list of available Lectures, please visit: www.morganclaypool.com/page/lifesci All lectures available as PDF. Access to the Collection is free for readers at institutions that license Colloquium. Please e-mail [email protected] for more information.

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Colloquium Series on the Developing Brain Editor Margaret M. McCarthy, Ph.D. Department of Pharmacology, University of Maryland School of Medicine The goal of this series is to provide a comprehensive state-of-the art overview of how the brain develops and those processes that affect it. Topics range from the fundamentals of axonal guidance and synaptogenesis prenatally to the influence of hormones, sex, stress, maternal care and injury during the early postnatal period to an additional critical period at puberty. Easily accessible expert reviews combine analyses of detailed cellular mechanisms with interpretations of significance and broader impact of the topic area on the field of neuroscience and the understanding of brain and behavior. Published titles (for future titles please see the website, http://www.morganclaypool.com/toc/dbr/1/1)

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Copyright © 2014 by Morgan & Claypool All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means—electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. Pattern Formation in the Cerebellum Carol Armstrong and Richard Hawkes www.morganclaypool.com ISBN: 9781615044566 paperback ISBN: 9781615044573 ebook DOI: 10.4199/C00096ED1V01Y201310DBR011 A Publication in the COLLOQUIUM SERIES ON THE DEVELOPING BRAIN Lecture #11 Series Editor: Margaret M. McCarthy, University of Maryland School of Medicine Series ISSN ISSN 2159-5194 print ISSN 2159-5208 electronic

Pattern Formation in the Cerebellum Carol Armstrong

Department of Biology, Mount Royal University

Richard Hawkes

Department of Cell Biology & Anatomy, Genes & Development Research Group and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Alberta, Canada

COLLOQUIUM SERIES ON THE DEVELOPING BRAIN #11

M &C

MORGAN

& CLAYPOOL LIFE SCIENCES

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ABSTRACT

Pattern formation has fascinated biologists since the time of Aristotle, but only recently have new tools begun to reveal the underlying mechanisms that create these patterns during development. In particular, the central nervous system is dynamically patterned and highly modular, ranging from nuclear cell clusters in the brain stem and spinal cord to the elaborate cytoarchitecture of the neocortex. Similar developmental processes divide brain structures such as the cerebral cortex, basal ganglia, superior colliculus, and cerebellum into these sub-compartments. The way neural modules form and the mechanisms that establish connectivity between these modules is one of the most complex problems in neuroscience and also one of the most important. This monograph focuses on pattern formation in the developing cerebellum.

KEYWORDS

pattern formation, cerebellar cortex, cerebellar architecture, ventricular zone, rhombic lip, cerebellar progenitors, Purkinje cell, Purkinje cell phenotype, Purkinje cell clusters, transverse zones, parasagittal stripes, granule cell, climbing fibers, mossy fibers, afferent topography

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Contents

List of Figures ��������������������������������������������������������������������������������������������������������������������� xi



Sidebars��������������������������������������������������������������������������������������������������������������������������������  xiii



Tables�����������������������������������������������������������������������������������������������������������������������������������  xiii



Abbreviations and Glossary���������������������������������������������������������������������������������������������� xv

1

Background and Rationale ������������������������������������������������������������������������������������������������� 1

2

Overview of Cerebellar Organization������������������������������������������������������������������������������ 5 2.1 Cerebellar Anatomy and Terminology ������������������������������������������������������������ 5 2.2 Histology and Cytology ���������������������������������������������������������������������������������� 7 2.2.1 Layers of the Cerebellar Cortex���������������������������������������������������������� 7 2.2.2 Neurons of the Cerebellar Cortex�������������������������������������������������������� 8 2.3 Cerebellar Afferents and Efferents������������������������������������������������������������������ 12 2.3.1 Cerebellar Afferents �������������������������������������������������������������������������� 12 2.3.2 Cerebellar Efferents, Cerebellar Nuclei, and the Corticonuclear Projection������������������������������������������������������������������������������������������ 14 2.4 Cerebellar Circuitry���������������������������������������������������������������������������������������� 15

3

The Modular Cerebellum�������������������������������������������������������������������������������������������������� 17 3.1 Zones�������������������������������������������������������������������������������������������������������������� 17 3.2 Stripes ������������������������������������������������������������������������������������������������������������ 22 3.3 Patches������������������������������������������������������������������������������������������������������������ 25 3.4 What is the Topographical Resolution of the Cerebellar Map? �������������������� 25

4

Overview of Cerebellar Development �������������������������������������������������������������������������� 27

5

Establishment and Organization of the Cerebellar Anlage ������������������������������������� 29 5.1 The Boundaries of the Cerebellar Anlage ������������������������������������������������������ 30 5.2 Products of the Cerebellar Anlage������������������������������������������������������������������ 32

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5.2.1 Products of the 4th Ventricle: Specification of GABAergic neurons (Purkinje Cells and Inhibitory Interneurons)���������������������� 33 5.2.2 Products of the Rhombic Lip: Specification of Glutamatergic Neurons �������������������������������������������������������������������������������������������� 34 5.2.3 The Role of the Rhombomere 1 Roof Plate�������������������������������������� 35 6

Development and Patterning of Purkinje Cells ���������������������������������������������������������� 37 6.1 Purkinje Cell Genesis ������������������������������������������������������������������������������������ 37 6.1.1 Specification of Purkinje Cell Subtypes�������������������������������������������� 38 6.1.2 The Role of Engrailed in Cerebellar Development �������������������������� 40 6.2 The Cluster Map�������������������������������������������������������������������������������������������� 42 6.2.1 Cluster Formation and Patterning ���������������������������������������������������� 42 6.2.2 Cluster Dispersal�������������������������������������������������������������������������������� 47 6.2.3 Purkinje Cell Migration�������������������������������������������������������������������� 49 6.3 From Clusters to Stripes �������������������������������������������������������������������������������� 49 6.3.1 Stripe Formation ������������������������������������������������������������������������������ 49 6.3.2 Purkinje Cell Stripes and Dendritogenesis���������������������������������������� 50

7

Development and Patterning of Granule Cells ����������������������������������������������������������� 53 7.1 Formation of the External Granular Layer from the Rhombic Lip �������������� 53 7.2 Formation of the Granular Layer from the External Granular Layer������������ 53 7.2.1 Proliferation in the External Granular Layer������������������������������������ 53 7.2.2 Granule Cell Migration from the External Granular Layer to the Granular Layer���������������������������������������������������������������������������� 55 7.3 Patterning of the Granular Layer ������������������������������������������������������������������ 56 7.3.1 Lineage Restriction Boundaries in the External Granular Layer and Granular Layer���������������������������������������������������������������������������� 56 7.3.2 Phenotype Restriction Boundaries in Mutant Mice�������������������������� 57 7.3.3 Gene Expression Domains in the Granule Cell Layer �������������������� 58

8

Development of Afferent Projections ���������������������������������������������������������������������������� 61 8.1  Climbing Fibers���������������������������������������������������������������������������������������������� 62 8.1.1 Climbing Fiber Development and Refinement �������������������������������� 62 8.1.2 Establishment of Climbing Fiber Topography���������������������������������� 63 8.2  Mossy Fibers�������������������������������������������������������������������������������������������������� 65 8.2.1 Mossy Fiber Development���������������������������������������������������������������� 65 8.2.2 Establishment of Mossy Fiber Topography�������������������������������������� 66

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Patterning of Other Cells in the Cerebellum: Inhibitory Interneurons, Unipolar Brush Cells, and Glia���������������������������������������������������������������������������������������� 67 9.1  Origin and Development of Inhibitory Interneurons ������������������������������������ 67 9.2  Patterning of Inhibitory Interneurons������������������������������������������������������������ 69 9.2.1 Basket/Stellate Cell Patterning���������������������������������������������������������� 69 9.2.2 Golgi Cell Patterning������������������������������������������������������������������������ 70 9.3  Origin and Development of Unipolar Brush Cells���������������������������������������� 71 9.4  Unipolar Brush Cell Patterning���������������������������������������������������������������������� 72 9.5  Origin and Development of Cerebellar Glial Cells���������������������������������������� 73 9.6  Glial Cell Patterning�������������������������������������������������������������������������������������� 75

10

Neuronal Cell Death in Normal Development ����������������������������������������������������������� 77 10.1  Programmed Purkinje Cell Death in Normal Development������������������������ 78 10.2  Patterned Purkinje Cell Death �������������������������������������������������������������������� 78



Conclusion/Summary�������������������������������������������������������������������������������������������������������� 81



References����������������������������������������������������������������������������������������������������������������������������� 83



Author Biographies����������������������������������������������������������������������������������������������������������  119

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List of Figures Figure 1.1 Stripes in the cerebellum. Figure 2.1 The gross anatomy of the cerebellum. Figure 2.2 The cerebellar cortex has three layers. Figure 2.3 Primary neurons in the cerebellar cortex. Figure 2.4 The Purkinje cell. Figure 2.5 The Purkinje cell dendritic arbor. Figure 2.6 Cell types in the cerebellar cortex. Figure 2.7 Cerebellar circuitry. Figure 3.1 Transverse zones in the cerebellar cortex. Figure 3.2 Zones and stripes. Figure 3.3 Mossy fiber afferents align with Purkinje cell stripes. Figure 3.4 Complexity within Purkinje cell stripes. Figure 3.5 Climbing fibers and mossy fibers both terminate in parasagittal stripes in the cerebellar cortex. Figure 3.6 The architecture of the cerebellar cortex. Figure 4.1 Key stages in cerebellar development. Figure 5.1 The neural tube. Figure 5.2 The isthmic constriction and rhombomere 1. Figure 5.3 Rotation of the cerebellar primordium. Figure 5.4 Germinal zones of the cerebellum. Figure 6.1 Birthdate in the ventricular zone determines a Purkinje cell’s stripe location. Figure 6.2 Ebf2 is expressed in stripes. Figure 6.3 Ebf2 determines Purkinje cell phenotype. Figure 6.4 Engrailed-2 and cerebellar development.

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Figure 6.5 What is the origin of cerebellar complexity? Figure 6.6 Transverse boundaries divide embryonic Purkinje cell clusters. Figure 6.7 Purkinje cell dispersal into a monolayer is triggered by reelin signaling. Figure 7.1 The sonic hedgehog (Shh) signaling pathway. Figure 7.2 Lineage boundaries in the cerebellum. Figure 8.1 Afferent innervation during development. Figure 9.1 Temporal sequence of the birth of neurons in the mouse cerebellar cortex. Figure 9.2 How do basket/stellate cells align with Purkinje cell stripes? Figure 9.3 Interneuron dendrites do not cross Purkinje cell stripe boundaries. Figure 9.4 The dual origin of unipolar brush cells. Figure 9.5 Subsets of unipolar brush cells. Figure 9.6 The first demonstration of molecular stripes in the cerebellar cortex. Figure 9.7 Ibogaine-activated glial cells are organized in parasagittal stripes.

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Sidebars An inside-out cerebellum Patterning defects in mouse mutants Lobes and lobule—foliation Granule cell proliferation and medullablastoma The matching game Pathological Purkinje cell death

Tables Table 3.1 Markers that are co-expressed with zebrin II-immunopositive or zebrin II-immunonegative Purkinje cells Table 6.1 Different subclasses of embryonic Purkinje cell clusters can be distinguished through the differential expression of numerous molecules Table 7.1 Transverse zone boundary markers

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Abbreviations and Glossary AldoC Ascl1

AZ BAX Bcl-2 BDNF BEN bHLH BMP CaBP CRF CZ E EAAT4 EBF2 EGL En2 Eph/Ephrins

Fas

aldolase C (= zebrin II) Achaete-scute homolog 1 (a.k.a. Mash1): a basic helix-loop-helix transcription factor that acts as a proneural gene to promote cell cycle exit and neuronal differentiation anterior zone a Bcl-2-associated that promotes apoptosis by antagonizing Bcl-2 B-cell lymphoma 2: a member of the Bcl family of mitochondrial proteins that block apoptosis brain-derived neurotrophic factor glycoprotein implicated in the establishment of cerebellar afferent topography basic helix-loop-helix: a protein structural motif characteristic of a family of transcription factors bone morphogenetic protein Calbindin: a 28kDa Purkinje cell marker in adults; a selective Purkinje cell cluster antigen in embryos corticotropin releasing factor central zone embryonic age (in days; refers to mouse unless stated otherwise) excitatory amino acid transporter 4: transports glutamate at the parallel fiber’Purkinje cell synapse Early B-cell Factor: the products of a family of 4 genes encoding bHLH transcription factors. Ebf1-Ebf3 are expressed in the cerebellum external granular layer Engrailed: a homeodomain transcription factor Ephrins are the ligands of the ephrin receptor. Ephrin receptors are a large family of receptor protein-tyrosine kinases. Both Ephs and Ephrins are membrane-bound proteins that mediate direct cell-cell interactions during embryogenesis ligand in a key pathway leading to apoptosis by binding to the Fas-receptor (FasR or APO-1)

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ABBREVIATIONS AND GLOSSARY

FGF GABA

fibroblast growth factor

GluRδ2 HNK1 HSP25

glutamate receptor δ2 subunit located in Purkinje cell dendritic spines human natural killer cell 1 25kDa heat shock protein, constitutively expressed in a small set of Purkinje cell stripes both in the adult and during development large family of homeobox genes whose gene products carry the LIM cysteine-rich zinc-binding domain methylazoxymethanol acetate: a neurotoxin that kills dividing cells in the brain see Ascl1 mesencephalon metencephalon metabotropic glutamate receptor a bHLH transcription factor family of bHLH proneural transcription factors related to the Drosophila gene atonal, involved in neuronal specification N-methyl-D-aspartate: a specific agonist of a subset of glutamate receptors— the NMDA receptors—important for glutamatergic neurotransmission and during development for granule cell migration neuronal nitric oxide synthase nodular zone postnatal age (in days; refers to mouse unless stated otherwise with P0 = day of birth) pituitary adenylate cyclase-activating polypeptide paired box gene 2: a transcription factor—PAX2 expression is a marker of interneuron precursors in the cerebellum 6kDa polypeptide that is expressed in all Purkinje cells in the adult cerebellum and in specific clusters during embryogenesis

GIRK2

LIM MAM Mash1 Mes Met mGluR NeuroD Neurog1/2 NMDA

nNOS NZ P PACAP PAX2 PEP 19 PLCβ3/4

Ptf1a

γ-aminobutyric acid: an inhibitory neurotransmitter G-protein coupled, inwardly rectifying K+ channel mutated in the spontaneous mouse mutant weaver

phospholipase Cβ4—a positive marker of the zebrin II-immunonegative Purkinje cell subset. PLCβ3—a positive marker of the zebrin II-immunopositive Purkinje cell subset bHLH pancreas transcription factor that defines the “cerebellar” ventricular zone in which all Purkinje cells are born

ABBREVIATIONS AND GLOSSARY

P1+ to P7+ P1- to P6PZ r Shh SPHK1a TrkB UBC VZ Zebrin II+/-

zebrin II-immunopositive Purkinje cell stripes zebrin II-immunonegative Purkinje cell stripes posterior zone rhombomere sonic hedgehog: a morphogen with multiple roles in patterning the nervous system sphingosine kinase 1a: a 48kDa splice variant that catalyzes the formation of sphingosine 1-phosphate. SPHK1a is co-expressed with zebrin II a BDNF receptor tyrosine kinase unipolar brush cell: a glutamatergic interneuron in the cerebellar cortex ventricular zone anti-zebrin II immunoreact

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1

CHAPTER 1

Background and Rationale Pattern formation has fascinated biologists since the time of Aristotle, but only recently have new tools begun to reveal the underlying mechanisms that create these patterns during development. In particular, the central nervous system is dynamically patterned and highly modular, ranging from nuclear cell clusters in the brain stem and spinal cord to the elaborate cytoarchitecture of the neocortex. Similar developmental processes divide brain structures such as the cerebral cortex, basal ganglia, superior colliculus, and cerebellum into these sub-compartments. The way neural modules form and the mechanisms that establish connectivity between these modules is one of the most complex problems in neuroscience and also one of the most important. This monograph focuses on pattern formation in the developing cerebellum. Cerebellar architecture is extraordinary. The architecture of the adult cerebellum is intricately patterned into a series of functional modules. In brief the cerebellar cortex is first divided into transverse zones, each of which is further subdivided into stripes. The stripe is the structural and functional “quantum” of the cerebellar cortex (Figure 1.1).

FIGURE 1.1: Stripes in the cerebellum. The functional and structural “quantum” of the cerebellar cortex is the parasagittal stripe: a subset of Purkinje cells that expresses common antigens and receives specific afferent input. In this figure, a wholemount (A) and a coronal section (B) through the mouse cerebellum immunostained for zebrin II illustrate this striped architecture.

Cerebellar nomenclature is unfortunately rather baroque, and the same words are used differently by different people. A brief review of this is found in Apps and Hawkes, 2009. In this monograph we have simplified it down to four elements—transverse zones, parasagittal stripes, patches and clusters. Each subdivision is reproducible between individuals and across species. All can be identified anatomically (e.g., through afferent connectivity), through the differential expression of molecular markers, and functionally by electrophysiological mapping:

2

PATTERN FORMATION IN THE CEREBELLUM

• Zones: regions of the cerebellar cortex from anterior to posterior, separated by transverse (i.e., from medial to lateral) boundaries. • Stripes: parasagittal subdivisions of the zones (a.k.a “bands”). Stripes do not cross zone boundaries. • Patches: subdivisions of the granular layer that subdivide stripes into still smaller elements. • Clusters: groups of Purkinje cells during embryogenesis that will subsequently transform into stripes. There are at least five transverse zones and several hundred stripes. Each stripe is built around a small group of Purkinje cells, of which there are many different subtypes. Indeed, the data imply that the cerebellar cortex is patterned with such exquisite precision that a typical module may comprise only a few hundred Purkinje cells!1 This emerging understanding has led to a revolution in thinking about the cerebellum, from it being treated as homogeneous and repetitious to highly patterned. Zone-and-stripe architecture influences all aspects of cerebellar biology. For example: i.  afferent projections to the cerebellar cortex use cerebellar architecture as a template to target their terminal fields to specific zones and stripes (Chapter 8); ii.  cerebellar interneurons are restricted at stripe boundaries, and are thought to use Purkinje cell cues to establish their topography (Chapter 9); iii.  functional boundaries in the cerebellum align with stripe boundaries (Chapter 11); iv.  cerebellar mutant phenotypes are restricted at stripe and zone boundaries (Chapter 3 and Lobes and lobule—foliation); v.  Purkinje cell death in cerebellar pathologies is almost always restricted to stripes (Pathological Purkinje cell death in Chapter 10); vi.  adult zone and stripe architecture is highly reproducible between individuals, conserved through evolution and appears to be insensitive to experimental manipulation (Chapter 3). Cerebellar development occurs over an extended period beginning mid-way through embryogenesis and continuing postnatally for up to three weeks in mice (and at least two years in humans). The elaborate sequence of events that generates cerebellar architecture is gradually becoming 1

The adult mouse cerebellum has ~160,000 Purkinje cells. Taking 200 stripes as a ballpark estimate we arrive at an average of 800 Purkinje cells per stripe. Some stripes are much smaller than this. Others are much larger, but there is reason to believe that the “large” stripes may be composites of several smaller ones (see Chapter 4).

BACKGROUND AND RATIONALE

clearer. Cerebellar architecture influences cytology, connectivity, function, and pathology, so is imperative to understand cerebellar pattern development. Furthermore, because similar developmental processes pattern many brain regions, a thorough understanding of patterning in one structure will illuminate others, and mechanisms identified in one case will apply to many. Two distinct processes interact to organize the brain and spinal cord. First, intrinsic mechanisms (signaling centers, morphogen gradients, migration pathways, etc.) establish coarse-grained maps. Secondly, extrinsic, activity-dependent mechanisms refine them. For example, in the neocortex intrinsic mechanisms create a protomap in the ventricular zone that specifies major cortical areas (e.g., visual or somatosensory), after which thalamocortical afferent projections further sculpt them into their characteristic adult forms (ocular dominance columns in the visual cortex: see Rakic, 2009; Feldheim and O’Leary, 2010 for reviews; whisker barrel fields in the somatosensory cortex: see Frostig, 2006 for review). Remarkably, all evidence to date suggests that the cerebellar stripe map develops independent of afferent input. Indeed, Purkinje cell architecture is expressed well before the cerebellar cortex has its functional circuitry. Once postmitotic Purkinje cells have migrated from the ventricular zone into clusters, no experimental intervention—afferent lesions, sensory deprivation, in vitro slice and dissociated cultures, cerebellar transplants, etc.—has altered a Purkinje cell phenotype. The cerebellum is therefore a beautiful model to study intrinsic mechanisms of brain development. All of these issues will be reviewed in the following chapters. By way of introduction we will begin with a brief review of cerebellar anatomy and organization, and then provide an overview of the main processes and timetable involved in developing and sculpting the patterns inherent in the mammalian cerebellum.  

3

5

CHAPTER 2

Overview of Cerebellar Organization 2.1

CEREBELLAR ANATOMY AND TERMINOLOGY

The cerebellum is located dorsal to the brainstem and inferior to the occipital lobes of the cerebral hemispheres. It has two major divisions—the cerebellar cortex and cerebellar nuclei. In non-mammalian vertebrates, the typical cerebellar cortex consists of two parts: the corpus cerebelli and the lateral auricles. In mammals, the cerebellar cortex consists of the vermis or midline region of the cerebellum that is derived from the corpus cerebelli, as well as the flocculus, located laterally and derived from the auricles, and the hemispheres, which are found only in mammals and birds (Figure 2.1; Larsell, 1948; Marzban et al., 2010). The cerebellar cortex overlies the cerebellar nuclei. From medial to lateral, these are the fastigial (or medial in human), anterior interposed (or emboliform), posterior interposed (or globose), and lateral (or dentate) nuclei. The cerebellar nuclei, together with some vestibular (or Deiter’s) nuclei, are the targets of the efferent projections from the cerebellar cortex. The specific topographical relationships between cerebellar cortex and nuclei have been mapped in detail (e.g., Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2007).

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PATTERN FORMATION IN THE CEREBELLUM

FIGURE 2.1: The gross anatomy of the cerebellum. (A) Dorsal view of the mouse cerebellum. The central vermis (V) is flanked on either side by hemispheres (H) and the flocculus/paraflocculus (F/pf ). (B) Sagittal section taken through the midline (dashed line in A) illustrates ten lobules of the cerebellar cortex, numbered in Roman numerals from I–X.

While the architecture of the cerebellar cortex is fundamentally a sheet, it is elaborately folded into lobes (the primary folds) that are further pleated into numerous smaller lobules. Lobes and lobules constitute the traditional way to describe cerebellar anatomy (Figure 2.2). A sagittal section through the vermis illustrates the division of the cerebellar cortex into the anterior and posterior lobes on either side of the primary fissure. These lobes can be subdivided into ten rostrocaudal lobules (I–X) as shown in Figures 2.1 and 2.2. While these anatomical divisions represent the traditional view of the cerebellum, numerous studies from the last few decades have shown that they do not necessarily reflect the functional organization. For instance, homologies between lobules in different species are often arbitrary and lobules do not represent restriction boundaries for cerebellar afferent projections or molecular expression restriction boundaries. Thus, rather than being key topographical units of cerebellar organization, it seems more likely that lobes and lobules may simply be an anatomical solution to the problem of how to pack the cerebellar cortical sheet into a small volume (for an alternative opinion, see Welker 1990).

OVERVIEW OF CEREBELLAR ORGANIZATION

FIGURE 2.2: The cerebellar cortex has three layers. (A) From outside to in, these are the molecular layer (ml), the Purkinje cell layer (pcl) and the granular layer (gl), as seen in this drawing of the rabbit cerebellum by Camillo Golgi (1882). (B) A sagittal section through the mouse cerebellum that has undergone calbindin-immunohistochemistry to label Purkinje cells, and been counterstained with cresyl violet illustrates the trilaminar organization.

2.2

HISTOLOGY AND CYTOLOGY

2.2.1

LAYERS OF THE CEREBELLAR CORTEX

Superficially, the cerebellar cortex comprises a simple and uniform three-layered structure and cytoarchitectonic areas similar to those in the cerebral cortex are not evident. However, as will be

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discussed below, this simplicity is misleading. A trilaminar cerebellar organization plus underlying white matter is characteristic of all vertebrates except for agnathans (e.g., lampreys: Lannoo and Hawkes, 1997) where the Purkinje cells are dispersed rather than in a monolayer. At the outside, beneath the pial surface, lies the molecular layer, which consists primarily of neurites—the dendritic trees of the Purkinje cells and the parallel-fiber axons of the granule cells. In addition to neurites, the molecular layer contains the cell bodies of numerous inhibitory interneurons that modulate Purkinje cell firing (see Chapter 2.3). The molecular layer is separated from the underlying granular layer by the Purkinje cell layer—a monolayer of large Purkinje cell somata. The granular layer consists primarily of the small, densely packed somata of granule cells together with the occasional somata of Golgi cells (Figure 2.2). The three laminae overlie the white matter tracts that contain the afferent and efferent axons of the cerebellar cortex. An inside-out cerebellum A review by Butts et al. (2011) that discusses the diverse cerebellar morphology across vertebrates suggests that one key difference that has arisen during evolution lies in the generation of the granule cell layer. In birds and mammals, granule cell precursors migrate tangentially before differentiating into granule cell neurons (which is different from lamprey, shark, zebrafish). This results in an “inside-out” cerebellum with the granule cell layer located closest to the pial surface.

2.2.2

NEURONS OF THE CEREBELLAR CORTEX

The cell types of the cerebellar cortex and their connections were essentially worked out by Ramón y Cajal in the late nineteenth century (Figure 2.3). The cerebellar cortex is organized around the Purkinje cell, discovered by the Czech anatomist Jan Purkinje in 1837 (Figure 2.4) and perhaps the first neuron to be recognized. From their somata in the Purkinje cell layer, axons extend through the granular layer to the white matter and then project to targets in the cerebellar and vestibular nuclei. Occasional recurrent axon projections provide feedback inhibition to other Purkinje cells and to cerebellar interneurons (Figure 2.3). Purkinje cells have elaborate and characteristic dendritic arbors in the molecular layer (Figure 2.5) that are flattened in the plane perpendicular to the fissures with the result that Purkinje cell dendrites and granule cell parallel-fiber axons form a regular orthogonal lattice in the molecular layer. The dendrites consist of both smooth shafts and dendritic spines, each with distinct synaptic connections: climbing fibers and stellate interneurons terminate on the smooth dendritic shafts, parallel fibers terminate on the dendritic spines (e.g., Ito, 1984). Although

OVERVIEW OF CEREBELLAR ORGANIZATION

morphology shows no differences between Purkinje cells, molecular evidence reveals multiple different Purkinje cell subtypes: this issue is taken up in greater detail in Chapter 6.

FIGURE 2.3: Neurons in the cerebellar cortex. (A) Santiago Ramon y Cajal’s (1852–1934) exquisite drawings are remarkably detailed and accurate including (B) this sketch of two Purkinje cells and numerous granule cells in the cerebellum of a pigeon, drawn in 1899.

The granule cells are the most numerous neurons of the cerebellum and outnumber Purkinje cells 1000-fold (indeed granule cells probably outnumber all other neurons in the brain—Lent et al., 2012). The somata of the granule cells are packed into the granular layer (Figure 2.2). Three to five short dendrites extend from the soma and terminate in synaptic glomeruli in the granular layer where they receive excitatory input from mossy fiber terminals and inhibitory input from Golgi cell axons. The granule cell axons extend into the molecular layer where they bifurcate and run mediolaterally as long parallel fibers that form glutamatergic synapses on the dendritic spines of the Purkinje cells. Although granule cells are very similar morphologically, several subtypes with topographically restricted distributions have been recognized. The development and patterning of the granular layer is taken up in Chapter 7.

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FIGURE 2.4: The Purkinje Cell. (A) Jan Evangelista Purkinje (1787–1869) was a Czech anatomist credited with the discovery of Purkinje cells in the cerebellum. This was the first neuron ever to be identified in the brain. He was also a fine artist, as seen (B) in his 1837 drawing of a section through the pigeon cerebellum.

In addition to granule cells, a second class of excitatory interneurons has recently been recognized—the unipolar brush cell (UBC: e.g., Mugnaini et al., 2011). Unipolar brush cells also use glutamate as their neurotransmitter. They receive excitatory input from mossy fiber afferents, which they relay to multiple granule cells, thereby amplifying the effects of specific mossy fiber pathways. The different UBC classes and their restricted distributions are discussed in Chapter 9.

OVERVIEW OF CEREBELLAR ORGANIZATION

FIGURE 2.5: The Purkinje cell dendritic arbor. Ramon y Cajal’s diagram of a Purkinje cell: the extensive dendritic tree is monoplanar.

Finally, the cerebellar cortex contains several classes of GABAergic inhibitory interneurons. Basket cells and stellate cells are confined to the molecular layer (they are described here as two distinct neuronal types, but it is very possible that they are a single class of neuron whose anatomy varies depending on their location in the molecular layer: Figure 2.6). Basket cells are small, multipolar interneurons located close to the Purkinje cell somata. Their dendrites extend through the molecular layer where they receive excitatory inputs from the parallel fibers. The basket cell axons envelop the Purkinje cell somata (the so-called “baskets”) and terminate both on the Purkinje cell somata and as curious “pinceaux” on the initial axon segments of the Purkinje cells. Stellate cells are small, star-shaped interneurons that have their somata further away from the Purkinje cell layer. Like basket cells, stellate cells are also excited by parallel fibers: they terminate on the shafts of the Purkinje cell dendrites.

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FIGURE 2.6: The Purkinje cell dendritic arbor. Ramon y Cajal’s diagram of a Purkinje cell: the extensive dendritic tree is monoplanar.

Two large inhibitory interneurons have their somata in the granular layer: Golgi cells and Lugaro cells. Golgi cell dendrites extend into the molecular layer to receive synaptic input from the parallel fibers. Their axons form GABAergic inhibitory synapses on granule cell dendrites within the synaptic glomerular complex. Multiple Golgi cell subtypes have been reported: the different Golgi cell classes and their restricted distributions are discussed in Chapter 9. Lugaro cells lie immediately beneath the Purkinje cell layer. They have been described only in mammals (reviewed in Ambrosi et al, 2007). They are inhibitory interneurons with widespread connections that span many Purkinje cells and seem to serve a prominent role in the integration of cerebellar activity (Lainé and Axelrad, 1998; Crook et al., 2007).

2.3

CEREBELLAR AFFERENTS AND EFFERENTS

2.3.1

CEREBELLAR AFFERENTS

Most afferent projections enter the cerebellum through two large fiber bundles: the inferior and middle cerebellar peduncles. There are two major afferent pathways—climbing fibers and mossy fibers (Figures 2.6, 2.7). The climbing fiber projection arises from the inferior olivary complex in the caudal medulla oblongata. Climbing fiber axons cross the midline at the base of the brainstem and enter the cerebellum via the inferior peduncle. Within the cerebellar cortex, they synapse directly on the apical portion of the Purkinje cell body and “climb” up the proximal dendritic tree (hence the name “climbing fibers”: Figure 2.7). There is a remarkable numerical matching between the inferior olive and the Purkinje cells such that each Purkinje cell is innervated by only one climb-

OVERVIEW OF CEREBELLAR ORGANIZATION

ing fiber. However, during development, climbing fibers from multiple olivary neurons innervate multiple Purkinje cells. As will be described later (Chapter 8), this innervation is systematically sculpted during postnatal cerebellar development by the elimination of supernumerary inputs by an activity-dependent process (reviewed in Sotelo and Chédotal, 2005; Watanabe and Kano, 2011).

FIGURE 2.7: Cerebellar circuitry. The cerebellar cortex receives two main afferents: mossy fibers, which synapse in the granular layer and climbing fibers, which synapse on Purkinje cells. Both afferents also send collateral projections to the cerebellar nuclei (CN). Purkinje cell axons are the sole output of the cerebellar cortex and synapse on neurons within the CN that in turn project to the thalamus, pons, and midbrain. Adapted from Ruigrok, 2011; climbing fiber from Regehr website.

The most populous afferent projections are the mossy fibers. Mossy fibers terminate in the granular layer in characteristic acellular “moss-like” islets called synaptic glomeruli where they form glutamatergic synapses on the dendrites of the granule cells (Figure 2.7). There are four principal sources of mossy fibers: 1. The spinocerebellar pathway from the dorsal spinal cerebellar nuclei and external cuneate nuclei. (For example, lobules I–V of the vermis receive prominent input from the spinocerebellar pathways, are involved in the regulation of limb movement, and are collectively known as the spinocerebellum.)

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2. Pontocerebellar projections from the pontine nuclei. (For example, lobules VI and VII in particular are innervated by pontocerebellar afferents, overwhelmingly from the cerebral cortex. They relay information for the planning, initiation, and timing of movements. For this reason, the pontocerebellar receiving area of the cerebellar cortex is sometimes known as the cerebrocerebellum). 3. Vestibulocerebellar projections from the primary vestibular root fibers. (For example, the flocculonodular lobe receives prominent input from the vestibular labyrinths and functions in vestibular reflexes. For this reason it is sometimes known as the vestibulocerebellum.) 4. Reticulocerebellar projections from the lateral reticular nuclei (similar to the spinocerebellar pathways). In general, ascending mossy fiber projections (spinal, reticular, and vestibular) terminate contralaterally, while the descending pontocerebellar mossy fibers are ipsilateral, although there are many exceptions to this rule. While these functional subdivisions of the cerebellar cortex are widely used, a more fundamental architecture underlies them—transverse zones. Cerebellar zonal architecture is taken up in Chapter 3. In addition to their projections to the cerebellar cortex, both mossy fibers and climbing fibers send projections to the cerebellar and vestibular nuclei (Figure 2.7). These projections converge on the cerebellar nuclear regions innervated by the Purkinje cells that they also excite. Thus, sub-regions of the inferior olive, Purkinje cell stripes and specific cerebellar subnuclei are linked together as a modular array of circuits. There are several other more diffuse cerebellar afferent systems, including noradrenergic projections from the locus coeruleus, cholinergic fibers from the pedunculopontine tegmental nuclei and the serotoninergic axons from the raphe nuclei, but little is known about their development and patterning.

2.3.2

CEREBELLAR EFFERENTS, CEREBELLAR NUCLEI, AND THE CORTICONUCLEAR PROJECTION

Purkinje cell axons—the corticonuclear projection—are the sole efferent projection from the cerebellar cortex. They extend through the granular layer into the white matter and terminate exclusively ipsilaterally on the cerebellar nuclei and some vestibular nuclei (Figure 2.7). In turn, axons from the cerebellar nuclei project to the rest of the brain via the superior peduncles. In general, efferent pathways to higher centers project contralaterally, whereas descending projections remain ipsilateral, but again there are prominent exceptions. The individual cerebellar nuclei have characteristic efferent targets. For example, the fastigial nuclei send both crossed and uncrossed efferent projections to the

OVERVIEW OF CEREBELLAR ORGANIZATION

vestibular nuclei, the reticular formation and the thalamus. The interposed nuclei project primarily to the contralateral red nucleus. The lateral nuclei project principally to the contralateral thalamus. We will not discuss the topography of the cerebellar nuclei (CN) in this monograph: too little is known about its development. However, it should be born in mind that the complex topography of the cerebellar cortex has its counterpart in the cerebellar and vestibular nuclei, which are the targets of the Purkinje cell axons (= corticonuclear projection). Three key points. First, within the CN a number of expression domains can be recognized, which both correlate with and extend the classical subdivisions (fastigial, interposed etc.: e.g., Chung et al., 2009a). Secondly, particular Purkinje cell stripes project to particular CN territories (e.g., Voogd and Ruigrok, 2004; Ruigrok, 2011; Sugihara, 2011). Thirdly, afferents frequently project both to a stripe in the cerebellar cortex and to the terminal field of that Purkinje cell stripe in the CN (e.g., Apps and Garwicz, 2005).

2.4

CEREBELLAR CIRCUITRY

A simplified view of the basic functional circuitry of the mature vertebrate cerebellum is described below (see also Figure 2.7). Both climbing fibers and mossy fibers generate an excitation in the cerebellar and vestibular nuclei. This excitation is modulated by interneurons in the cerebellar cortex. In the cerebellar cortex, climbing fiber activation leads to glutamate release at synapses on the dendritic shafts of Purkinje cells. This results in large unitary excitatory postsynaptic potentials in the Purkinje cell dendrite that propagates as a calcium-dependent spike to the somata where they trigger a series of sodium-dependent action potentials in the form of a “complex spike.” Thus, activation of the inferior olive generates all-or-nothing climbing fiber spike bursts in the Purkinje cell. Mossy fibers form glutamatergic synapses on granule cell dendrites. Mossy fiber excitation passes through the granule cell/parallel pathway to excite Purkinje cell dendrites. In contrast to the climbing fiber input, mossy fiber excitation generates a graded synaptic response at the Purkinje cell to drive “simple” sodium spike discharge. The convergent afferent pathways to the cerebellar cortex are modulated by the γ-aminobutyric acid (GABA)-ergic inhibitory interneurons, principally the stellate/basket and the Golgi cells, which receive excitatory input from the parallel fibers. Stellate/basket cells inhibit the Purkinje cells directly. Golgi cells inhibit the mossy fiber pathway via synapses on the granule cell dendrites and thereby terminate the mossy fiber volley. Finally, because the Purkinje cell axons from the cerebellar cortex to the cerebellar nuclei are inhibitory the upshot is that climbing fiber plus mossy fiber excitation in the cerebellar and vestibular nuclei is modulated by the Purkinje cell pattern of inhibition.  

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CHAPTER 3

The Modular Cerebellum The general overview of the cerebellum provided above—histology, afferents, and circuits—is a preamble to the major issue of this monograph, the development of cerebellar patterning. Imagine the mammalian cerebellar cortex flattened out as a simple sheet. By using differential expression maps and the restricted terminal fields of the cerebellar afferents, two orthogonal sets of boundaries can be identified: one set running from medial to lateral which divides the cerebellum into a string of transverse zones, the second, running from rostral to caudal, which divides each zone into long, narrow parasagittal stripes.

3.1

ZONES

The cerebellar cortex can be divided from rostral to caudal into transverse zones (Ozol et al., 1999). The boundaries between the zones do not align with traditional anatomical boundaries such as lobules and fissures, but form well before lobules appear and are generally thought to represent a more fundamental level of organization (Figures 2.2, 3.1). The boundaries between transverse zones can be identified in numerous ways. For example, as mentioned above (Chapter 2.3.1), mossy fiber afferents terminate in specific regions of the cerebellar cortex. Likewise, gene expression boundaries are evident in sagittal sections through the cerebellum (e.g., Figure 3.4). For example, the expression of the small heat shock protein HSP25 in Purkinje cells extends through lobule X to a predictable and reproducible boundary in the middle of lobule IXc that separates the posterior zone from the nodular zone.2 Finally, many mouse mutations result in cerebellar defects with zonal restrictions (see Chapter 7.2). A conserved architecture with at least five transverse zones is seen in numerous mammals (e.g., Sillitoe et al., 2005; Marzban et al., 2008; Figure 3.1). The anterior zone (AZ) is roughly equivalent to lobules I-V with a boundary about three-quarters of the way up the primary fissure on the caudal side (Wassef et al., 1985; Ozol et al., 1999). The central zone (CZ) is roughly equivalent to lobules VI and VII (by using immunohistochemistry for neurofilament-associated antigen, the CZ can be further divided into an anterior CZa (~lobules VIa and VIb) and a posterior CZp (~lobule VII: Marzban et al., 2008). The posterior zone (PZ) is equivalent to lobule VIII (rostral boundary at the base of the prepyramidal fissure) and the dorsal half of lobule IX (Armstrong et 2

The boundaries between transverse zones are not sharp. Rather there is extensive interdigitation and overlap. For example, the boundary between the AZ and CZ in the mouse interdigitates through most of the length of the caudal face of the primary fissure (lobules V/VI: Ozol et al., 1999).

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al., 2000). The nodular zone (NZ) includes the ventral half of lobule IX and lobule X (Armstrong et al., 2000). There is a consistent difference between mammals and birds, with both embryonic and adult avian species appearing to have an additional lingular zone anterior to the AZ in lobule I (LZ—Pakan et al. 2007; Iwaniuk et al., 2009; Marzban et al., 2010;3 Gilbert et al., 2012: Figure 3.1).

FIGURE 3.1: Transverse zones in the cerebellar cortex. The traditional organization of the cerebellum is into lobes (anterior, posterior, flocculonodular) and lobules (I-X). However, gene expression boundaries, the topographic restriction of afferent terminals, and the phenotypes of mouse cerebellar mutants suggest a more fundamental organization into at least four transverse zones anterior (AZ: lobules I-V (red)), central (CZ: lobules VI and VII (green)), posterior (PZ: lobule VIII and the dorsal half of lobule IX (blue)), and nodular (NZ: ventral half of lobule IX and lobule X (yellow)). Birds and bats also have a lingular zone (LZ) anterior to the AZ in lobule I. Zonal boundaries are not crisp, but rather extensively interdigitate (from Marzban et al., 2010).

To illustrate the zonal architecture, Figure 3.2 shows three cartoons of the adult mouse cerebellum. The first illustrates the distribution of the antigen zebrin II (Brochu et al. 1990: zebrin II = aldolase C (AldoC)—Ahn et al. 1994). Zebrin II is a Purkinje cell antigen with expression restricted to a Purkinje cell subset (= zebrin II+; non-immunoreactive Purkinje cells are designated zebrin II-). In the vermis, four transverse domains in the anterior-posterior axis are identified by 3

The one exception is the bats, which also have a lingular zone. It has been speculated that the LZ is a cerebellar adaptation to flight (Kim et al., 2009).

THE MODULAR CEREBELLUM

zebrin II expression in a subset of Purkinje cells: the striped AZ (~lobules I-V), the uniformly zebrin II+ CZ: ~lobules VI-VII – includes both CZa and CZp), the striped PZ (~lobules VIII-dorsal IX), and the uniformly zebrin II+ NZ (~lobules IX ventral and X). A similar alternation of zones is seen in the hemispheres. The second drawing illustrates the expression pattern of the small heat shock protein HSP25. HSP25 is only expressed in a subset of Purkinje cells in the CZ, NZ, and flocculus/paraflocculus: the same zones where zebrin II fails to reveal any Purkinje cell heterogeneity (Armstrong et al., 2000). In addition to these two antigens, immunoperoxidase-staining for the antigen phospholipase Cβ4 (PLCβ4: Sarna et al., 2006) illustrates that PLCβ4+ Purkinje cells are complementary to the zebrin II+ subset—thus all Purkinje cells in the CZ and NZ are entirely PLCβ4-/zebrin II+ (Figure 3.2).

FIGURE 3.2: Zones and stripes. Cartoon drawings of the intact cerebellum illustrate the zone and stripe architecture as revealed by using immunohistochemistry for zebrin II (A), HSP25 (B), and PLCβ4 (C). Note that HSP25 expression reveals a stripe organization in the CZ, NZ, and flocculus/ paraflocculus—regions in which all Purkinje cells express zebrin II uniformly. PLCβ4 expression is restricted to zebrin II-immunonegative Purkinje cells and thus shows the opposite pattern to that in A.

The zones identified by the use of molecular markers are also reflected in the restriction of afferent terminal fields in the cerebellar cortex. Both climbing fiber and mossy fiber terminals are restricted at transverse zone boundaries. For example, mossy fiber projections from the spinal cord (the spinocerebellar projection) terminate in stripes in the AZ and PZ (e.g., Ji and Hawkes, 1994; Voogd and Ruigrok, 1997 etc.). Similarly, a subset of mossy fiber afferents that is somatostatin-28 immunopositive is restricted to the CZ, NZ, and the paraflocculus/flocculus (Armstrong et al., 2009: Figure 3.3). The restriction of mossy fiber terminal fields at transverse zone boundaries is reviewed in Marzban et al. (2010)(Figure 3.6). Climbing fiber and mossy fiber terminal fields and their relation to the Purkinje cell architecture have been mapped in multiple species (e.g., Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2007; Sugihara and Quy, 2007; Ruigrok, 2011). In addition to afferent topography and differential protein expression, the presence of transverse zones in the cerebellar cortex is supported by evidence from mutant mice that have phenotypic abnormalities that are restricted by zone (Figure 3.2: see Patterning defects in mouse mutants).

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FIGURE 3.3: Mossy fiber afferents align with Purkinje cell stripes. The mediolateral extent of somatostatin 28 (Sst28)-immunopositive mossy fiber terminal stripes in the granule cell layer directly aligns with the HSP25-immunpositive subset of Purkinje cells (see Armstrong et al., 2009).

Patterning defects in mouse mutants Spontaneous and engineered neurological mouse mutants are an important tool to begin to dissect the interplay between genes, the nervous system, and behavior. Interestingly, one of the most common phenotypes described among spontaneous mutants is motor ataxia. This is not surprising as animal handlers can readily detect the abnormal motor behavior of a mouse while changing cages or during casual observation of the animal’s behavior. Thus, there exist a large number of mouse mutants with ataxic phenotypes that correlate with cerebellar defects (e.g., Sidman et al., 1965; Bodenstein and Sidman, 1987). These strains have served as important tools to examine genetic determinants of cerebellar development and the effects that these cerebellar sensitive gene mutations have on events down-stream of the primary genetic insult. A few examples from the rich supply of mice with abnormal cerebellar patterning are: Reeler (rl/rl): The reeler gene encodes the signaling molecule reelin (D’Arcangelo et al., 1995). In the rl/rl mouse there is no reelin and Purkinje cells fail to disperse from their embryonic clusters but nevertheless express their normal zebrin II+/- phenotypes in ectopic locations (Edwards et al., 1994: see Chapter 6.1.1) Apoer2/Vldlr: Apoer2/Vldlr genes code for the lipoprotein receptors on Purkinje cells that bind reelin. The targeted Apoer2/Vldlr double null mimics the reeler phenotype (Trommsdorff et al., 1999). Single mutants have selective dispersal phenotypes (Larouche et al., 2008: see Chapter 6.2).

THE MODULAR CEREBELLUM

Disabled/scrambler (Dab1): Two mutations in the adaptor protein disabled (DAB1), which lies downstream of the Apoer2/Vldlr receptor. Each mouse phenocopies reeler (Howell et al., 1997; Goldowitz et al., 1997: see Chapter 6.2). Ebf2-/-: Targeted deletion of Ebf2 causes the ectopic expression of zebrin II+ markers in Purkinje cell stripes that are normally zebrin II- (Croci et al., 2006). Despite this, the underlying stripe architecture is not greatly affected (Chung et al., 2008: see Chapter 6.1.1). Engrailed1/2 (en/en): Deletion of en1 and en2 results in abnormal cerebellar morphology, gene expression patterns, and afferent connectivity (e.g., Sillitoe and Joyner, 2007; Sillitoe et al., 2008, 2010: see Chapter 6.1.2). NeuroD-/-: NeuroD is a bHLH transcription factor. Targeted deletion of NeuroD generates a mouse without a normal granular layer with the defect restricted to the AZ (Miyata et al., 1999: see Chapter 7.3.2). Weaver (wv/wv): A mutation in a GIRK2 channel results in the failure of dispersion of a small subset of Purkinje cells—the others disperse normally (Eisenman et al., 1998; Armstrong and Hawkes, 2001). Cerebellar deficient folia (cdf/cdf), Rostral Cerebellar Malformation (rcm/rcm), and Meandertail (mea/mea): three spontaneous mutations that result in selective cerebellar defects restricted to the AZ (mea—Ross et al., 1990; cdf—Bierebach et al., 2001; rcm—Eisenman and Brothers, 1998). Dreher (Lmx1adr-J): A spontaneous mutation of Lmx1a, a LIM homeodomain protein (Millonig et al., 2000). The cerebellar morphological phenotype of the spontaneous neurological mutant mouse dreher (Lmx1adr-J) results from a failure of cerebellar midline fusion: despite the reduction in volume and abnormal foliation of the cerebellum, the patterning of the vermis is present, and the normal stripe array is still present, albeit distorted (Sillitoe et al., 2012). Note that while all these mutants reflect profound failures of Purkinje cell dispersal during embryonic development or ectopic gene expression, none have yet been reported with fundamental alterations in the underlying cerebellar pattern.

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Thus, in the mammalian cerebellum ~20 different zones are known.4 The mouse cerebellum contains ~160,000 Purkinje cells so a typical transverse zone in the mouse comprises fewer than 10,000 Purkinje cells. The analysis of transverse zones as functional groupings is still in its infancy, but presumably they underpin to some extent the traditional compartmentation of the cerebellum (spinocerebellum, pontocerebellum, etc.: Chapter 2.3.1).

3.2

STRIPES

Each transverse zone is secondarily subdivided mediolaterally into parasagittal stripes.5 Subsets of Purkinje cells express different proteins and these alternating immunonegative and immunopositive subsets form parasagittal stripes. The initial discovery of the Purkinje cell “stripe” markers, zebrin I (Hawkes et al., 1985), and zebrin II (Brochu et al., 1990) demonstrated that while Purkinje cells may appear identical morphologically, subtypes can be differentiated on the basis of zebrin II expression. Zebrin II+ Purkinje cells align into stripes interspersed by zebrin II- Purkinje cell stripes in both the AZ and PZ of the vermis, while in the CZ and NZ Purkinje cells are entirely zebrin II+ (Figure 3.2). Purkinje cell stripes are reproducible between individuals and symmetrically distributed about the midline (Hawkes et al., 1985; Hawkes and Leclerc, 1987; Brochu et al., 1990). Zebrin II+ stripes are numbered as P1+ - P7+ from the midline laterally, and the intervening zebrin II- stripes are numbered with reference to the medial zebrin II+ stripe (i.e., P1- lies immediately lateral to P1+: see Figure 3.6). Over the past 20 years, zebrin II has been detected in Purkinje cells from fish (e.g., Meek et al., 1992) to primate (e.g., Sillitoe et al., 2004), and a conserved array of zebrin II+/- stripes has been reported in a wide variety of mammalian (e.g., Sillitoe et al., 2003a, b, 2004, 2005; Kim et al., 2009; reviewed by Apps and Hawkes, 2009) and avian (Pakan et al., 2007; Iwaniuk et al., 2009; Marzban et al., 2010) species. Numerous molecular markers are co-localized with either the zebrin II+ or zebrin II- Purkinje cells (Table 3.1).

4

Why twenty zones? The cerebellum develops from twin anlagen each side of the midline, which fuse late in embryogenesis. Thus zones are duplicated either side of the midline. There also appears to be a duplication of zones between vermis and hemisphere. Five zones in the vermis and five in the hemisphere, on either side of the midline, equals twenty. 5 Why “secondarily”? Because stripes are not continuous across transverse boundaries it is assumed that the sequence is: first divide into zones, then subdivide the zone into stripes.

THE MODULAR CEREBELLUM

TABLE 3.1: Markers that are co-expressed with zebrin II-immunopositive or zebrin II-immunonegative Purkinje cells

Co-expressed with zebrin IIimmunopositive: zebrin I

sphingosine kinase 1a (SPHK1a) cdk5 P39 activator phospholipase Cß3 excitatory amino acid transporter (EAAT)4 metabotropic glutamate receptor (mGluR)1b

Reference(s)

Hawkes et al., 1985; Hawkes and Leclerc, 1987 Terada et al., 2004 Jeong et al., 2003 Sarna et al., 2006 Dehnes et al., 1998 Mateos et al., 2000

integrin ß1 Murase and Hayashi, 1996 GABABR2 receptor Chung et al., 2007 Co-expressed with zebrin II-immunonegative: phospholipase Cß4 Sarna et al., 2006 neuroplastin Marzban et al., 2003 early B-cell factor (EBF)2 Croci et al., 2006; Chung et al., 2008

The pattern identified through zebrin II+/- (and co-expressed molecules) is not the limit of cerebellar complexity. First, in those zones without striped zebrin II expression (CZ, NZ), a striped architecture is revealed by other markers. For example, HSP25 is expressed in parasagittal Purkinje cell stripes in the CZ, NZ, and flocculus/paraflocculus—zones in which zebrin II is homogeneously expressed by all Purkinje cells (Figure 3.4: Armstrong et al., 2000). Six mediolateral stripes of HSP25-immunopositive Purkinje cells are present in the CZ and 3–5 stripes can be seen in the NZ (Armstrong et al., 2000: see Figure 3.4). Secondly, individual zebrin II+/- stripes can be further subdivided into narrower bands. For example, phospholipase Cß4 expression reveals that the first and second zebrin II- stripes from the midline (P1- and P2-) of the AZ in the mouse are each comprised of three embryonic clusters of Purkinje cells that fuse during development (Marzban et al., 2007), and expression of an L7/Purkinje cell protein2-lacZ transgene in the AZ is restricted to the medial subdivision of P2- (Ozol et al., 1999: Figure 5.1). Thirdly, comparisons between zebrin II and HNK1 reveal that although HNK1 expression is generally low in the zebrin II+ stripes and high in the zebrin II- (Eisenman and Hawkes, 1993), and in some places discrete Purkinje cell populations strongly express both antigens (e.g., the P3+ stripe in the PZ: Marzban et al., 2004).

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FIGURE 3.4: Complexity within Purkinje cell stripes. (A, B)The architecture of zebrin II-immunopositive (red) and -immunonegative stripes is well documented but is not the limit of the complexity of cerebellar organization. The small heat shock protein, HSP25 (green), is expressed in parasagittal stripes of Purkinje cells in the CZ and NZ. In some lobules, HSP25 expression subdivides zebrin II-immunopositive stripes. (C, D) Likewise, the expression of an L7/Purkinje cell protein2-lacZ transgene in the AZ is restricted to the medial subdivision of one zebrin II-immunonegative stripe (P2-). From Armstrong et al., 2001 and Ozol et al., 1999.

Just as climbing fiber and mossy fiber afferent projections are restricted at transverse zone boundaries (above—Chapter 3.1) a similar restriction is also found to specific stripes (e.g., the spinocerebellar projection and the corticotropin releasing factor immunoreactive climbing fiber projection: Figure 3.5). References to the detailed topography of both the olivocerebellar projection and various mossy fiber projections are provided in Chapter 3.1. Stripes do not only involve Purkinje cells and afferents; as discussed below expression markers also reveal zone and stripe restrictions associated with granule cells (Chapter 7), inhibitory and excitatory interneurons, and glial cells (Chapter 9).

THE MODULAR CEREBELLUM

FIGURE 3.5: Climbing fibers and mossy fibers both terminate in parasagittal stripes in the cerebellar cortex. (A) Corticotropin-releasing factor-immunopositive climbing fibers form bilateral stripes in lobule VIII of the mouse cerebellum that align with zebrin II-immunopositive stripes (B). Zebrin II-positive stripes (P1+, P2+) align with spinocerebellar terminal fields (T1, T2, T3: C) and cuneocerebellar terminal fields (Cu2, Cu3: D). Adapted from Sawada et al., 2008 and Ji and Hawkes, 1995.

3.3

PATCHES

Individual stripes may be further subdivided into “patches” reminiscent of peas in a pod. Multiple electrophysiological studies—notably those of Welker (e.g., Shambes et al., 1978a, b) and Bower (e.g., Bower et al., 1981)—have used electrophysiology to reveal a patchy organization of the granular layer (in particular associated with somatosensory tactile projections) and comparisons between patch boundaries and Purkinje cell stripe boundaries show substantial congruence (Chockkan and Hawkes, 1994; Hallem et al., 1999). In addition, antigenic and histochemical markers have revealed a complex, patchy organization in register with the overlying Purkinje cell stripes (see Chapter 7). Finally, if ethanol-fixed cerebellum is paraffin-embedded and sectioned, when the sections are rehydrated an elaborate, reproducible array of “blebs” is revealed, of similar size to patches revealed by using other methods and similarly aligned with the zebrin II+/- stripe topography (e.g., reviewed in Hawkes, 1997; Apps and Garwicz, 2005; Apps and Hawkes 2009).

3.4

WHAT IS THE TOPOGRAPHICAL RESOLUTION OF THE CEREBELLAR MAP?

A summary of cerebellar modular organization in the mouse is provided in Figure 3.6. First, the cerebellar cortex is subdivided into transverse zones, each comprising ~104 Purkinje cells. Next, each transverse zone is divided into stripes of ~103 Purkinje cells. Finally, the stripe array may be further subdivided into thousands of functional patches of only ~100 Purkinje cells each (reviewed in Hawkes, 1997). This astonishing topographical resolution appears to be highly reproducible be-

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tween individuals and across species. It also drives cerebellar circuitry—the afferent projections and the interneurons. What are the developmental mechanisms that create it?

FIGURE 3.6: The architecture of the cerebellar cortex. The cerebellar cortex is subdivided into ~101transverse zones (Z) each of which contains ~104 Purkinje cells (PC). Each transverse zone is further divided into ~102 parasagittal stripes of ~103 Purkinje cells each. Each stripe may be further subdivided into functional patches of only ~100 Purkinje cells. Thus the cerebellar cortex may be divided into several thousand distinct functional and structural units. Adapted from Apps and Hawkes, 2009.

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CHAPTER 4

Overview of Cerebellar Development

FIGURE 4.1: Key stages in cerebellar development.

Figure 4.1 summarizes the timetable for neuronal development in the cerebellum. Several distinct stages—albeit somewhat artificial—can be identified: E7–E9: All cerebellar neurons derive from the cerebellar primordium located in the most anterior segment of the hindbrain, rhombomere 1 (r1), normally patterned by the combinatorial action of distinct Hox genes. Rhombomere 1 is bounded by the midbrain rostrally and a Hox2a expression domain caudally, and formed under the influence of the isthmic cells of the midbrain–hindbrain boundary ( Joyner, 1996; Wingate and Hatten, 1999; Wang and Zoghbi, 2001). E10–E13: Purkinje cells are born (embryonic day (E) 10-E13 in mouse; e.g., Miale and Sidman, 1961) in the ventricular zone of the 4th ventricle and stack in the “cortical plate.” By this time, the Purkinje cell subtype phenotype is probably already established

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(see Chapter 6). Whether the positional identity of each stripe is determined at this time is unclear. E14–E18: Postmitotic Purkinje cells migrate to the cerebellar anlage to form a stereotyped array of clusters (reviewed in Herrup and Kuemerle, 1997; Oberdick et al., 1998; Armstrong and Hawkes, 2000; Larouche et al., 2006; Sillitoe and Joyner, 2007). These clusters are the forerunners of the adult stripes, so much positional information has already been acquired (see Chapter 6). During the embryonic cluster phase, the major afferent pathways enter the cerebellum and contact specific Purkinje cell subsets (see Chapter 8). It is thought that the Purkinje cells form a template around which afferent topography is constructed (see Chapter 8). Also, the external granular layer (EGL) forms during this period. The EGL is the germinal epithelium that generates the granule cells. The patterning of the EGL and the granular layer are reviewed in Chapter 7. E18–P20: Purkinje cell embryonic clusters disperse, triggered by Reelin signaling, to form the mature pattern of stripes. Granule cells are produced by the EGL and migrate through the immature Purkinje cell layer to form the granular layer. Most inhibitory interneurons also form during this stage (see Chapter 9).  

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CHAPTER 5

Establishment and Organization of the Cerebellar Anlage During embryogenesis, the neural tube is divided into three primary brain vesicles: the prosencephalon, mesencephalon, and rhombencephalon. As development proceeds, the prosencephalon is further subdivided into the telencephalon and diencephalon and the rhombencephalon into the metencephalon and myelencephalon for a total of five secondary brain vesicles. Each vesicle gives rise to specific structures in the adult brain and spinal cord: the telencephalon to the cerebral cortex, the diencephalon to the thalamus, epithalamus and hypothalamus, the mesencephalon to the midbrain, the metencephalon to the pons and cerebellum and the myelencephalon to the medulla oblongata and the spinal cord (Figure 5.1).

FIGURE 5.1: The neural tube. Following neurulation, the neural tube divides into five secondary vesicles: the telencephalon (tel: pink) that will develop into the cerebral cortex, the diencephalon (di: light blue) that will develop into the thalamus, hypothalamus, and epithalamus, the mesencephalon (mes: dark blue) that will develop into the midbrain, the metencephalon (met: light green) that will develop into the pons and cerebellum, and the myelencephalon (mye: dark green) that will develop into the medulla oblongata and spinal cord.

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5.1

THE BOUNDARIES OF THE CEREBELLAR ANLAGE

All cerebellar neurons derive from the anterior part of the metencephalon or hindbrain. At one time the interpretation of embryological studies using quail-chick chimeras suggested that the cerebellum develops from around the “isthmus,” an indentation in the neural tube located near the interface between the posterior mesencephalon (midbrain) and anterior metencephalon—the mesencephalon/metencephalon (mes/met) boundary. By this interpretation, Purkinje cells derive from both the midbrain and hindbrain and granule cells derive solely from the hindbrain (e.g., Hallonet et al., 1990). However, this interpretation was misleading because the isthmus moves with respect to the mes/met boundary. Instead, later studies have shown that all Purkinje cells derive from a domain in the most anterior part of the metencephalon (rhombomere (r)1—Hallonet and Le Douarin, 1993), bounded by nested domains of Otx gene expression anteriorly and a cluster of Hox gene expression in the hindbrain, with the posterior limit of Otx expression marking the anterior boundary of the cerebellum and the onset of Hox2a expression defining the caudal boundary (between rhombomeres 1 and 2—Lumsden and Krumlauf, 1996; Wingate and Hatten, 1999; for an overview of rhombomeres and hindbrain segmentation, see Alexander et al., 2009: Figure 5.2). Studies of X-inactivation mosaic embryonic stem cell chimeras suggest that Purkinje cell patterning is not yet specified at this stage (Baader et al., 1996; Hawkes et al., 1998).

FIGURE 5.2: The isthmic constriction and rhombomere 1. In the neural tube of the developing mouse the isthmus is located near the posterior border of Otx2 expression (yellow) and the anterior border of Gbx2 expression (blue) in the developing neural tube (its position moves with respect to the expression boundary, which was a significant source of confusion until it became understood). Engrailed-2 (En2: green) is expressed on either side of the isthmic constriction (as seen in in situ hybridization). Rhombomere 1 (r1) will develop into the cerebellar anlage (boxed area). Adapted from Davis and Joyner, 1988.

ESTABLISHMENT AND ORGANIZATION OF THE CEREBELLAR ANLAGE

Numerous studies reveal the importance of these hindbrain boundaries. For example, disruption of genes expressed specifically at the mes/met boundary, such as members of the fibroblast growth factor (FGF) family, leads to the loss or severe reduction in size of the cerebellum (Brand et al., 1996; Reifers et al., 1998; Martinez et al., 1999; Xu et al., 2000; Matsumoto et al., 2004; Koster and Fraser, 2006), possibly due to downregulation of Otx (Foucher et al., 2006; Sato and Joyner, 2009). Likewise, disruption of the caudal r1/r2 boundary in Hox2a null mice, results in a caudal expansion of the cerebellum into the hindbrain, while the mes/met boundary was unaffected (Gavalas et al., 1997). Indeed, it is generally the case that deletions of genes whose expression domains overlap rhombomere 1 disrupt cerebellar neurogenesis (e.g., Wnt1—McMahon and Bradley, 1990; Gbx2—Wassarman et al., 1997; Engrailed-1—Würst et al., 1994). The Engrailed (En) homeobox transcription factor family plays a key role in establishing the cerebellar anlage. En-1 and En2 are expressed from E8, and prior to E15.5 expression is uniformly localized at the midbrain-hindbrain border (Davis and Joyner, 1988; Sgaier et al., 2007; Figure 5.2). Loss-of-function experiments established that En1 is required for the specification of the cerebellar primordium and En-1 null mutant mice die at birth with a significant midbrain-hindbrain deletion (Würst et al., 1994). In contrast, En2 null mutants are viable but have a smaller cerebellum with abnormal foliation (Millen et al., 1994). Interestingly, the En1 mutant phenotype can be rescued by using an En2 gene insertion (Hanks et al., 1995). An intriguing study by Sgaier et al. (2005) used genetic fate mapping to demonstrate that the cerebellar primordium (r1) in the mouse, undergoes a 90° rotation during development. Two distinct cell domains were observed in r1, a rostral domain whose boundary coincides with the isthmic boundary and a caudal domain demarcated by En2 gene expression (Davis and Joyner, 1988). Cells within these domains do not mix and appear to have different rates of proliferation. As the posterior cells swing laterally, the rostrocaudal axis at E9.5 is converted to a mediolateral axis by E12.5 (Sgaier et al., 2005: Figure 5.3).

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FIGURE 5.3: Rotation of the cerebellar primordium. During embryonic development, the cerebellar primordium rotates such that the caudal portion of rhombomere 1 (r1) swings laterally while the rostral portion of r1 (arrows) becomes medial. Adapted from Sgaier et al., 2005.

5.2

PRODUCTS OF THE CEREBELLAR ANLAGE

The cerebellar anlage is comprised of two complementary proliferative zones from which all cerebellar neurons are generated: the ventricular zone (VZ) lining the dorsal aspect of the 4th ventricle, which generates the inhibitory neurons, and the rhombic lip at the dorsal portion of rhombomere 1, which generates excitatory neurons (Figure 5.4: Hoshino et al., 2005; Machold and Fishell, 2005; Pascual et al., 2007). This distinction between the GABAergic lineage (e.g., Purkinje cells) and the glutamatergic lineage (e.g., granule cells) is conserved across vertebrates, although the range of mature cell types varies (Butler and Hodos, 1996). The development and patterning of cerebellar interneurons will be discussed further in Chapter 9.

ESTABLISHMENT AND ORGANIZATION OF THE CEREBELLAR ANLAGE

FIGURE 5.4: Germinal Zones of the Cerebellum. All cerebellar neurons and interneurons are derived from one of two germinal zones: a Ptf1a-expressing domain in the ventricular zone (orange) that generates inhibitory neurons including Purkinje cells and GABAergic interneurons, and a Mash1-expressing rhombic lip (purple) that generates excitatory neurons including granule cells, neurons of the cerebellar nuclei and most unipolar brush cells.

5.2.1

PRODUCTS OF THE 4TH VENTRICLE: SPECIFICATION OF GABAERGIC NEURONS (PURKINJE CELLS AND INHIBITORY INTERNEURONS) The ventricular zone (VZ) generates neuronal precursors fated to adopt GABAergic phenotypes— that is, the inhibitory neurons of the cerebellum. The cerebellar VZ is delineated by the selective expression of the bHLH pancreas transcription factor Ptf1a (Figure 5.4). Within the Ptf1a+ domain, the proneural genes Achaete-scute homolog 1 (Ascl1), and neurogenins 1 and 2 (Neurog1 and Neurog2) show further restriction into subdomains, but whether these domains specify different regional identities or phenotypes is unclear (Figure 5.4: Chizhikov et al., 2006; Zordan et al., 2008: reviewed by Consalez et al., 2011). Deletion of Ptf1a in the mutant mouse cerebelless results in the loss of the entire cerebellar cortex (Hoshino et al., 2005), and genetic fate mapping shows that all Purkinje cells derive from Ptf1a+ precursors (Hoshino et al., 2005; Hoshino, 2006). Within the pool of GABAergic progenitors, two Ptf1a targets, Nephrin and Neph3, are expressed by all GAB-

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Aergic progenitors (Nishida et al., 2010). However, progenitors for Purkinje cells are distinguished from interneuron progenitors by the differential expression of E-cadherin (in cycling progenitors: Mizuhara et al., 2010) and the transcriptional co-repressor Corl2 (in postmitotic precursors: Minaki et al., 2008). Cell fate specification has also been explored within the Ascl1, Neurog1, and Neurog2 domains. At the onset of cerebellar neurogenesis (~E11), Ascl1 is expressed through the entire Ptf1a+ VZ and remains restricted here until E13.5 (Zordan et al., 2008). Genetic fate mapping studies confirm that Ascl1+ progenitors are restricted to the cerebellar VZ with a high degree of overlap with Ptf1a+, suggesting that Ascl1 is restricted to the GABAergic neuronal progenitors in the VZ (Kim et al., 2008). Ascl1 gene disruption and overexpression studies showed that Ascl1+ progenitors exit the VZ into the immature cerebellar white matter tracts to eventually settle in the cerebellar cortex where they give rise to inhibitory interneurons and oligodendrocyte precursors, but not most glutamatergic neurons (a subset of unipolar brush cells may be an exception—see Chapter 9.3), astrocytes, or Bergmann glial cells: the loss of Ascl1 leads to a dramatic reduction of interneuron and oligodendrocyte precursors, and Ascl1 overexpression promotes an interneuron fate and suppresses an astrocytic fate (Grimaldi et al., 2009). Neurog2+ progenitors appear to contribute primarily to the Purkinje cell population although NEUROG2 overexpression does not affect Purkinje cell number, suggesting that it is not required for Purkinje cell specification (Florio et al., 2012).

5.2.2

PRODUCTS OF THE RHOMBIC LIP: SPECIFICATION OF GLUTAMATERGIC NEURONS The rhombic lip germinal zone generates the glutamatergic neurons of the cerebellum including granule cells, most unipolar brush cells, and glutamatergic projection neurons of the cerebellar nuclei (Machold and Fishell, 2005; Englund et al., 2006; Chung et al., 2009a). The rhombic lip is a thickening of the roof plate of the 4th ventricle in the posterior part of rhombomere 1 and forms part of the germinal trigone (Figure 5.4: Wingate, 2001). Most rhombic lip-derivatives cells adopt neuronal fates, but the rhombic lip also contributes to the 4th ventricle roof plate and the hindbrain choroid plexus (Landsberg et al., 2005; Hunter and Dymecki, 2007; Rose et al., 2009). Rhombic lip progenitors express the proneural gene Atoh1/Math1 (Machold and Fishell, 2005; Wang et al., 2005) and targeted disruption of Atoh1 ablates virtually all cerebellar glutamatergic neurons ( Jensen et al., 2004; Wang et al., 2005). Three sets of cerebellar glutamatergic neurons are derived from the rhombic lip-EGL lineage: i.  glutamatergic projection neurons of the cerebellar nuclei: these are the first cells to migrate from the rhombic lip (~E10.5). They enter the cerebellar primordium and form a transient structure, the nuclear transitory zone, which subsequently becomes part of the cerebellar nuclei (Machold and Fishell, 2005).

ESTABLISHMENT AND ORGANIZATION OF THE CEREBELLAR ANLAGE

ii.  granule cells: because granule cells are so numerous—greater than 99% of the neurons in the cerebellum—the rhombic lip needs to generate a massive amplification of cell numbers. This is achieved through the formation of a secondary germinal epithelium, the external granular layer (EGL). To this end, a cohort of rhombic lip-derived glutamatergic progenitors disperses tangentially over the cerebellar surface to generate the EGL between E11–E14. These cells are fated to give rise to granule cells (Hallonet et al., 1990; Alvarez Otero et al., 1993). Starting shortly before birth, granule cell progenitors undergo a massive clonal expansion in the EGL (~E17-P20), under the control of signals secreted by Purkinje cells (Smeyne et al., 1995; Dahmane and Ruiz-i-Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999; Lewis et al., 2004), and generate all the granule cells (to be discussed in Chapter 7). iii.  unipolar brush cells (UBCs): the third population of glutamatergic neurons originating in the rhombic lip/EGL between E15 and E17 are UBCs (Englund et al., 2006). Most UBC precursors descend tangentially from the EGL and invade the white matter of prospective lobule X (=NZ) from whence they populate the future granular layer. The subtypes and regionalization of the UBC populations is discussed further in Chapter 9.

5.2.3 THE ROLE OF THE RHOMBOMERE 1 ROOF PLATE The initial organization of the cerebellar anlage is regulated by signaling factors from the rhombomere 1 roof plate (Chizhikov et al., 2006). The roof plate is an embryonic signaling center derived from the lateral edges of the neural plate. During embryological development it is located at the dorsal midline (~E9: Lee and Jessell 1999; Monuki et al., 2001; Chizhikov and Millen, 2005; Wilson and Maden, 2005; Cheng et al., 2006; Hébert and Fishell, 2008). Later (~E12) the roof plate epithelium contributes to the 4th ventricle choroid plexus (Awatramani et al., 2003; Currle et al., 2005; Chizhikov et al., 2006; Hunter and Dymecki, 2007). The roof plate and choroid plexus secrete signaling factors that regulate development and cell proliferation within the cerebellar anlage (Chizhikov et al., 2006). For example, in E12.5 roof plate–ablated embryos, the cerebellar Ptf1a expression domain in the r1 VZ is normal but the number of neurons generated (e.g., Purkinje cells) is significantly decreased (Chizhikov et al., 2006), suggesting that signals from the roof plate are critical for normal cerebellar GABAergic progenitor proliferation. Likewise, inactivation of the rhombomere 1 roof plate transcription factor Lmx1a, leads to a reduction in size of the 4th ventricle roof plate and the hindbrain choroid plexus, which is also associated with reduced VZ proliferation at E12.5, and a small cerebellum in the adult (Mishima et al., 2009). Roof plate signals that regulate proliferation of VZ progenitors in early development likely include both bone morphogenetic protein (BMP: e.g., Panchision et al., 2001; Qin et al., 2006)

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and Wnt (indirectly, via the rhombic lip and the roof plate: e.g., Landsberg et al., 2005; Chizhikov et al., 2006). Similarly, sonic hedgehog (Shh) secreted from the hindbrain choroid plexus is an important regulator of later VZ development (post E14: Huang et al., 2010). For example, embryos from which Shh signaling is conditionally ablated in all neural cells have a thin cerebellar VZ and reduced proliferation of VZ progenitors whereas conversely, ectopic Shh signaling leads to enhanced proliferation in the cerebellar VZ and increased numbers of GABAergic interneuron precursors (Huang et al., 2010). As is the case for the VZ, the development of rhombic lip-derived progenitors is also controlled by signals from the roof plate (Alder et al., 1999; Millonig et al., 2000; Chizhikov et al., 2006: see Chapter 5.1) and ablation of the 4th ventricle roof plate in early embryos results in a complete loss of the rhombic lip (Chizhikov et al., 2006).

 

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CHAPTER 6

Development and Patterning of Purkinje Cells 6.1

PURKINJE CELL GENESIS

Purkinje cells are born—that is, undergo terminal mitosis—in the Pft1a+ ventricular zone between E10-E13 (in the mouse—Miale and Sidman 1961; Hashimoto and Mikoshiba, 2003; Namba et al., 2011). As described in Chapter 5, Neurog1, Ascl1, and Neurog2-expressing progenitor cells can all differentiate into Purkinje cells but it is the Neurog2+ subset that primarily contributes to the Purkinje cell population (Figure 6.1; Florio et al., 2012). Birthdating studies further reveal a fascinating fact—a direct correlation between the birthdate of a Purkinje cell and its final mediolateral location in the cerebellar cortex. Two distinct Purkinje cell populations can be delineated—an early-born cohort (E10-E11.5) destined to become zebrin II+ and a late-born cohort (E11.5-E13) destined to become zebrin II- (Karam et al. 2000; Hashimoto and Mikoshiba, 2003; Larouche and Hawkes 2006: Figure 6.1). This finding implies that Purkinje cells acquire both subtype specification and positional information at or shortly after their terminal differentiation in the VZ. It is not known whether positional information and phenotype are specified at the same time or by the same process.6

6

Each stripe has two properties within the overall cerebellar pattern—a position and a phenotype. Phenotype is established in development at the time the Purkinje cells are born in the VZ (E10-E13). The biology underlying phenotype specification is starting to clarify (see Chapter 6.1.1). Positional information is not at all understood. Do Purkinje cells have a particular positional identity (that is, are they destined to contribute to a particular stripe—just because we know the Purkinje cell is in a particular stripe does not mean that the Purkinje cell knows)? Are positional cues and phenotype specification the same thing? Is there a cerebellar protomap in the VZ? We do not know.

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FIGURE 6.1: Birthdate in the ventricular zone determines a Purkinje cell’s stripe location. Birthdating studies reveal a direct correlation between the birthdate of a Purkinje cell and its mediolateral location in the cerebellar cortex. In the cerebellar anlage (A), Purkinje cells stack such that the earliest-born cells (~E10) are farthest from the 4th ventricle (V4) and later-born cells (~E12) are closer to the ventricle. Phenotypically (B), the earlier-born cells (before E11.5) are calbindin-immunopositive (green) and the later-born cells PLCβ4+ (=zebrin II-negative). Finally, virally infected progenitor cells exposed at E12.5, align into common Purkinje cell stripes by P20 (C: adapted from Hashimoto and Mikoshiba, 2003).

6.1.1 SPECIFICATION OF PURKINJE CELL SUBTYPES A central issue in developmental neurobiology is to understand the regulatory networks that control neuronal fate specification. In many regions in the central nervous system, patterns are the result of patterned neuronal activity (e.g., the retinotectal system—reviewed in Ruthazer and Cline, 2004). However, this does not appear to be the case in the cerebellum, as multiple experiments have shown that Purkinje cell phenotype specification and stripe formation seem to be activity-independent (Leclerc et al., 1988; Wassef et al. 1990; Seil et al., 1995). First, zebrin+ Purkinje cells are more numerous in cultures of posterior cerebellum than anterior cerebellum, consistent with the expression pattern seen in vivo (Leclerc et al., 1988; Hawkes, unpublished data). Next, if Purkinje cell phenotype in the developing cerebellar cortex is specified by patterned afferent input then deafferentation should alter the zone and stripe architecture—but this is not the case (e.g., zebrin I—Leclerc et al., 1988; HSP25—Armstrong et al., 2001). Similarly, cerebellar anlagen can be taken from embryos at E12-E15 and transplanted into ectopic locations in adult hosts—for example, into the anterior chamber of the eye—such that they do not receive normal afferent innervation (Wassef et al. 1990). Nevertheless, in such grafts both zebrin II+ and zebrin II- Purkinje cells are plentiful. Finally, Seil et al. (1995) showed that Purkinje cells in newborn slice cultures express both zebrin+

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

and zebrin- phenotypes, and drugs that block electrical activity in the slice or deplete granule cells and glia did not change this. Thus, it seems that subtype specification is Purkinje cell intrinsic.7 If Purkinje cell phenotype is indeed intrinsic, when and how are subtypes specified? A key candidate in the process is EBF2, a helix-loop-helix transcription factor highly conserved in evolution (reviewed in Dubois and Vincent, 2001; Liberg et al., 2002) that has been shown to couple cell cycle exit to the onset of neuronal differentiation and migration (Garcia-Dominguez et al., 2003). In the normal adult mouse EBF2 expression is seen in the zebrin II- subpopulation of Purkinje cells (Figure 6.2). Ebf2 null mice have small cerebella, probably due to the apoptotic cell death of many Purkinje cells. Perhaps more important, at least as far as the specification of Purkinje cell subtypes is concerned, was the striking result that a major fraction of surviving Purkinje cells in the Ebf2 null cerebellum are transdifferentiated into a zebrin II+/zebrin II- phenotype (i.e., these cells coexpress markers normally restricted to either zebrin II+ or zebrin II- subsets: Croci et al., 2006; Chung et al., 2008).

FIGURE 6.2: Ebf2 is expressed in stripes. (A) Double labeled transverse section showing zebrin II+/- stripes (immunoperoxidase - brown). Expression of the EBF2-lacZ transgene (β-gal-stained: green) is restricted to the zebrin II- stripes. (B) Immunofluorescent-double labeling shows that “transdifferentiated” Purkinje cells in the Ebf2 null express both zebrin II (green) and EBF2-lacZ (red). (Adapted from Croci et al., 2006; Chung et al., 2008).

7 What is the advantage of Purkinje cell phenotype being intrinsic rather than determined by afferent projections? The most obvious gain is that system maturation need not proceed linearly. In a neuronal chain in which target topography passes from member to member, trans-synaptic determination requires that development occur serially and proceed in the descending direction. The converse, where the target topography is independently determined, permits elements of the chain to mature independently. Such a mechanism also avoids a second problem with linear determination—that errors high in the chain will disrupt the topographic organization at all lower levels. Thus, in the cerebellum, the descending efferent pathways can mature earlier than the cerebellum itself without compromising anatomical topography (Hawkes and Gravel, 1991).

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As noted earlier, early-born Purkinje cells (E10–E11.5) are primarily destined to become zebrin II+, while late-born Purkinje cells (E11.5–E13) become zebrin II-. The current working hypothesis is that the early-born Ptf1a+ cohort expresses neither Neurog1/2 nor Ebf2 and therefore becomes zebrin II+ in the adult (E10–E11.5: Figure 6.2). Soon after E11, Ptf1a directly upregulates Neurog1/2 in late-born Purkinje cell progenitors (a similar upregulation of Neurog1 and 2 by Ptf1a has been demonstrated in dorsal spinal cord, Henke et al., 2009). In turn, Neurog1/2+ precursors upregulate and stabilize Ebf2 expression in the late-born Purkinje cell progenitors (Croci et al., 2006: in keeping with findings from Xenopus, where the Neurog1/2 homolog induces and maintains Ebf2 expression during neurogenesis (Chitnis and Kintner, 1996; Dubois et al., 1998; Pozzoli et al., 2001)). As a result, Ebf2 represses the zebrin II+ phenotype and the late-born Purkinje cells adopt a zebrin II- phenotype in the adult (Croci et al., 2006; Chung et al., 2008). Following Ebf2 deletion, Purkinje cells express both markers of the zebrin II- phenotype and those associated with zebrin II+). Thus, in summary a Ptf1a’Neurog1/2’Ebf2 regulatory network represses the zebrin II+ phenotype in the late-born Purkinje cell cohort (Figure 6.3).

FIGURE 6.3: Ebf2 determines Purkinje cell phenotype. Purkinje cells destined to express zebrin II undergo terminal cell division in the Ptf1a+ region of the ventricular zone 1–2 days before the Purkinje cells that are destined to be zebrin II-negative. In the Ebf2 null mouse, Purkinje cells express both zebrin II and markers of the zebrin II-negative subset (e.g., PLCB4: see Croci et al., 2006; Chung et al., 2008).

Nothing is known about the specification of other Purkinje cell subtypes.

6.1.2 THE ROLE OF ENGRAILED IN CEREBELLAR DEVELOPMENT As described in Chapter 5.1, the En genes En-1 and En2 are uniformly expressed at the midbrain-hindbrain border from E8 to E15.5 (Davis and Joyner, 1988; Sgaier et al., 2007). Then, from E15–E17.5, parasagittal domains of expression appear: En1 is expressed preferentially at the

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

midline and En2 expression reveals five distinct mediolateral Purkinje cell clusters (Millen et al., 1994). Engrailed genes are implicated in the control of multiple cerebellar processes during development. First, in gain-of-function studies, Baader et al. (1999) examined the expression patterns of “early onset” (e.g., L7/pcp2-β-gal and cadherin-8) and “late onset” (e.g., zebrin II) Purkinje cell markers following the targeted misexpression of En2 in all Purkinje cells. They found no difference in the expression of early markers (see Figure 6.8) but the expression of late markers was disrupted (Baader et al., 1999: Figure 6.4). In an effort to determine whether the mediolateral expression pattern of En1 and En2 establishes or influences the parasagittal stripes of Purkinje cells seen in the adult cerebellum, Sillitoe et al. (2008) manipulated En expression in mutant mice and examined the resultant expression of adult Purkinje cell markers zebrin II and HSP25. They concluded that the patterning code is dependent on both En1 and En2 in a dose-dependent manner. Furthermore, En1 and En2 are involved in determining the location of fissures during lobule formation (Orvis et al., 2012). En1 and/or En2 were inactivated in the rhombic lip and ventricular zone, either in progenitor cells at E10.5 or in descendent cells at E15.5. They then analyzed the resultant morphology of the adult mouse cerebellum (Orvis et al., 2012). The upshot was that En2 is required in progenitor cells for normal cerebellar foliation, but not later in development, and that conditional loss of both engrailed genes early in cerebellar formation resulted in fissure patterning defects (Orvis et al., 2012). Finally, En genes have also been shown to play a role in the establishment of the specific topography of mossy fiber afferents to the cerebellar cortex (Sillitoe et al., 2010): En2 null mutant mice have only mild defects in the topographic organization of the spinocerebellar mossy fibers (Vogel et al., 1996) but there is significant disruption of the parasagittal stripe pattern in En1/2 double mutants (Sillitoe et al., 2010). Mossy fiber patterning is discussed in Chapter 8.2.2.

FIGURE 6.4: Engrailed2 and cerebellar development. Prolonged gain-of-function expression of Engrailed2 in Purkinje cells affects cerebellar size and cell number but does not change the parasagittal stripe pattern revealed by using histochemistry for an L7/pcp2-lacZ transgene. From Baader et al., 1999.

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6.2

THE CLUSTER MAP

6.2.1 CLUSTER FORMATION AND PATTERNING Postmitotic Purkinje cells migrate dorsally out of the VZ, in part along radial glia processes (e.g., Morales and Hatten, 2006), and stack in the cerebellar anlage (~E13) with the earliest-born Purkinje cells located most dorsally (Figure 6.5) and becoming zebrin II+ in the adult—and the youngest ventrally, becoming zebrin II- (and also expressing EBF2 at this stage—Croci et al., 2006). It is not known if Purkinje cells in these lamina are destined to end up in a specific stripe (it is clear from birthdating studies—cited above—that stripe identity correlates with birthdate, but this could be explained as secondary to phenotype specification together with a non-specific tendency for cells located more medially in the embryo to end up medial in the adult, etc.). Whatever the case, a remarkable topographical order rapidly emerges as the Purkinje cells in the cerebellar plate undergo a complex reorganization (e.g., Miyata et al., 2010), possibly involving cell-signaling molecules including cadherins (e.g., Neudert and Redies, 2008; Redies et al., 2011) and ephrins (e.g., Karam et al., 2000; Sentürk et al., 2011), to yield a stereotyped array of Purkinje cell clusters (E14–E18: Figure 6.5).

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

FIGURE 6.5: What is the origin of cerebellar complexity? The intricate parasagittal organization of the adult Purkinje cells into zones and stripes does not seem to be consistent with the number of Purkinje cell clusters in the embryonic cerebellum. Two models that may explain this are shown: one, the “hidden complexity” model suggests that Purkinje cell clusters are more complex than they appear, perhaps due to secondary differentiation within individual clusters. The second, suggests that single, uniform clusters may undergo complex dispersal pathways to yield multiple stripes.

By E18, numerous Purkinje cell molecular markers show restriction to subsets of clusters (Table 6.1). The expression profiles of Purkinje cell clusters are of four kinds. First, there are molecules that are selectively expressed at some time during embryogenesis but subsequently disappear and are not expressed in the adult (e.g., neurogranin—Larouche et al., 2006). Secondly, some molecules are selectively expressed during embryogenesis but are expressed by all Purkinje cells in the adult (e.g., calbindin—Wassef et al., 1985). Thirdly, a few molecules are selectively expressed by Purkinje cell subsets both in the embryo and the adult (e.g., PLCβ4—Marzban et al., 2007); and finally, in some cases expression reveals one pattern in the neonate and a completely different one in the adult (e.g., HSP25—Armstrong et al., 2001). The accumulated data from expression mapping of single markers suggest the possibility of a straightforward embryonic architecture: all known early markers appear to be restricted to the same schema with no more than ~10 clusters on each side of the midline. So why do there appear to be many more adult Purkinje cell stripes than there are embryonic clusters (a few hundred stripes vs. a few dozen clusters)?

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TABLE 6.1: Different subclasses of embryonic Purkinje cell clusters can be distinguished through the differential expression of numerous molecules

Antigen calbindin (Calb1)

olfactory marker protein-lacZ transgene (OMP-lacZ): phospholipase Cβ4 (PLCβ4) Engrailed-2 (En2) cadherins Purkinje cell protein 2-lacZ transgene (L7/ pcp2-lacZ) synaptotagmin IV (Syt IV) neurogranin (Nrgn)

inositol 1,4,5-trisphosphate (IP3) receptor-lacZ transgene (IP3RnlslacZ) early B-cell factor 2 (EBF2) PEP-19 ephrin type-A receptor 4 (EphA4)

Cluster pattern a large cluster on either side of the midline and a smaller cluster more lateral 6 mediolateral clusters

Age E16

Reference(s) Wassef et al., 1985; Larouche et al., 2006

E14.5-P0

Nunzi et al., 1999

two clusters on either side of midline

P3

Marzban et al., 2007

E17.5

Millen et al., 1994

E12– perinatal P0

Redies et al., 2011

?

P0–P4

Berton et al., 1997

4 thin clusters on either side of midline: 3 medial and one lateral 2 broad lateral clusters at E15 that disperse into 6 clusters arranged symmetrically across the cerebellum by E17

E17

Larouche et al., 2006

E15–P0

Furutama et al., 2010

a single band at midline with two symmetrical bands laterally Multiple clusters 2 bands on either side of midline

3 small clusters on either side of E17.5 midline multiple clusters around the P1 midline a cluster on either side of E18.5 midline and two bilaterally

Ozol et al., 1999

Croci et al., 2006 Herrup and Kuemerle, 1997 Hashimoto and Mikoshiba 2003

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

heat shock protein 25 (HSP25) cyclic GMP-dependent protein kinase (cGK) Wnt7b

6 mediolateral clusters

P3

2 lateral clusters

E17-P3

a cluster on either side of midline and two bilaterally (same as EphA4)

Armstrong et al., 2001 Wassef et al., 1985 Hashimoto and Mikoshiba 2003

The apparent increase in cerebellar complexity from embryonic clusters to adult stripes is a mystery at the heart of cerebellar pattern formation (Larouche et al., 2006; reviewed by Dastjerdi et al., 2012). The key question is whether embryonic clusters are the precursors of the adult stripes. Are embryonic clusters mixtures of multiple Purkinje cell subtypes that segregate into neighboring stripes of different phenotypes, or are clusters homogeneous? Is there one cluster for each adult stripe or do embryonic clusters split and interdigitate to each yield multiple stripes? To date there is preliminary evidence for both of these scenarios: Marzban et al. (2007) demonstrated that perinatal clusters of PLCβ4–immunopositive Purkinje cells fused together into a single, broad zebrin IIstripe in the adult. Sillitoe et al. (2009) tagged Purkinje cell clusters with a pcp2-CreER-IRES-hAP transgene and found that many (but not all) tagged cells ended up in stripes that were zebrin II+. One explanation is that clusters are much more complex than is generally appreciated. By this view, the elaborate adult topography arises because each “simple” embryonic cluster in fact comprises multiple sub-clusters. In some cases there may be internal partitions (e.g., a medial vs. a lateral component of a cluster, each becoming a separate stripe in the adult); in other cases, Purkinje cells of different phenotypes may be intermingled within a cluster but segregate into separate stripes as the cluster transforms into stripes. Alternatively, each embryonic cluster may be homogeneous and additional complexity introduced into the adult map because individual clusters disperse into multiple stripes of the same adult phenotype. For example, in the weaver mouse two clusters either side of the midline fail to disperse and six adult stripes are missing, all of the zebrin II+/ HSP25+ phenotype (Armstrong and Hawkes, 2001). Sillitoe et al. (2009) reveal a similar story by using a pcp2-CreER-IRES-hAP transgene to tag three bilateral clusters on approximately E15 and show they yielded zebrin II+ Purkinje cells of nine adult stripes. On the other hand, in some cases multiple embryonic clusters merge to form a single stripe. The clearest example are the zebrin II-/ PLCβ4+ stripes in the vermis of the AZ, which arise from the fusion of three perinatal PLCβ4+ clusters (Marzban et al., 2007: in this case, and others, the “fusion” of three clusters into one stripe may be an illusion due to a lack of appropriate stripe subtype markers because these apparently uniform adult stripes are seen to be subdivided into triplets by the pattern of mossy fiber innervation ( Ji and Hawkes, 1994).

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An answer to the enigma—few embryonic clusters, many adult stripes—is that the number of embryonic clusters has been significantly underestimated. First, while there appear to be only ten or so clusters each side of the midline, the early presence of transverse boundaries (e.g., Ozol et al., 1999; Dastjerdi et al., 2012 and unpublished data: Figure 6.6) has not been systematically taken into account. In fact, at least five embryonic transverse boundaries can be identified in the newborn mouse, corresponding to those previously identified in the adult (Figure 7.2). This implies—to simplify—that there are not ten clusters each side of the midline, but sixty8 (ten clusters, each divided into six by the five transverse boundaries). Secondly, Sugihara and his colleagues recently conducted an exhaustive anatomical mapping of clusters of the E17–P6 mouse cerebellum and reproducibly identify over fifty clusters that can be correlated with their counterparts in the adult (Fujita et al., 2012).

FIGURE 6.6: Transverse boundaries divide embryonic Purkinje cell clusters. Six expression domains can be seen in the perinatal cerebellum by using immunohistochemistry for Forkhead box protein 2 (FoxP2), calbindin (CaBP), the small heat shock protein 25 (HSP25), and phospholipase Cβ4 (PLCβ4).

In conclusion, with minor exceptions where clusters fuse or split, positional information regarding the adult stripes and zones seems to be fully encoded in the architecture of the embryonic clusters. Is positional information already specified at the VZ stage? There are two “extreme” 8

Each of the ~10 clusters is subdivided by five transverse boundaries into six sub-clusters: 10 x 6 = 60.

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

models. Akin to the “protomap” model of the neocortex, whereby the regional identity of pyramidal neurons along the tangential plane is specified in the VZ (e.g., Rakic, 1988), a cerebellar protomap hypothesis would suggest that different Ptf1a+VZ microdomains specify aspects of cerebellar regional identity, and indeed the Ptf1a+VZ is not homogenous and gene expression differences do further subdivide it into microdomains (e.g., Chizhikov et al., 2006; Zordan et al., 2008). By the protomap hypothesis, Purkinje cells from different VZ microdomains would be restricted to specific regions in the adult (e.g., vermis vs. hemisphere), or even to particular stripes. Alternatively, the two laminae in the E13 cortical plate (early-born/EBF2-/destined to form zebrin II+ stripes and late-born/EBF2+/destined to form zebrin II- stripes) are indeed homogeneous and the elaborate embryonic cluster pattern is sculpted by the environment through which they disperse.

6.2.2 CLUSTER DISPERSAL Whatever the origin of the cluster architecture, the subsequent transformation of clusters into a monolayer is much better understood, in large part due to the availability of mouse mutants with Purkinje cell dispersal phenotypes (Patterning defects in mouse mutants). Notably, when the Reelin pathway is disrupted by mutation of Reelin (reeler: e.g., reviewed in Tissir and Goffinet, 2003), Reelin receptors (Trommsdorff et al., 1999), or the Disabled1 docking protein (Dab1-/-: Goldowitz et al., 1997; Howell et al., 1997; Sheldon et al., 1997; Gallagher et al., 1998), cluster dispersal is blocked. For example, in the homozygous reeler mutant mouse, only 5% of Purkinje cells are in the correct location between the molecular layer and the internal granular layer, whereas 10% are trapped in the granular layer and the remaining 85% fail to disperse and remain clumped together in ectopic clusters (Heckroth et al., 1989). Starting at around E18, the clusters disperse to form a monolayer, triggered by Reelin signaling (Figure 6.7: D’Arcangelo et al., 1997; Miyata et al., 1997; Tissir and Goffinet, 2003). Reelin secreted by the external granular layer binds two Purkinje cell receptors—the apolipoprotein E receptor 2 (Apoer2) and the very low-density lipoprotein receptor (Vldlr: Trommsdorff et al., 1999; Hiesberger et al., 1999). Reelin binding induces receptor clustering (Strasser et al., 2004) and activates a protein kinase cascade leading to tyrosine phosphorylation of the docking protein Disabled1 (Dab1: Goldowitz et al., 1997; Howell et al., 1997; Sheldon et al., 1997; Gallagher et al., 1998; Rice et al., 1998). Downstream of Dab1 are multiple kinases (Bock and Herz, 2003; Kuo et al., 2005) with the end result being a drop in mutual Purkinje cell-Purkinje cell adhesion, thereby freeing the embryonic clusters to disperse into stripes. However, in the specific context of cerebellar patterning there are still aspects of the ReelinDab1 pathway that are obscure. For example, in contrast to the full reeler phenotype with no cluster dispersal in the Apoer2-/-:Vldlr-/- double null, mutations in the Reelin receptors (Apoer2-/-; Vldlr-/-; Apoer2+/-:Vldlr+/- i.e., double heterozygous for both receptors) result in a partial reeler

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phenotype—most embryonic clusters disperse normally into stripes but a few remain ectopic in the cerebellar core (Larouche et al., 2008: no Purkinje cell ectopia is present in mice heterozygous for either receptor alone). This selective ectopia is strange because it is thought that all Purkinje cells express both receptors. Furthermore, Larouche et al. (2008) showed that specific subsets of Purkinje cells are differentially affected. By examining the expression pattern of zebrin II in mice that lacked either Apoer2 or Vldlr, it was found that Purkinje cell dispersal and migration defects were largely confined to the zebrin II- Purkinje cells in mice lacking the ApoER2 receptor (Larouche et al., 2008). In contrast, in mice lacking the Vldlr, both zebrin II- and zebrin II+ Purkinje cells were ectopic (Larouche et al., 2008). Clearly, there are complexities in the Reelin pathway that are yet to be uncovered.

FIGURE 6.7: Purkinje cell dispersal into a monolayer is triggered by reelin signaling. Reelin secreted from the external granular layer (EGL) binds to a Vldlr/Apoer2 receptor complex on Purkinje cells. Receptor binding activates an intracellular signaling pathway via tyrosine phosphorylation of the adaptor protein disabled-1. In turn, this leads to a downstream signaling cascade that includes multiple intermediates (cdk5, p35, p39 etc.) that results in the downregulation of Purkinje cell surface adhesion molecules (cadherins, integrins) and the upregulation of key elements of the neuronal cytoskeleton (e.g., tau). The upshot is thought to be a drop in Purkinje cell–Purkinje cell adhesion that allows the embryonic cluster to disperse into stripes. In mutants that disrupt reelin signaling, the embryonic cluster architecture is retained in the adult.

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

These studies focused on Purkinje cell dispersal. Similar considerations apply to the granule cells. The specific influence of granule cell precursors in the external granular layer on Purkinje cell migration and positioning was examined further by Jensen et al. (2002), who used the math1 null mutant mouse in which the external granular layer does not form, and therefore there is no external granular layer-derived Reelin protein. However, unlike the mass Purkinje cell ectopia seen in the reeler mutant, Jensen et al. (2002) found that most Purkinje cells migrate successfully in the Math1 null, with the remainder located ectopically, either deep in the cerebellar cortex, anteriorly in the inferior colliculus, or stuck at the midline ( Jensen et al., 2002). These findings imply that additional sources of Reelin, aside from the external granular layer are present.

6.2.3 PURKINJE CELL MIGRATION In 1985, Altman and Bayer used 3H-thymidine labeling to determine the path, direction, and timing of Purkinje cell migration in rats. Their results demonstrate regional differences with respect to both the time taken for migrating cells to reach their destination in the Purkinje cell layer and the sequence in which the Purkinje cell clusters disperse. For example, Purkinje cells that will comprise lobule IX settle in the Purkinje cell layer as early as E17 while anterior Purkinje cells (e.g., lobules II, III) do not settle until E21 (Altman and Bayer, 1985). Consistent with this, the Purkinje cell clusters become a monolayer in a caudal-to-rostral sequence from lobule IX (E17), to lobule VIII (E18), lobule VI (E19), lobule V and IV (E20) and lobules II, III (E21: e.g., Altman and Bayer, 1985).

6.3

FROM CLUSTERS TO STRIPES

6.3.1 STRIPE FORMATION As the embryonic clusters disperse into stripes the Purkinje cells spread to form the adult monolayer. Because this dispersal occurs primarily in the anteroposterior plane, as the lobules of the cerebellum form (see Lobes and lobule—foliation), the rostrocaudal length of the cerebellum increases ~25-fold while the width increases only ~1.5-fold (Gallagher et al., 1998). As a result the clusters string out into long parasagittal stripes. Most adult stripe markers are first expressed during this period. A few already show more-or-less adult patterns of restriction by around P5 (e.g., PLCβ4— Marzban et al., 2007) but most—including zebrin II (Lannoo et al., 1991)—are first expressed at around this time but go through a “global expression” phase in which they are expressed by all Purkinje cells (e.g., HSP25—Armstrong et al., 2001; zebrin II—Lannoo et al., 1991a, Rivkin and Herrup, 2003; Olfactory Marker Protein (OMP)-lacZ, Nunzi et al., 1999) before they are selectively downregulated and the stripe architecture matures by P20. Various attempts have been made to link these expression phases with concomitant developmental processes such as refinement of

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climbing fiber synapses, onset of motor movement, opening eyes, etc. (e.g., Armstrong et al., 2001) but to date there has been no satisfactory explanation as to why Purkinje cells undergo this elaborate and dynamic expression during postnatal development. Lobes and lobules—foliation Numerous studies on the functional organization of the cerebellum have illustrated that there is disparity between anatomical boundaries and functional boundaries, which leads to the question of why the cerebellar cortex is so intricately divided into distinct lobes and lobules? The ten lobules of the cerebellum can be further subdivided into smaller units (e.g. lobule IXa, IXb, IXc) that vary in size and shape but are remarkably consistent among individuals. So what is the point of lobes and lobules and how do they link to cerebellar development and patterning? Recent work in the Joyner lab describes the base of each fissure as an “anchoring center” for the evagination of lobules. The growth of an individual lobule may be determined by granule cell migration along Bergmann glia (Sudarov and Joyner, 2007).

6.3.2 PURKINJE CELL STRIPES AND DENDRITOGENESIS Purkinje cells are easily recognizable by their beautiful and elaborate dendritic trees, highlighted by Golgi and Ramon y Cajal in their drawings of the cerebellum (e.g., Figures 2.3, 2.5, 2.6). Mature Purkinje cell dendrites receive two excitatory afferent inputs: a single climbing fiber from the inferior olivary nucleus and multiple parallel fibers inputs from granule cells. During the first postnatal week, Purkinje cells extend multiple dendrites in “random orientations” before a single primary dendrite is determined during the second postnatal week (reviewed by Sotelo and Dusart, 2009). The first postnatal week of development is characterized by elongation of the dendrite and extensive branching (Figure 6.8). There are multiple processes occurring during the second postnatal week including granule cell migration from the EGL to the granular layer, formation of parallel fibers and parallel fiber-synapses on the developing Purkinje cell dendrite, elimination of supernumerary climbing fiber afferents and the “capuchon” and dendritic phases of climbing fiber innervation (the translocation of afferent terminals from the Purkinje cell soma to the dendritic tree: see Chapter 8.1.1). The dendritic tree does not reach its mature state until approximately four weeks of age (Altman, 1972; McKay and Turner, 2007).

DEVELOPMENT AND PATTERNING OF PURKINJE CELLS

FIGURE 6.8: Purkinje cell dendritogenesis. A: In the first postnatal week (P0–P7), Purkinje cells extend dendritic processes in all directions. (A) single, primary dendrite is selected during the second postnatal week that elongates and branches in multiple planes. This extensive branching is subsequently remodeled until it becomes restricted into a single plane by approximately three weeks of age, concomitant with the maturation of the climbing fiber innervation. (B) A PLCβ4-immunopositive Purkinje cell harvested at E18 and grown for 21 days in vitro (from McKay et al., 2007 and Marz-

ban et al., 2010).

Purkinje cell dendritogenesis is sensitive to changes in afferent input and developmental influences. For example, elimination of either climbing fibers (e.g., Kawaguchi et al., 1975) or granule cells (e.g., Altman and Anderson, 1972; Hartkop and Jones, 1977) results in abnormal dendrites with fewer branches and spines. On the other hand, the basic architecture is much more intrinsic— in cerebellar primary cultures in vitro, where afferent input is either very abnormal or altogether absent, a classic Purkinje cell morphology is easily recognized (e.g., Figure 6.8; Marzban et al., 2007). From the perspective of cerebellar stripes, the puzzling issue is the mechanism that results in the orientation of the dendritic arbor parallel to the stripe (and orthogonal to the parallel fibers). The mature planar form is achieved through extensive remodeling during postnatal development (Kaneko et al., 2011), suggesting a role for cerebellar functioning in the elimination of unwanted dendritic branches. Consistent with this, mice with abnormal climbing fiber activity do not remodel properly. In the absence of parallel fibers (e.g., ectopic Purkinje cell mouse mutants such as reeler (reln/reln)) the planar dendritic tree is lost, which might suggest that parallel fibers or ascending granule cell axons constrain the lateral spread of the dendrites but any mechanism is obscure (e.g., Altman and Anderson, 1972).  

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CHAPTER 7

Development and Patterning of Granule Cells 7.1

FORMATION OF THE EXTERNAL GRANULAR LAYER FROM THE RHOMBIC LIP

All granule cells arise from the rhombic lip. Because granule cells are so abundant, the developing cerebellum forms a transient secondary germinal epithelium from progenitor cells in the rhombic lip to amplify their production—the external granular layer (EGL). As described in Chapter 5, progenitor cells in the rhombic lip express the transcription factor Ascl1 (Figure 5.4; Machold and Fishell, 2005). EGL progenitors begin to proliferate in the mouse embryo by ~E10 and by ~E13 Ascl1-expressing precursors leave the rhombic lip and migrate tangentially from lateral to medial and posterior to anterior until they cover the surface of the embryonic cerebellum to form the EGL (reviewed in Chédotal, 2010). How this process is regulated is still largely unknown but may include an attractive chemokine released by the meninges (CXCL12: Zhu et al., 2002) as well as BMP signaling (Alder et al., 1999; targeted deletion of BMP1 receptors leads to a severe loss of granule cells: Qin et al., 2006). The formation of the EGL is complete by birth. From ~E15 until P20, by which time it is largely spent, the EGL generates all the granule cells of the cerebellar cortex.

7.2

FORMATION OF THE GRANULAR LAYER FROM THE EXTERNAL GRANULAR LAYER

7.2.1 PROLIFERATION IN THE EXTERNAL GRANULAR LAYER The external granular layer (EGL) is a neurogenic niche that supports the proliferation of granule cells. It is estimated that more than a million granule cell precursors are present in the mouse cerebellum at the peak of proliferation (P5–P8: Hatten, 1985: Granule cell proliferation and medullablastoma). By the time the EGL has completely covered the surface of the cerebellum, a number of signaling pathways are active that promote granule cell proliferation. The most studied is sonic hedgehog (Shh: reviewed by Vaillant and Monard, 2009). Shh is expressed in developing Purkinje cells as early as E18 (Wechsler-Reya and Scott, 1999; Wallace, 1999). At the same time a

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Shh receptor, the transmembrane protein Patched, is expressed by granule cell precursors. In turn, Patched inhibits the downstream effector, Smoothened. Thus, Shh secreted by Purkinje cells binds Patched and releases the inhibition of Smoothed (Figure 7.1). This in turn allows the downstream expression of transcription factors that control granule cell proliferation (e.g., Gli1, Gli2, and Gli3: Corrales et al., 2004; Lee et al., 2010). Granule cell proliferation and medullablastoma Medulloblastoma is a fast-growing tumor of the cerebellum that is believed to arise from granule cell precursors in the EGL. Haldipur et al. (2012) examined the expression pattern of Shh, Patched, and Smoothened receptors as well as the downstream Gli1 and Gli2 transcription factors in the human cerebellum at various times from 10 weeks of gestation to 5 yrs of age and found that the expression patterns were equivalent to those seen in mice with two exceptions: Shh is initially secreted within the external granule cell layer prior to Purkinje cell expression, and Shh is downregulated postnatally. These findings are also clinically relevant as deregulation of Shh signaling can lead to uncontrolled proliferation and subsequently to the formation of medullablastoma, the most common malignant nervous system tumors in children (reviewed by Hatten and Roussel, 2011 and Rousell and Hatten, 2011). Granule cell development is particularly susceptible to damage because development is protracted—starting during embryogenesis and not finishing until a few weeks (or years in humans) after birth—and because of the tightly controlled but massive amount of proliferation that occurs.

FIGURE 7.1: The sonic hedgehog (Shh) signaling pathway. In the absence of Shh, the Patched (Ptc) receptor inhibits the Smoothened (Smo) receptor. The binding of Shh to Patched leads to activation of the Smoothened receptor (Smo) and transactivation of downstream Gli, Gli2, Gli3 genes, which leads in turn to granule cell proliferation.

DEVELOPMENT AND PATTERNING OF GRANULE CELLS

7.2.2

GRANULE CELL MIGRATION FROM THE EXTERNAL GRANULAR LAYER TO THE GRANULAR LAYER During the first three postnatal weeks, granule cell precursors in the EGL exit the cell cycle and move to a location between the EGL and the developing molecular layer. The transition from proliferative to postmitotic to migratory appears to require the movement of the cell out of the EGL, and away from the influence of Shh (e.g., Choi et al., 2005). Postmitotic newborn granule cells rest in the EGL for one-two days before they begin to migrate into the granular layer. At this location (a.k.a. the premigratory zone) several events occur. First, granule cell somata move mediolaterally: this is not well understood but has been speculated to balance granule cell numbers across different cerebellar stripe compartments (Komuro et al., 2001). Secondly, they extend processes both medially and laterally—these will become the parallel fibers. Thirdly, the granule cell somata descend into the molecular layer until they reach the immature granular layer. The migration of granule cells has been the topic of intense research. The most widely accepted theory is that migration is guided by Bergmann glial fibers, which extend from the cerebellar surface to the Purkinje cell layer. Bergmann glia are unipolar astrocytes with their cell somata in the Purkinje cell layer and long straight processes extending through the molecular layer to the pial surface. Numerous studies have shown that Bergmann glial cells are critical for normal cerebellar development (reviewed in Yamada and Watanabe, 2002; Hoogland and Kuhn, 2010). Hatten and Liem (1981) were among the first to examine the interactions between migrating neurons and astrocyte fibers. They cultured single cell suspensions from embryonic and postnatal mouse cerebellum and demonstrated that astrocytes extend a template of fibers that act as a “glial monorail” (Hatten, 1990) along which immature neurons migrate to their destination (Hatten and Liem, 1981: for a critical review of this issue, see Chédotal, 2010). The direction of migration, from the EGL and towards the granular layer, may be mediated by BDNF: post-mitotic granule cells in the external granule cell express the BDNF receptor TrkB, while granule cells in the internal granular layer express both the ligand and the receptor, suggesting that a BDNF gradient may attract granule cells towards the granular layer (Zhou et al., 2007: for other candidates, see Chédotal, 2010). The interaction of migrating granule cell and Bergmann fiber is still contentious, and many candidates have been put forward. The rate at which granule cells migrate through the molecular layer is closely controlled. Komuro and Rakic (1995) demonstrated that the average rate of granule cell migration increases with development resulting in a similar overall time of migration despite a concurrent increase in the depth of the molecular layer postnatally. In addition, Bergmann fibers may also play a regulatory role. For example, migrating granule cells express N-methyl-D-aspartate (NMDA) receptors, and blocking these significantly slowed the migration of the newborn granule cells (reviewed by Komuro and Rakic, 1998). This led to the idea that dynamic interactions with Bergmann glia modulate the

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rate of granule cell migration as well as the trajectory. The termination of migration is linked to the intrinsic cessation of fluctuations in intracellular calcium (Kumada and Komuro, 2004). Once the granule cell has reached the granular layer it moves into the most superficial part of the granular layer, adjacent to the Purkinje cell layer, and extends four-five short dendrites. These receive presynaptic input from the excitatory afferent mossy fiber pathways and the inhibitory Golgi cell axons in a complex synaptic structure known as a synaptic glomerulus (reviewed in Ito, 2006). The development of the mossy fiber pathway is reviewed in Chapter 8.2. As a result of this migration mechanism, granule cells stack in the granular layer with the earliest born closest to the white matter and the last born closest to the Purkinje cell layer. Similarly, in the molecular layer the parallel fibers are also stacked with the youngest nearest to the cerebellar surface (e.g., Espinosa and Luo, 2008). This may account for gene expression patterns in which only half the width of the granular layer is immunoreactive (e.g., Sillitoe et al., 2003).

7.3 7.3.1

PATTERNING OF THE GRANULAR LAYER

LINEAGE RESTRICTION BOUNDARIES IN THE EXTERNAL GRANULAR LAYER AND GRANULAR LAYER As described in Chapter 6, the adult cerebellar cortex is elaborately patterned through transverse zones and parasagittal stripes that are largely established by the Purkinje cell architecture. In contrast, it is usually supposed that the granule cells represent a homogeneous population but this is not the case (reviewed by Ozol and Hawkes, 1997). In fact, the granular layer comprises several distinct embryological lineages and several distinct molecular phenotypes. In fact, the restriction into transverse zones and parasagittal stripes characteristic of the external granular layer and the granular layer is apparent both through lineage restriction boundaries, mutant phenotypes, and patterns of differential gene expression. Analysis of embryonic stem cell chimeras identified two consistent lineage boundaries in the adult cerebellum: one located posterior to the AZ/CZ Purkinje cell boundary between lobules VI and VII and a second posterior to the PZ/NZ boundary between lobules IX and X (Hawkes et al., 1999: Figure 7.2). Similar evidence of distinct lineage compartments in the EGL also comes from analysis of murine chimeras (e.g., Unc5h3 X +/+—Goldowitz et al., 2000; Sey/SeyNeu X +/+—Swanson and Goldowitz, 2011; M. musculus X M. caroli—Goldowitz, 1989; weaver X +/+; Smeyne and Goldowitz, 1989 etc.), all of which reveal consistent EGL lineage boundaries. Consistent with this, it has been established by genetic fate mapping that early-born granule cell progenitors (E12.5–E15.5) migrate preferentially into the anterior vermis, whereas later-born ones distribute more evenly along the AP axis, and only late-born ones (circa E17) populate lobule X (Machold and Fishell, 2005). Jensen et al. (2004) showed “temporal cohorts” of post-mitotic

DEVELOPMENT AND PATTERNING OF GRANULE CELLS

granule cells leaving the external granule layer. In addition, Chizhikov et al. (2010) showed that the “last-born” granule cell population is uniquely characterized by the expression of Lmx1. Thus it is likely that the cerebellar granular layer has multiple lineage histories and derives from multiple distinct precursor pools either side of lineage boundaries within the rhombic lip.

FIGURE 7.2: Lineage boundaries in the cerebellum. Analysis of embryonic stem cell chimeras identified one lineage boundary posterior to the AZ/CZ Purkinje cell boundary (between lobules VI and VII) and another posterior to the PZ/NZ boundary between lobules IX and X. Adapted from Hawkes et al., 1999.

The fact that different granule cell lineages end up in reproducible locations within the cerebellum implies that the migration pathways from the rhombic lip are more complex than a simple lateromedial dispersal implies, but the specific details are yet to be teased out. Because transverse boundaries in the granular layer align with the overlying Purkinje cell transverse zones, it is plausible to speculate that granular layer architecture is established by the topography of the Purkinje cell landscape over which the external granular layer disperses.

7.3.2 PHENOTYPE RESTRICTION BOUNDARIES IN MUTANT MICE Restriction boundaries are seen in the phenotypes of many mutant mice. For example, in rostral cerebellar malformation in which the cerebellum extends rostrally beyond the normal anterior boundary, there is a “discontinuity” in the granule cell layer on the anterior side of the primary fissure (Eisenman and Brothers, 1998), and in the NeuroD-/- mutant, there is a reduction in the granule cell layer in the anterior cerebellum and no granule cell layer at all in the posterior cerebellum, with

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a transition near the AZ/CZ boundary (Miyata et al., 1999). Finally, in disabled a distinct discontinuity is present in the granular layer at the AZ/CZ boundary (Gallagher et al., 1998).

7.3.3 GENE EXPRESSION DOMAINS IN THE GRANULE CELL LAYER In the same way as the Purkinje cell layer, differential gene expression patterns reveal boundaries that divide the granular layer into transverse zones (Table 7.1). Whether the mutant phenotypes and expression boundaries are lineage restricted or due to secondary interactions with the local environment is unclear. At that stage, both intrinsic lineage differences between granule cell populations and interactions with Purkinje cells or mossy fiber afferents (inter alia) could sculpt patterns of granule cell gene expression. TABLE 7.1: Transverse zone boundary markers

Zonal boundary AZ/CZ

PZ/NZ

Gene acidic fibroblast growth factor, receptor protein tyrosine phosphatase Otx-1 nNOS (= NADPH) Otx1 En2 Tlx3 Lmx1a+

Reference McAndrew et al., 1998

Frantz et al., 1994 Hawkes and Turner, 1994 Frantz et al., 1994 Millen et al., 1994 Logan et al., 2002 Chizhikov et al., 2010

Beyond the restriction of granule cell subtypes to transverse zones, several markers reveal a more elaborate parcellation into parasagittal stripes (e.g., the expression patterns of acetylcholinesterase (Marani and Voogd, 1977; Boegman et al., 1988), cytochrome oxidase (Hess and Voogd

DEVELOPMENT AND PATTERNING OF GRANULE CELLS

1986; Leclerc et al., 1990), and neuronal nitric oxide synthase (nNOS: Yan et al., 1993; Hawkes and Turner, 1994; Schilling et al., 1994). The relative contributions of lineage restriction and secondary epigenetic interactions in granular layer stripe formation are not known. However, it is difficult to imagine a mechanism by which granule cell stripes form through the targeted migration of granule cell subtypes to hundreds of destinations, so it is more plausible that stripe phenotypes among granule cells are secondary responses to local cues. This is consistent with the demonstration in vitro that ingrowing mossy fibers may downregulate nNOS expression and thereby contribute to the generation of granule cell subtypes (Schilling et al., 1994).  

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Development of Afferent Projections The cerebellum receives two major afferent projections—climbing fibers from the inferior olivary complex and mossy fibers from the pons, medulla, and spinal cord (Chapter 2.3). In many brain regions, afferent projection maps reflect the spatial segregation of incoming fibers (e.g., the patch-matrix architecture of the striatum (Gerfen, 1984), the columnar organization of the neocortex (Rakic, 1988), the lamination of the dorsal lateral geniculate nuclei (Sretavan and Shatz, 1984 etc.)). The same is true in the cerebellum: as described in Chapters 3.1 and 3.2 a critical feature of cerebellar afferent projections is that they terminate in a elaborate array of stripes that are restricted at transverse zone boundaries and are in register with the stripes of Purkinje cells. We will argue below that both climbing fibers and mossy fibers develop their mature topography in similar ways, based on specific chemoaffinity between the projections—the incoming growth cones—and the targets—embryonic Purkinje cell clusters. Thus, when cluster dispersal takes place (see Chapter 6.2) and clusters stretch out into long stripes, the afferent fibers go along with them and hence in the adult, Purkinje cell stripes and afferent fiber terminal fields are in register (The matching game). The matching game The detailed anatomy of the mossy fiber pathway in the cerebellar cortex is confusing. The great divergence of the granule cell projection to the Purkinje cell—each parallel fiber synapses with hundreds of Purkinje cells—is in sharp contrast to the precise topography of the mossy fiber terminal fields. It is not well understood why the mossy fiber terminals are highly focused at narrow bands of granular layer when the granule cells immediately spread the pattern of excitation very widely. However, despite the superficial appearance given by the anatomy, it is the Purkinje cells that immediately overlie a mossy fiber terminal field that respond preferentially when that terminal field is excited; that is, the signal dispersion via the parallel fibers is not as prominent as the cytology might suggest. Part of the explanation may be that the ascending portions of the granule cell axons are especially important in the establishment of Purkinje cell firing patterns. The parallel fiber contribution to the pattern of excitation may then be to provide “context” by informing a Purkinje cell band or patch about the activity of its mediolateral neighbors.

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8.1  CLIMBING FIBERS 8.1.1 CLIMBING FIBER DEVELOPMENT AND REFINEMENT Ramón y Cajal (1890) outlined three stages in the postnatal development of climbing fiber innervation: a “pericellular nest” stage, a “capuchon” stage, and a “young dendritic” stage. By P5, during the pericellular nest stage, climbing fibers from several olivary neurons form synaptic connections with Purkinje cell somata (Mason et al., 1990). Climbing fibers are not seen “climbing” Purkinje cell dendrites until about P8 in the capuchon (or “hooded”) stage during which multiple climbing fiber terminals become concentrated on the apical portion of the Purkinje cell somata and the proximal dendrites (Mason et al., 1990). Finally, during the dendritic stage (P9–15) the mature configuration is achieved as the climbing fiber synapses translocate from the Purkinje cell somata to the dendritic shafts. Transient synaptic connections between climbing fibers and Purkinje cells may be present as early as E19 (Mason et al., 1990; Chédotal and Sotelo, 1992; Wassef et al., 1992; Morara et al., 2001). Although at birth each Purkinje cell receives input from multiple cells in the inferior olive, in the mature cerebellum an individual Purkinje cell is innervated by only one climbing fiber, while each inferior olivary neuron contacts on average seven Purkinje cells (Schild, 1970). This one-to-one relationship is the result of postnatal elimination of supernumerary climbing fibers, which occurs during the second and third postnatal weeks (e.g., Crépel et al., 1976; Mariani and Changeux, 1981). The elimination of excess climbing fibers is activity-dependent. For example, two complementary studies—one that altered neuronal activity in the inferior olive (Andjus et al., 2003) and one that altered Purkinje cell activity (Lorenzetto et al., 2009)—both resulted in persistent multiple climbing fiber innervation. Synapse elimination occurs mainly during the second postnatal week when a single dominant climbing fiber is selected such that there is one “strong” input and several weaker ones (Hashimoto and Kano, 2003; Kano and Hashimoto, 2011: a selection mediated by Purkinje cell Ca2+ influx—Kano and Hashimoto, 2011). The strong climbing fiber translocates from the soma to the dendritic arbor and only then does the elimination of the other inputs proceed (Hashimoto et al., 2009a). During the same period as climbing fiber elimination is proceeding, newborn granule cells are migrating through the molecular layer, leaving their parallel fiber axons in their wake to synapse on Purkinje cell dendritic spines. It seems that granule cell–Purkinje cell interactions also play a role in climbing fiber elimination. For example, numerous studies of cerebella deficient in granule cells also reveal the preservation of climbing fiber multi-innervation (e.g., either experimentally induced granule cell elimination—Crépel and Delhaye-Bouchard, 1979; or mutant mice that lack a normal granule cell complement, such as staggerer (Rorasg: e.g., Crépel et al., 1980) or reeler (Reln: Mariani et al., 1977)). The mechanism(s) by which excess synapses are eliminated is unclear but seems to

DEVELOPMENT OF AFFERENT PROJECTIONS

be mediated by glutamate receptors on Purkinje cell dendritic spines. In a mutant mouse deficient in the glutamate receptor δ2 subunit (GluRδ2) the single dominant climbing fiber is selected normally and the initial elimination of excess climbing fibers begins but subsequently the process stalls (Hashimoto et al., 2009b). In addition, the number of parallel fiber-Purkinje cell dendrite synapses is halved (Kurihara et al., 1997). Thus, Hashimoto et al. (2009b) conclude that climbing fiber synapse elimination is initially independent of parallel fiber-Purkinje cell dendrite formation but then becomes dependent on it for final refinement as parallel fibers and climbing fibers compete for synaptic territory. The elimination of supernumerary climbing fibers is also regulated by GABAergic inhibition (Nakayama et al., 2012). By combining electrophysiology and immunohistochemistry, it was shown that multiple climbing fiber innervation persisted in mutant mice with diminished GABAergic transmission, and that this effect could be reversed by enhancing the sensitivity of GABAA receptors (Nakayama et al., 2012).

8.1.2 ESTABLISHMENT OF CLIMBING FIBER TOPOGRAPHY Correct brain functioning is critically dependent on precise connections between neurons and their targets, and topographic maps are the means by which sensory and motor systems process afferent information. As discussed in Chapter 2, there is a strict and reproducible topographical relationship between subnuclei of the inferior olivary complex and specific Purkinje cell stripes and zones in the cerebellar cortex. Because climbing fibers enter the cerebellar cortex before the cerebellar circuit becomes functionally active, it is believed that afferent axons find their correct target cells guided by positional cues that are intrinsic to the Purkinje cell architecture. The earliest evidence of olivocerebellar topography is already seen during the Purkinje cell embryonic cluster stage (~E14–E18: e.g., Wassef et al., 1992; Paradies & Eisenman 1993: reviewed by Sillitoe and Joyner, 2007; see Chapter 4). During this period—sometimes referred to as the climbing fiber “creeper” stage (Mason et al., 1990)—climbing fiber axons from the contralateral inferior olivary complex enter the cerebellar cortex via the inferior cerebellar peduncles and ramify amongst the Purkinje cells in the cluster, with up to 100 “creeper” fibers arising from each olivocerebellar neuron (Sugihara, 2005; reviewed in Watanabe and Kano, 2011). Once the Purkinje cell clusters disperse into adult stripes, the climbing fiber terminals go along with their targets and thus the terminal field becomes a long stripe in register with a specific Purkinje cell stripe. This hypothesis is supported by the fine structure of the olivocerebellar connection—while each Purkinje cell receives only one climbing fiber, each projection neuron in the inferior olive contacts multiple Purkinje cells. These are arranged in parasagittal stripes, consistent with the idea that an initially symmetrical cluster of target cells has been drawn out longitudinally during postnatal development (Figure 8.1).

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FIGURE 8.1: Afferent innervation during development. Mossy fibers are seen in the embryonic cerebellar cortex as early as E13–E15 where they associate with clusters of Purkinje cells. From birth (P0), when Purkinje cells are dispersing into a monolayer, to about postnatal day 10 (P10), “combination fibers” are seen, which share morphological characteristics of both climbing fibers and mossy fibers and which are seen in both the Purkinje cell layer (PCL) and the granular layer (GL). By ~P12, the adult organization of afferents is present with climbing fibers innervating Purkinje cell directly and mossy fibers confined to the granular layer.

To explain the specificity of Purkinje cell clusters and subsets of climbing fiber afferents, Sotelo espoused the chemoaffinity model of Sperry (1963) and hypothesized that climbing fiber projection neurons and their Purkinje cell targets organize the projection map by matching shared positional cues: the “matching hypothesis” (e.g., Sotelo and Wassef, 1991; Wassef et al., 1992a, b; Sotelo, 2004; Sotelo and Chédotal, 2005). Consistent with this idea, subtle alteration of Purkinje cell patterns via manipulation of Engrailed2 seems to result in circuitry defects (Sillitoe et al., 2010). In a landmark in vitro study using explants taken from E7.5–E8 chick embryos, Chédotal et al. (1997) demonstrated that the embryonic cerebellum contains topographic cues that guide developing inferior olivary axons: experimental rotation of the cerebellar target region resulted in a corresponding rotation of the climbing fiber projection. It thus seems that the alignment of Purkinje cell stripes and climbing fiber terminal fields is achieved by direct chemospecific interactions. The molecular interactions that mediate the precise matching between climbing fiber projections and Purkinje cell clusters are still unclear. Candidates include the cell adhesion molecule BEN and Ephs and Ephrins. BEN is transiently expressed in a subset of neurons in the inferior olive, a subset of Purkinje cells, and a subset of the deep cerebellar nuclei at E7–8 chick cerebellum (Chédotal et al. 1996). EphA tyrosine kinase receptors are expressed in specific domains of the inferior olive, climbing fiber subsets target Purkinje cell domains of ephrinA expression (Karam et al., 2000;

DEVELOPMENT OF AFFERENT PROJECTIONS

Nishida et al., 2002), and ephrinA2 overexpression disrupts the topography of the olivocerebellar map (Nishida et al., 2002).9 What is the role of climbing fiber synapse elimination? It is characteristic of much of the nervous system development that ordered patterns are sculpted from initially homogeneous arrays by selective elimination (e.g., Changeux and Danchin, 1976; Law and Constantine-Paton, 1980; O’Leary et al., 1986). Indeed, in theoretical models of map formation afferent terminal stripes emerge naturally when inputs interact competitively (Willshaw and von der Malsburg, 1976; Constantine-Paton and Law, 1982). These might serve either to ensure topographic mapping despite poor aim (i.e., improve topographical fidelity) or to ensure complete filling of the target. However, the data do not support a significant role for collateral elimination in the refinement of olivocerebellar topography (Crépel, 1982; Chédotal and Sotelo, 1992). However, target filling—ensuring that each target cell gets an input—is clearly crucial in the cerebellum, where each Purkinje cell receives exactly one climbing fiber input in the adult. Thus, the most plausible role for excess climbing fiber collaterals and collateral elimination is not to pattern the olivocerebellar projection but rather to ensure that each Purkinje cell receives its climbing fiber input.

8.2  MOSSY FIBERS As described in Chapter 2, mossy fibers do not synapse directly on Purkinje cells in the adult but rather mossy fiber terminals synapse in the granular layer with granule cell dendrites and Golgi cell axons (Figure 2.7; e.g., Palay and Chan-Palay, 1976). The mossy fiber pathway then excites Purkinje cells via granule cell parallel fiber synapses on Purkinje cell dendrites. However, despite this apparent difference between climbing fibers and mossy fibers, the underlying processes that pattern the topography are similar.

8.2.1 MOSSY FIBER DEVELOPMENT The identification of mossy fibers in the developing cerebellum is complicated because their anatomy appears different than in the adult (where mossy fibers and climbing fibers are morphologically distinct). In the embryo many afferents—likely mossy fibers—share morphological characteristics of both climbing fibers and mossy fibers and that extend into both the granular layer and the Purkinje cell layer (referred to as “combination fibers”: Mason and Gregory, 1984). Manzini et al. (2006) showed that these combination fibers form synaptic connections onto both granule cells and Purkinje cells in postnatal mice. It is unclear whether mossy fibers form functional synapses 9

An alternative model is that olivocerebellar topography is mediated by a third party—the cerebellar nuclei. Anatomical studies have shown that Purkinje cell axons and climbing fiber afferents share a common target territory in the cerebellar nuclei. It might be that the cerebellar nuclei are the place that the initial matching takes place and that the projection to the cerebellar cortex forms subsequently, perhaps guided by the different Purkinje cell axon subsets from the cerebellar cortex (Hawkes and Gravel, 1991).

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on Purkinje cells. That being said, mossy fiber innervation of the cerebellar cortex can reliably be identified during the last week of embryonic development: vestibulocerebellar mossy fiber axons are present in the rat cerebellum as early as E14 (Morris et al., 1988; Ashwell and Zhang, 1998), spinocerebellar mossy fibers are seen in the mouse cerebellum at E13–14 (Grishkat and Eisenman, 1995), and pontocerebellar fibers are seen in Purkinje cell clusters at birth (Mason et al., 1990).

8.2.2 ESTABLISHMENT OF MOSSY FIBER TOPOGRAPHY The important point, from the perspective of pattern formation, is that mossy fibers innervate the cerebellar anlage in a patterned way before their granule cell targets have developed. The usual interpretation is that mossy fiber topography forms in the same way as for climbing fibers: embryonic Purkinje cell clusters are the initial targets. Subsequently, as the granule cells are born and migrate to the granular layer the mossy fibers terminals displace from the Purkinje cells (hence the transient combination fibers) to eventually synapse with their adult targets, the granule cell dendrites. Additional data in support of this hypothesis include: • Ji and Hawkes (1994) found a positive (transient) association between calbindin-immunopositive clusters of Purkinje cells and spinocerebellar mossy fibers in the perinatal mouse cerebellum. • Mouse mutants in which granule cells do not form nonetheless have striped terminal fields (see review by Sotelo, 1990), suggestive of a patterning role for Purkinje cells (but there may be a secondary role in sharpening and refining the projection—see Ji and Hawkes, 1996). • As for climbing fibers, there is no evidence of activity-dependent sculpting of mossy fiber terminal fields or competition for target territory. For example, lesioning the spinocerebellar tract (and thus removing a subset of afferents) did not lead to the expansion of adjacent (cuneocerebellar) terminal fields suggesting that competition between mossy fibers for Purkinje cell clusters is not setting up cerebellar topography ( Ji and Hawkes, 1995). As for climbing fibers the molecules that mediate matching between mossy fiber growth cones and Purkinje cell clusters are not well understood.

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CHAPTER 9

Patterning of Other Cells in the Cerebellum: Inhibitory Interneurons, Unipolar Brush Cells, and Glia As reviewed in Chapter 2, the cerebellar cortex contains multiple inhibitory interneurons that modulate the neuronal activity of Purkinje cells and granule cells. These inhibitory interneurons include basket/stellate cells with somata in the molecular layer and Golgi cells with somata in the granular layer. Additional interneurons, such as Lugaro cells, have also been described but much less is known about their development (reviewed in Schilling et al., 2008). This chapter reviews the putative mechanisms by which inhibitory interneurons develop their mature topography by interacting with the Purkinje cell zone and stripe scaffolding. The patterning of unipolar brush cells (UBCs) is also described here and it seems likely that the mechanisms that result in their specific stripe locations are common.

9.1  ORIGIN AND DEVELOPMENT OF INHIBITORY INTERNEURONS The GABAergic inhibitory neurons of the cerebellar cortex—Purkinje cells, basket/stellate cells, and Golgi cells—all derive from the Pft1a-expressing ventricular zone (Figure 6.4). Within this zone, subdomains are evident based on the expression of proneural genes Ascl1 or Neurog1 and Neurog2 (Chapter 5.2.1). A common precursor (Leto et al., 2006), expressing the proneural gene Neurog1 (Chizhikov et al., 2006; Zordan et al., 2008; Lundell et al., 2009; Kim et al., 2009), gives rise to all interneurons (reviewed in Consalez et al., 2011; Leto and Rossi, 2012; Leto et al., 2009). There appears to be a lineage relationship between inhibitory interneurons and astrocytes, since in Ascl1 null mutant mice, Grimaldi et al. (2009) found that Ascl1 ablation results in a fate switch from interneuron to astrocyte. The basket/stellate cells are born during late embryonic and early postnatal life. Interneuron progenitors delaminate from the ventricular zone and migrate into the developing white matter tracts, which represent a specific neurogenic niche (Leto et al., 2010: Figure 9.1) Disruption of the presumptive white matter by ablation of oligodendrocytes at the time basket/stellate cells are being born prevents their migration into the molecular layer (Mathis et al., 2003).

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FIGURE 9.1: Temporal sequence of the birth of neurons in the mouse cerebellar cortex. Purkinje cells (Pc) are the first neurons in the cerebellar cortex to become postmitotic. They are born in the ventricular zone of the 4th ventricle (VZ) between E10–E13. Unipolar brush cells are born between E14–P2, Golgi cells from E15 to P5 and basket/stellate interneurons arise perinatally (from the late embryonic period to P2). Granule cells are the last neurons to be born—between P0–P21.

Within the presumptive white matter, GABAergic interneurons can be identified through the transient expression of PAX2 (Maricich and Herrup, 1999). In the prospective white matter, the precursor population undergoes multiple further divisions (Zhang and Goldman, 1996; Yamanaka et al., 2004) to generate successive populations of interneurons. Postmitotic interneurons migrate out from the white matter and settle in the developing cerebellar cortex. The eventual choice of phenotype depends upon on stage-specific extracellular cues (Leto et al., 2006; Leto et al., 2009) since PAX2-immunopositive GABA interneuron progenitors transplanted into wild-type recipients develop into interneurons characteristic of the age of the host rather than of the donor (e.g., early progenitors transplanted into older hosts adopt the stellate rather than basket cell morphology: Leto et al., 2009). The timing of terminal cell division correlates with both phenotype and location in the cerebellar cortex. Three quarters of all inhibitory interneurons are generated in the first postnatal week with a maximal increase at P5 (Weisheit et al., 2006). Golgi cells and Lugaro cells are the earliest born inhibitory interneurons (~E15–P5: Figure 9.1: Miale and Sidman, 1961; Altman and Bayer, 1997; Morales and Hatten, 2006; Carletti and Rossi, 2008) and their somata settle in the immature granular layer. Next, the basket/stellate cells are born (~E19–P10) with the earliest-born settling close to the Purkinje cell layer and becoming basket cells and the later born migrating deep into the molecular layer and becoming stellate cells. The origin of Golgi cells is more controversial as experimental evidence supports both the EGL and the VZ as sites of origin. For example, early studies by Popoff (1896) and Athias (1897)

PATTERNING OF OTHER CELLS IN THE CEREBELLUM

suggested the EGL was the origin of Golgi cells. In support of this, Hausmann et al. (1985) observed “typical Golgi cells” following ectopic explantation of the EGL. Furthermore, Chung et al. (2011) identified a small population of ZAC1-immunopositive Golgi cells, restricted to the posterior zone of the cerebellum, which appears to derive from the EGL between E13–E16. In contrast, Ramón y Cajal (1911) and Altman and Bayer (1977) both concluded that Golgi cells derive from the ventricular neuroepithelium, and this conclusion is supported by retroviral lineage tracing data (Zhang and Goldman, 1996). It is possible that both explanations are correct, and that two lineage-distinct Golgi cell populations are present in the adult cerebellum.

9.2  PATTERNING OF INHIBITORY INTERNEURONS 9.2.1 BASKET/STELLATE CELL PATTERNING There is anatomical and physiological evidence that basket/stellate cells are restricted by zone and stripe boundaries. For example, Chen and Hillman (1993) showed that a protein kinase Cδ-immunoreactive basket/stellate cell subset in the rat cerebellum is strongly concentrated in the AZ. Furthermore, both the axons and the dendrites of basket and stellate cells tend to be oriented parasagittally with Purkinje cell stripes (e.g., Eccles, 1967, Rakic, 1972, King et al., 1993). Consistent with the morphology, physiological studies both in vitro and in vivo confirm that the inhibitory fields of basket/stellate cells are confined to a single stripe, with molecular layer inhibition restricted parasagittally (e.g., Ekerot and Jörntell, 2001, 2003; Jörntell and Ekerot, 2002; Gao et al., 2006; Dizon and Khodakhah, 2011; reviewed by Jörntell et al., 2010). Cerebellar afferent terminal fields become restricted to particular stripes through embryonic interactions with Purkinje cell clusters. However, the same hypothesis is not compelling for basket/ stellate cells as they are born postnatally—too late to interact at the cluster stage (and there is little evidence of basket/stellate subtypes). Rather, the parasagittal orientation of basket cell axonal arbors might be secondary to Purkinje cell cluster dispersal. Molecular layer interneurons appear to invade the immature molecular layer randomly. Once in the molecular layer they contact a local cluster of some 40 Purkinje cells. As lobulation develops, the Purkinje cell layer extends rostrocaudally more than 10-fold with almost no change in width. As a result, the basket/stellate cell terminal field, initially symmetrical, becomes a short, parasagittal stripe (Figure 9.2). By this hypothesis, Purkinje cell cluster dispersal would result in a continuum of terminal field shapes: the earliest-born interneurons enter the molecular layer first and therefore develop the most extended parasagittal terminal fields (basket cells); the later-born interneurons have progressively more symmetrical terminal fields (stellate cells: e.g., Sultan and Bower, 1998).

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FIGURE 9.2: How do basket/stellate cells align with Purkinje cell stripes? Basket cells enter the molecular layer and synapse on a small group of Purkinje cells. As the Purkinje cell clusters disperse parasagittally to form adult stripes, the basket/stellate cells extend with them so that their dendritic arbors are stretched out in the parasagittal plane. Stellate cells enter the molecular layer later in development, after much of the cluster dispersal is over and as a result, their dendritic arbors are less spread out. From Consalez and Hawkes, 2012.

9.2.2 GOLGI CELL PATTERNING Five distinct classes of Golgi cell have been identified based on morphology and differential expression patterns of metabotropic glutamate receptor2, ZAC1 and neurogranin (Simat et al., 2007; Chung et al., 2011). With the exception of the restriction to the PZ of the ZAC1+ subpopulation, nothing is known of the localization of Golgi cell subtypes to particular transverse zones or lobules.

PATTERNING OF OTHER CELLS IN THE CEREBELLUM

However, an additional form of restriction, reminiscent of that of cerebellar afferent terminal fields is seen in Golgi cells. Sillitoe et al. (2008) demonstrated that the apical dendrites of Golgi cells are restricted at Purkinje cell stripe boundaries (i.e., Golgi cell dendrites are confined within territories defined by Purkinje cell stripes; in fact, less than 3% of Golgi cell dendrites cross a Purkinje cell stripe boundary: Figure 9.3). The mechanisms that restrict Golgi cell dendritic arbors are speculative. On the one hand, structural barriers may be present, such as those reported in the somatosensory cortex (Faissner and Steindler, 1995). Alternatively, Golgi cell dendritic restriction may arise naturally as the Purkinje cell matures (e.g., Hekmat et al., 1989; Nagata and Nakatsuji, 1991). In this model, a newly born Golgi cell migrates into the cerebellar cortex via the white matter and contacts a Purkinje cell cluster. As for cerebellar afferents, chemospecific interactions would target Golgi subtypes to particular clusters. Therefore, as the clusters disperse into stripes the Golgi cells would move with them, just as for mossy fiber afferent terminal fields, and therefore also become restricted to specific stripes and zones. Subsequently, they would mimic the mossy fibers and relocate from the Purkinje cell to the granular layer as granule cell migration proceeds. This model also explains the restriction of Golgi cell apical dendrites to particular stripes.

FIGURE 9.3: Interneuron dendrites do not cross Purkinje cell stripe boundaries. The dendrites of neurogranin-immunopositive Golgi cells (red) are restricted at the boundaries between with parasagittal stripes of zebrin II+ (green) and zebrin II– (blue) Purkinje cells. Adapted from Sillitoe et al., 2008.

9.3  ORIGIN AND DEVELOPMENT OF UNIPOLAR BRUSH CELLS Unipolar brush cells (UBC) are glutamatergic interneurons of the granular layer. They are born between E14 and P2 (Mugnaini and Floris, 1994; Abbott and Jacobowitz, 1995; Sekerková et al., 2004; Chung et al., 2009b, 2009c) in the rhombic lip (reviewed in Hevner et al., 2006). If the rhombic lip is removed in embryonic slice cultures, the overall number of UBCs is significantly reduced (Englund et al., 2006). Similarly, UBC number is decreased in the Math1 null cerebellum (Math1 is a prerequisite for the development of rhombic lip derivatives: Machold and Fishell, 2005;

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Wang et al., 2005). Their maturation is protracted—Morin et al. (2001) describe four stages of differentiation extending over a month—and the brush-like dendrite is not morphologically mature until around P28. UBC migration paths from the rhombic lip to the granular layer are complex. Most UBCs migrate into the developing cerebellar anlage, starting at ~E14 (Abbott and Jacobowitz, 1995), and disperse via the presumptive white matter tracts, presumably guided by cues associated with Purkinje cell axons or afferent projections. A second, small population of UBCs reaches the granular layer by following the same dorsoventral migratory route as the granule cells (Abbott and Jacobowitz, 1995; Chung et al., 2009c: Figure 9.4).

FIGURE 9.4: The dual origin of unipolar brush cells (UBCs). UBCs arise in two ways—one population of UBCs arises from the ventricular zone and a second, larger population arises from the rhombic lip. Adapted from Hevner et al., 2006.

9.4  UNIPOLAR BRUSH CELL PATTERNING Three expression markers have been used to identify subpopulations of UBCs—calretinin (CR+), metabotropic glutamate receptor 1α (mGluR1α), and PLCβ4. Double and triple immunolabeling studies with these markers conclude that there are at least three distinct UBC classes in the mouse cerebellum: CR+/PLCβ4−/mGluR1α−, PLCβ4+/mGluR1α−/CR−, and mGluR1α+/PLCβ4+/ CR− (e.g., Figure 9.5: Nunzi et al., 2002, Chung et al., 2009b). Each class has a distinct topographical distribution and there is a clear alignment between UBC subtypes and Purkinje cell stripes. It is hypothesized that this alignment arises by the same three-stage process way proposed for Golgi cells—initial association with specific Purkinje cell clusters, dispersal with the clusters into stripes,

PATTERNING OF OTHER CELLS IN THE CEREBELLUM

and finally displacement from the cluster to the nascent granular layer beneath (reviewed in Consalez and Hawkes, 2012).

FIGURE 9.5: Subsets of unipolar brush cells. Several distinct classes of unipolar brush cells (UBCs) are present in the cerebellar cortex of an adult mouse. (A) Calretinin (CR) is a marker of all UBCs and CR-immunoreactivity reveals UBC morphology. (B) Anti-mGluR1α immunoreactivity in UBCs. (C) Anti-PLCβ4 immunoreactivity in UBCs. (D) Double immunofluorescence staining for CR (red) and PLCβ4 (green) shows no co-expression. Scale bar: E = 5 μm (A–E). From Chung et al., 2009b.

9.5  ORIGIN AND DEVELOPMENT OF CEREBELLAR GLIAL CELLS In addition to Purkinje cells and interneurons, the glial cells also deserve attention in any discussion of cerebellar pattern development (see Buffo and Rossi, 2013 for review). In fact, as described below, ablation of glia during development has severe and widespread effects on neuronal proliferation, differentiation and migration. There are three main glial cells in the cerebellar cortex: astrocytes, Bergmann glia (specialized astrocytes), and oligodendrocytes.

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Following PC genesis (~E10–E13), the progenitor cells in the Ptf1a+ VZ gives rise to inhibitory interneurons and UBCs (Figure 9.2) as well as cerebellar astrocytes, Bergmann glia, and oligodendrocytes (Sudarov et al., 2011; reviewed by Buffo and Rossi, 2013). Numerous studies have explored the possibility that glial cells also arise from the rhombic lip/EGL and the evidence is conflicting. While a majority of studies show that EGL progenitors are committed to a granule cell fate, the results of a few studies indicate that EGL progenitors have the potential to develop into glia (reviewed by Buffo and Rossi, 2013). For example, progenitor cells isolated from the EGL and exposed to Shh and BMP2 lose their neuronal markers and express GFAP, an astrocytic marker in vitro (Okano-Uchida et al., 2004). However, the classical view is that astrocytes and oligodendrocytes arise from the VZ germinal zone in the late embryonic period (reviewed in Buffo and Rossi, 2013). The role of the proneural gene Ascl1/Mash1 (that is transiently expressed in the VZ as described in Chapter 5.2.1) in glial differentiation was investigated by Grimaldi et al. in 2009 using Ascl1 null mutant mice. Ascl1 ablation results in a significant decrease in oligodendrocytes and also a significant decrease in interneurons that are “rewired” to change their fate and differentiate into astrocytes (Grimaldi et al. in 2009). Thus Ascl1 is believed to be required for oligodendrocyte differentiation and to inhibit astrocyte differentiation. Oligodendrocyte loss induced during the first postnatal week leads to widespread and dramatic effects on cerebellar development: the overall size of the cerebellum was reduced and fissure formation was altered; Purkinje cells did not disperse into a monolayer but remained clumped into a “multicellular” layer (likely due to the misalignment and disorganization of Bergmann glia); Purkinje cells dendrites were shorter and “misoriented’; interneurons did not migrate into the cerebellar cortex, and the depth of the internal granule cell layer was significantly reduced (Mathis et al., 2003). Loss of oligodendrocytes during the first postnatal week leads to a 50% decrease in Shh expression in Purkinje cells, a decrease in reelin expression concomitant with decreased numbers of granule cell precursors in the external granule cell layer and altered BDNF/Trk signaling (Collin et al., 2007). Bergmann glia—the specialized astrocytes discovered by Bergmann in 1857 and named by Golgi—are derived from radial glia that undergo morphological changes such that their cell body is close to Purkinje cell soma and multiple processes extend through the molecular layer to the pial surface. During neurogenesis, radial glia processes extend from the ventricular zone towards the pial surface and may act as a “glial monorail” (Hatten, 1990) for immature neurons to migrate along (see Chapter 7.2.2).

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9.6  GLIAL CELL PATTERNING Glial cells have a special place in the history of cerebellar patterning because the first molecular evidence that the cerebellar cortex is striped came from Scott (1963) who reported the dramatic anterior-posterior “banded distribution” of the astroglial enzyme 5′-nucleotidase in the mouse cerebellum (Figure 9.6). A detailed atlas of 5′-nucleotidase expression in the mouse cerebellum has been published (Marani, 1982). 5′-nucleotidase is a cell surface–associated ectoenzyme, associated inter alia with Bergman glial fibers in the molecular layer of the cerebellum. It catalyses the dephosphorylation of 5’-nucleotides into ribonucleosides but whether its role in cerebellar metabolism is the generation of nucleosides (e.g., adenosine) for purinergic signaling is unclear.

FIGURE 9.6: The first demonstration of molecular stripes in the cerebellar cortex. Scott (1963) described differences in staining intensity following histochemistry for 5’-nucleotidase activity. (A) A sagittal section taken from an adult mouse cerebellum shows darker staining in the molecular layer of the posterior lobules (VIb-X) compared to anterior lobules. (B) A coronal section illustrates the parasagittal stripe pattern of 5’nucleotidase activity. From Scott, 1963.

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Indeed, the relationship between the modular stripes seen in cerebellar neurons and the organization of cerebellar glia remains uncertain. However, a direct correlation has been shown between the distribution of 5′-nucleotidase and zebrin I (Eisenman and Hawkes, 1989). Furthermore, it seems likely that the expression of 5′-nucleotidase is induced by the local Purkinje cell environment. For example, in Purkinje cell degeneration (pcd/pcd) and nervous (nr/nr) mice, where there is substantial Purkinje cell loss, stripes of 5′-nucleotidase also disappear except in regions where Purkinje cells survive (Hess and Hess, 1986). Likewise, in reeler (reln/reln) mice 5′-nucleotidase activity is associated with the ectopic Purkinje cell clusters (Eisenman, 1988). How the induction occurs is not known but enzyme activity does seem to be dependent on electrical activity in Purkinje cells and is enhanced by harmaline treatment, which induces tremor by inducing climbing fiber bursting (Balaban et al., 1984: note also that 5′-nucleotidase is expressed at climbing fiber synapses during postnatal development; Schoen et al., 1991). There are other examples of glial responses to the local Purkinje cell environment. For example, parasagittal stripes of activated OX-6- and OX-42-immunopositive microglia and GFAP-immunopositive astrocytes appear in the vermis of the rat cerebellum following an injection of the alkaloid ibogaine (O’Hearn et al., 1993a, b: Figure 9.7). This distribution presumably is secondary to the underlying Purkinje cell architecture probably reflects selective Purkinje cell loss (for more on Purkinje cell death in stripes, see Chapter 10).

FIGURE 9.7: Ibogaine-activated glial cells are organized in parasagittal stripes. When rats are treated with ibogaine, a striped subset of Purkinje cells is believed to die. As they die, local glial cells are activated, and so stripes of glial activation develop in the cerebellar cortex. From O’Hearn et al., 1993.

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Neuronal Cell Death in Normal Development A key part of cerebellar development is neuronal cell death (reviewed by Vogel, 2002; Dusart et al., 2006; Jankowski et al., 2011). Generally, cell death during development ensures that the correct numbers of cells are present, that projection neurons are numerically matched with their targets, and also that cells that are damaged or project incorrectly are removed. Failure to remove these cells may lead to oncogenesis (e.g., see The matching game on granule cell proliferation and medulloblastoma). Thus, once the basic trilaminar structure of the cerebellar cortex has been built, the focus shifts to maintaining precise Purkinje cell and granule cell numbers in order that the mature circuitry can be established. During normal cerebellar development, there is limited evidence for apoptosis in the granular layer (e.g., Lossi et al., 1998, 2002, 2004) and amongst interneurons in the molecular layer (e.g., Yamanaka et al., 2004). However, both effects are small and little is known of their role or consequences. In contrast, much more is understood regarding Purkinje cell death. The matching game The detailed anatomy of the mossy fiber pathway in the cerebellar cortex is confusing. The great divergence of the granule cell projection to the Purkinje cell—each parallel fiber synapses with hundreds of Purkinje cells—is in sharp contrast to the precise topography of the mossy fiber terminal fields. It is not well understood why the mossy fiber terminals are highly focused at narrow bands of granular layer when the granule cells immediately spread the pattern of excitation very widely. However, despite the superficial appearance given by the anatomy, it is the Purkinje cells that immediately overlie a mossy fiber terminal field that respond preferentially when that terminal field is excited; that is, the signal dispersion via the parallel fibers is not as prominent as the cytology might suggest. Part of the explanation may be that the ascending portions of the granule cell axons are especially important in the establishment of Purkinje cell firing patterns. The parallel fiber contribution to the pattern of excitation may then be to provide “context” by informing a Purkinje cell band or patch about the activity of its mediolateral neighbours.

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10.1  PROGRAMMED PURKINJE CELL DEATH IN NORMAL DEVELOPMENT The evidence for early developmental cell death among Purkinje cells is somewhat circumstantial. First, pyknotic cells and microglia were identified throughout the cerebellar cortex in the embryonic (E13) to postnatal (P10) mouse cerebellum with a peak at about P3 (Ashwell, 1990). Subsequently, Krueger et al. (1995) found widespread pyknosis, primarily in the white matter and granular layer of P3-P14 rat pups, and report that most of these cells are astrocytes, which the authors suggest may reflect “competition for limiting amounts of survival factors” (Krueger et al., 1995). Secondly, transgenic mice that overexpress Bcl-2, an oncogene that inhibits apoptosis, have 30–40% more Purkinje cells than wildtype littermates (Zanjani et al., 1996). The evidence that programmed Purkinje cell death occurs in a perinatal developmental window from E15–P6 is reviewed in Dusart et al. (2006). Additional evidence for Purkinje cell death during normal development comes from Jankowski et al. (2011) who confirmed that many Purkinje cells undergo apoptotic cell death during the first postnatal week, with a peak at P3. Purkinje cell death was not random, but in symmetrically disposed clusters on either side of the midline ( Jankowski et al., 2011). A key cell signaling pathway in maintaining a balance between cell survival and apoptosis in the cerebellar cortex involves activation of the Fas receptor (reviewed by Raoul et al., 2000). The protein Lifeguard antagonizes Fas-mediated cell death by interfering with caspase 8 activation (but not the recruitment to the Death-induced Signalling Comples (DISC)) thus preventing apoptosis (Somia et al., 1999; Hurtado de Mendoza et al., 2011). In the adult cerebellum, Lifeguard is expressed in Purkinje cells as well as throughout the molecular layer and internal granular layer (Schweitzer et al., 1988, 1989). A recent study on the effect of reducing or eliminating Lifeguard expression levels using shRNA lentiviral transgenesis or knockout mice found that P7 mice with reduced Lifeguard expression had significantly smaller cerebella and a thinner granular layer than wildtype (Hurtado de Mendoza et al., 2011). However, both these defects attenuate over the following weeks until the adult cerebellum was only slightly smaller and no difference was seen in the thickness of the granular layer (Hurtado de Mendoza et al., 2011).

10.2  PATTERNED PURKINJE CELL DEATH It is clear that significant Purkinje cell death occurs during the perinatal period. Does this play a role in the sculpting of cerebellar topography? Two complementary hypotheses can be considered. First, the study by Jankowski (2011) of naturally occurring cell death in the cerebellum described above, identified a spatial organization to Purkinje cell apoptosis that correlates with stripe boundaries in the adult, and proposed the interesting hypothesis that cell death may sharpen the acellular raphes between clusters. Secondly, naturally occurring cell death could be an error-correction

PATTERNING OF OTHER CELLS IN THE CEREBELLUM

mechanism. A striking feature of adult cerebellar topography is its high reproducibility between individuals and its attendant low error rate (e.g., zebrin II+ Purkinje cells are very rarely seen in zebrin II- stripes). If stripes derive from clusters, and stripes have no errors, then either clusters have no errors (and migration from the ventricular zone into clusters is close to perfect), or errors that occur during cluster formation are subsequently eliminated. In this context it is interesting that many Purkinje cells—perhaps as many as a third—undergo cell death by apoptosis during the perinatal period (Dusart et al, 2006; Jankowski et al., 2009). This suggests the paired hypotheses that perinatal apoptosis might both sculpt stripe boundaries and eliminate Purkinje cells that wind up in the wrong embryonic cluster. Purkinje cell ectopia is not lethal per se: for example, clusters that fail to disperse normally do not die (e.g., reeler (Goffinet, 1983; Edwards et al., 1994), weaver (wv/wv: Armstrong and Hawkes, 2001), Vldlr-/-:Apoer2-/- (Larouche et al., 2008), Dab1-/- (Howell et al., 1997)), and Purkinje cells located ectopically in the molecular or granular layers survive indefinitely. Rather, one might evoke a community effect (à la Yang et al., 2002), such that being in the wrong cluster leads to apoptosis (possibly via a local insulin-like growth factor 1 pathway—Croci et al., 2011; see also Jung et al., 2008). However, preliminary data on the Bax-/- mutant cerebellum do not support these interpretations. BAX is a Bcl-2/BH3–associated protein expressed in normal Purkinje cells perinatally that promotes apoptosis by binding to and antagonizing the anti-apoptotic Bcl-2 protein (Gillardon et al., 1995a, b). In Bax-/- mice, perinatal Purkinje cell death is inhibited with the result that the cerebellum has many more Purkinje cells (a 30% excess compared to controls: White et al., 1998; Fan et al., 2001; Vogel, 2002), as well as Purkinje cell ectopia, suggesting Purkinje cell migration problems ( Jung et al., 2008). Similarly, in transgenic mice with pan-neuronal overexpression of the Bcl-2 there is a 27–43% increase in Purkinje cell number (Zanjani et al., 1996). This increase was attenuated when anti-apoptotic proteins were overexpressed specifically in Purkinje cells (Goswami et al., 2005). Behaviorally, mice with excess Purkinje cells show no difference in motor coordination at three months of age but significantly motor control deficits by six months (Goswami et al., 2005). If apoptosis refines cerebellar topography, one would expect many more errors in stripe architecture (e.g., ectopic zebrin II+ Purkinje cells in zebrin II- stripes) in mice with disrupted apoptosis pathways. However, this is not the case—for example, while Bax-/- stripes are wider than normal, there is no significant increase in topographical errors (Wang and Hawkes—unpublished data). Any role for Purkinje cell death in the establishment of cerebellar stripe patterning thus remains speculative (Pathological Purkinje cell death).

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Pathological Purkinje cell death The role of cell death in normal development is still unclear but there is a second manifestation of patterned Purkinje cell death—that which occurs in many pathologies—that is better understood. The cerebellum shows different patterns of striped Purkinje cell loss in response to injury or insult—ranging from ibogaine intoxication (a treatment for heroin addiction), to selective mutation, to prion diseases, to epilepsy (reviewed in Sarna and Hawkes, 2003). Likewise, zebrin II- Purkinje cells were recently shown to be selectively sensitive to cell death, requiring Igf1 to promote their survival (Croci et al., 2011). This suggests that different Purkinje cell subtypes exhibit differential susceptibility to perinatal damage. Recognizing which stripes have particular sensitivities is the first step towards the rational development of neuroprotective and restorative strategies (Sarna and Hawkes, 2003).

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Conclusion/Summary “The significance of this unusual pattern of localization is not immediately apparent and any attempt at explanation must be conjectural.” Scott (1963) Pattern formation is an elaborate process that involves all cerebellar cell types, projection neurons and interneurons, glial cells and afferent inputs, and extends over at least a month of development in the mouse (before E9 to after P20). First the cerebellar territory is specified in the neural tube and the anterior end of the hindbrain. Then Purkinje cells are born with different phenotypes, and these migrate from the VZ to form a cluster array. This array is the scaffold around which interneuron and afferent topography is established. Thus, by around birth, all the key features of cerebellar patterning are in place. Postnatally, the embryonic cerebellum undergoes an elaborate morphological transformation, which converts the cluster architecture into the adult array of zones and stripes. The upshot is that the cerebellar cortex is divided into hundreds of reproducible stripes, each with specific inputs, and possibly into thousands of reproducible patches. While it is beyond the scope of this review, a few words on the possible functional significance of cerebellar architecture are not out of place. At this stage we can only speculate—very little concrete is known (e.g., Hawkes and Gravel, 1991; Hawkes, 1997; Ebner et al., 2012; etc.). Perhaps multiple stereotyped circuits of the kind we have described are a substrate for the parallel processing of motor information and motor commands. Individual stripes or patches could be devoted to specific tasks or subtasks and the segregation of afferents could achieve the positional coding of sensory information, so that minor inputs could be focused and appropriately assessed and used. Furthermore, stripes could be customized such that different sensory inputs are processed differently—the extensive molecular differences between stripes and zones certainly point in that direction. One example of this might be the long-term depression at the parallel fiber-Purkinje cell synapse that is mooted to underpin aspects of motor learning in the cerebellum. Conjunctive stimulation of the parallel fiber and climbing fiber pathways produces a long-lasting depression of synaptic transmission at the synapses between the parallel fibers and Purkinje cells. The observed decrease in synaptic strength results from a reduction in the sensitivity of postsynaptic AMPA receptors (e.g., Ito and Kano 1982). Some of the molecular basis of long-term depression is understood, and it is striking that many components are distributed in stripes—nNOS (Chapter 7), mGluR1 (Mateos et al., 2000), excitatory amino acid transporter 4 (Dehnes et al., 1998), corticotropin releasing

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factor (Sawada et al., 2008), PLCβ3/4 (Chapter 3), the inositol triphosphate receptor (Furutama et al., 2010), protein kinase C (Barmack et al., 2000), etc. This suggests the hypothesis that long-term depression at the parallel fiber-Purkinje cell synapse is different in different stripes.

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Author Biographies Dr. Carol Armstrong completed her undergraduate degree in Biological Sciences from the University of Calgary and a Masters Degree in Anatomy and Neurobiology at Dalhousie University under the mentorship of Dr. David Hopkins. She returned to the University of Calgary as a CIHR-funded doctoral student and completed a Ph.D. in Neuroscience with Dr. Richard Hawkes, focusing her studies on heat shock proteins and pattern formation in the developing cerebellum. Following three years as a CIHRfunded postdoctoral fellow in Dr Dennis O’Leary’s Molecular Neurobiology lab at the Salk Institute for Biological Studies (La Jolla, CA), Carol accepted a position as an Assistant Professor in Biomedical Science at the Ontario Veterinary College in Guelph, ON (2005–2010). She is currently an Associate Professor in Biology at Mount Royal University (Calgary, AB) with an adjunct appointment in the Faculty of Medicine at the University of Calgary. Richard Hawkes, Ph.D., graduated from University College London and the University of Hull in the U.K., and completed postdoctoral fellowships at the Hebrew University of Jerusalem, the University of Oregon U.S. and the Friedrich Miescher Institut in Basel Switzerland, before taking up an academic appointment at Université Laval in Québec, Canada in 1982. It was during that time he discovered the markers of cerebellar compartmentation known as zebrins. He moved to the Faculty of Medicine, University of Calgary, Canada in 1989, where he served first as Head of Cell Biology and Anatomy, subsequently as Associate Dean Graduate Studies, Senior Associate Dean Research, and Associate Vice President Research, and latterly as doting grandfather. In 2005 he was awarded Doctor of Medicine (honoris causa) from Buenos Aires, Argentina. He has served on multiple editorial boards, granting panels, and advisory committees to both government and the private sector. He is currently Professor of Cell Biology and Anatomy, and a member of the Hotchkiss Brain Institute and the Alberta Children’s Hospital Research Institute at the University of Calgary. His research for over 25 years has focused on pattern formation during cerebellar development. His 300-odd publications have been cited over 10,000 times.