Molecular Approaches to Supracellular Phenomena [Reprint 2016 ed.] 9781512806441

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Molecular Approaches to Supracellular Phenomena

University of Pennsylvania Press D e v e l o p m e n t a l B i o l o g y Series Stephen Roth, Editor Department of Biology University of Pennsylvania

Molecular Approaches to Supracellular Phenomena Edited by Stephen Roth

Ujljl University of Pennsylvania Press

Philadelphia

Copyright © 1990 by the University of Pennsylvania Press All rights reserved Printed in the United States of America

Library of Congress Cataloging-in-Publication Data Molecular approaches to supracellular phenomena / edited by Stephen Roth. p. cm. — (Developmental biology series) Includes bibliographical references. Includes index. ISBN 0-8122-8251-5 (cloth) 1. Cell differentiation. 2. Molecular biology. I. Roth, Stephen, 1942— . II. Series: Developmental biology series (Philadelphia, Pa.) [DNLM: 1. Cell Differentiation—physiology. 2. Extracellular Matrix—physiology. QH 607 M718] QH 607.M65 1990 574.87'612—dc20 DNLM/DLC for Library of Congress 90-12412 CIP

Contents

Introduction Regulation of Timing in Dictyostelium Morphogenesis and Other Developing Systems David R. Soil Role of Oncogenes in Differentiation

vii

1 83

David Boettiger Embryonic Origins of Segmented Nervous Systems

137

Charles B. Kimme I Cell Adhesion Molecules

175

Urs Rutishauser Glycosyltransferases as Effectors of Cell Recognition

201

Stephen Roth Contributors Index

225 227

Introduction

Developmental biologists are at, or just over, the threshold of exciting discoveries and new principles, and marriage to molecular genetics has carried us here. Although that marriage, like all marriages, is so full of promise, it would nevertheless be prudent to ask some important questions. How much further can we expect to be carried? What can we contribute to the union? Can we, and should we, attempt to maintain our spousal identity as developmental biologists? What do we want our children to be like? No book, or series of books, will answer such questions for all developmental biologists. We hope here, however, to present some of the most fascinating and complex developmental problems, not in a descriptive and archival fashion, but as specific and experimentally accessible hypotheses. As scientists, the greatest hope we can have for our hypotheses is that they are sufficiently exciting and vulnerable as to be eliminated by rigorous, elegant experiments. They will then be reborn, in modified forms, to risk once again the challenge of elimination by experiment and observation. This is the process that eventually yields a close approximation to what scientists call truth. This first volume contains five chapters, each of which presents specific, conceptual frameworks for five very important developmental problems that, by their natures, resist conventional molecular approaches. The first chapter, by David Soli, deals with the single dimension that best characterizes developmental phenomena—time. When we strive to explain what it is that makes developmental biology different from, say, physiology, or biochemistry, or cell biology, the closest we can come to a satisfactory answer is that developmental events occur only once in the lifetime of a cell or organism, and that these events occur vectorially and irreversibly. Gastrulation, neurulation, and organogen-

vlll

Introduction

esis h a p p e n only once, a n d , a l t h o u g h they can be arrested, usually they cannot be reversed. To the extents that regeneration, metamorphosis, carcinogenesis, a n d evolution are temporally vectorial, they may also be t e r m e d developmental. Despite the central significance of time to developmental biology, it has stubbornly resisted analysis. Using the slime mold Dictyostelium as a model, Soli describes m e t h o d s f o r d e t e r m i n i n g w h e t h e r temporally linked events are also causally linked, a n d , if so, w h e t h e r the linkages are serial or parallel. In addition to methodology a n d strategy, this c h a p t e r discusses o t h e r developmental systems whose t e m p o r a l aspects have been successfully attacked using the techniques described. T h e second chapter, by David Boettiger, presents several possible functions f o r proto-oncogenes d u r i n g development. A critical question concerning proto-oncogenes is, of course, why are they there? O n e answer that is becoming m o r e a n d m o r e attractive is that protooncogenes allow cells to behave in ways that are necessary f o r embryogenesis b u t that are relatively f o r b i d d e n postembryonically. For example, embryonic cells o f t e n must u n d e r g o rapid mitoses even t h o u g h s u r r o u n d e d by o t h e r cells; some embryonic cells also migrate large distances, sometimes t h r o u g h whole organs. Neither of these behaviors is typical of adult cells, a l t h o u g h both behaviors are typical of malignant cells. Wholesale changes in differentiation states a n d modifications to m e m b r a n e biochemistry are o t h e r p h e n o t y p e s shared by embryonic a n d cancerous cells. For molecular geneticists, proto-oncogenes could prove to be the most direct inroad to mechanisms of embryogenesis. T h e r e m a i n i n g t h r e e chapters deal with d i f f e r e n t aspects of what I think is the most difficult of the developmental p h e n o m e n a to quantitate and, t h e r e f o r e , to deal with analytically—morphogenesis, the acquisition of biological f o r m . A l t h o u g h it is clearly hereditary at the level of the species, a n d even the individual, we can still say that n o g e n e p r o d u c t in any organism has yet been identified that is responsible for these morphological characteristics. At the genetic level, in o t h e r words, we d o not know the basis of t h e difference between a d o g a n d a cat. Still less d o we u n d e r s t a n d the differences between how genes specify an a r m a n d a leg, or a right h a n d a n d a left h a n d . All o r g a n systems are m o r p h o g e n e t i c marvels, b u t n o n e has fascinated biologists as m u c h as the typical nervous system. Bilaterally symmetric, intricately wired, a n d responsible ultimately f o r o u r investigations into its own n a t u r e , the central a n d peripheral nervous systems have attracted f a r m o r e attention t h a n any o t h e r single system. Charles Kimmel puts forth a mechanism f o r the generation of nervous systems by segmental iteration, a n d Urs Rutishauser describes recent

Introduction

Ix

progress and problems with the neural cell adhesion molecule, NCAM. These chapters propose that, at both cellular organization and biochemical levels, the cells that make up vertebrate nervous systems are more closely related than previously thought. Certainly, the possible clonal relationship among cells at similar positions, but different segments, could also explain the widespread presence of similar cell adhesion molecules, and the ways in which so few cell adhesion molecules might effect so many different processes could also be more easily understood. The last chapter presents my own hypothesis for an evolutionary continuum of enzymes that initially, perhaps, only synthesized complex carbohydrates. Later, on the cell surface, the enzymes would recognize these same carbohydrates and may have evolved into primitive immunoproteins, and, eventually, immunoglobulins themselves. All of these chapters present models that try to account in cellular or biochemical terms for supracellular or histological phenomena. The hypotheses will be successful if they stimulate future experimentation. They will be valid if they survive that experimentation. For me, at least, it will be enough if they are only successful. I thank the contributors for the thoughtfulness and care with which they prepared their chapters, and I thank Tom Rotell of the University of Pennsylvania Press for his interest in this project. Any errors of language, style, or presentation, however, are mine. Stephen Roth

Philadelphia October 1989

Chapter 1

Regulation of Timing in Dictyostelium Morphogenesis and Other Developing Systems David R. Soli

Introduction Whether an individual cell is differentiating, or a group of cells is undergoing morphogenesis, the major developmental aspect shared by each system is phenotypic change. It is, therefore, surprising that so little attention continues to be paid to the fundamental question of why events happen when they do, a problem we will refer to as developmental timing. There may be several reasons for this inattention (Soli, 1979, 1983). First, many biologists interested in biorhythms believe that the formal problem of timing is exclusive to circadian rhythms. Similarly, many biologists interested in endocrine systems believe that timing is a problem of hormonal release. The main reason that developmental timing has been a neglected problem, however, stems from the developmental biologists' conception of timing. When the timing of stages accompanying differentiation is considered, there is commonly an underlying assumption that because stages, by definition, are in temporal order, the causal events for these stages must be in the same temporal order. In turn, it is assumed that causal events are timing events. This simplistic view of developmental timing appears to have led to a trivialization of the problem of timing regulation. In the discussion that follows, I hope to demonstrate that timing regulation is a fundamental problem in development, that there are very specific modes of conceptualizing and discussing the timing aspects of a developmental system, and that there are procedures for discriminating and dissecting those molecular pathways or processes that determine when events happen in developing systems. I will begin by formulating a set of methods for dissecting the complexity and relationships of timing pathways in a developmental program without

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knowledge of their molecular identities (Soil, 1979, 1983, 1987), and then describe the results obtained when these methods are applied to the morphogenetic program of Dictyostelium discoideum (Soli, 1979; Mercer and Soli, 1980; Varnum et al., 1983; Soil and Finney, 1987), the early development of sea urchin development (Matsumoto et al., 1988), and water-mold sporulation (Peralta and Lodi, 1988). In addition, I will consider the possibilities that particular developmental pathways may have evolved as developmental cues, that the regulatory regions of differentially expressed genes tell time, and that variations in developmental timing may be effected by transposons. Finally, I will consider the relationship of developmental timing pathways and the evolutionary hypothesis of heterochrony. What D o We M e a n by D e v e l o p m e n t a l Timing? In each developing system, a sequence of events can be ordered in time. For instance, in bacterial sporulation, a cell in the growth phase differentiates into a highly specialized, resistant spore (Fitz-James and Young, 1969). T h e stages in sporulation can be temporally ordered, and the sequence includes (1) the transition to the axial stage, (2) forespore development, (3) cortex development, (4) coat-protein deposition, (5) dehydration of the spore protoplast and accumulation of dipicolinic acid and calcium, (6) refractility, and (7) spore release. In addition to temporal order, these stages can also be timed. T h e difference between ordering and timing is an important one. In many developing systems, the order of most stages may be invariant under a wide range of conditions, but timing may vary dramatically and differentially. In our discussion of timing regulation, our interest will be to understand why stages occur when they do under a single set of environmental conditions. T h e ordering of events and distinctions of dependence and independence (Pringle, 1978) are basic to an understanding of causality in a developing system, but knowing why events happen at a particular time brings us into the realm of developmental timing, a distinction that will become less esoteric in the following sections in which we attempt to develop a formal conceptual framework for timing analyses. Although I have begun a discussion of timing with an example of prokaryotic differentiation, the same considerations are applicable to both lower and higher eukaryotic differentiation. For example, in the water mold Blastocladiella emersonii, a motile zoospore progresses through four phenotypes, or stages, during germination (Soll et al., 1969; Soli and Sonneborn, 1971): (1) the initial zoospore stage, characterized by a posterior single flagellum, an ameboid cell body, a single

Regulation of Timing In Developing Systems

3

nucleus, a single nuclear cap containing all the cytoplasmic ribosomes, and a single giant mitochondrion with lipid side bodies embedded in it; (2) the round cell I, which is spherical with a flagellar axoneme retracted into the cell body and an incipient cell wall; (3) the round cell II, which includes a fragmented and branched mitochondrion, released ribosomes, and disintegrated nuclear cap; and (4) the germling, which possesses a germ tube (Figure 1-1). Under constant environmental conditions (Soil and Sonneborn, 1969), these stages are rigidly ordered and timed. Again, there are two timing characteristics that we have noted, temporal order and actual timing (Figure 1-1). In terms of order, the zoospore stage precedes the round cell I stage, the round cell I stage precedes the round cell II stage, and the round cell II stage precedes the germling stage. In terms of timing, upon initiation of differentiation under rigidly defined laboratory conditions, the cells remain, on average, as zoospores for 10 minutes, then rapidly form the round cell II phenotype in the next 10 minutes, and the germling phenotype in the next 8 minutes. In total developmental time, the round cell I, round cell II, and germling phenotypes occur after approximately 12, 20, and 28 minutes. In the context of order, we may first ask if the sequence of stages is rigid. Because stages represent a combination of organellar changes, we may ask whether the organellar changes that comprise each stage are obligative temporal partners or whether they can be temporally dissociated. This consideration leads in turn to the dependence and independence of the individual changes associated with each stage. For instance, can the disintegration of the nuclear cap occur if the flagellar axoneme is not retracted? Or can the germ tube form if the disintegration of the nuclear cap does not occur? These are questions of order and causality, and they impinge upon timing regulation. In the context of timing regulation, we may ask why the particular stages occur when they do. Why does it take 10 minutes before the flagellum is retracted and the incipient cell wall synthesized? Why does it take 10 subsequent minutes to form round cell II and 8 subsequent minutes to form a germling? If the period to round cell I were contracted, would the total times to round cell II and germling be contracted by the same amount of time? In other words, are the ratelimiting processes for successive stages in sequence? It is clear that in considering the regulation of developmental timing in sporulation or zoospore germination, we can formulate questions that are related to order and causality, and we can formulate separate questions related to timing, or why things happen when they do. The same consideration of timing can be applied to higher eukaryotic, single-cell differentiation, such as the differentiation of a proerythroblast to a reticulocyte (Rifkin, 1974) after erythropoietin

Figure 1-1. T h e stages of zoospore germination in the water mold Blastocladiella emersonii. T h e zoospore represents a stable phenotype that can be induced to differentiate to a vegetative thallus by addition of monovalent cations at high concentration or removal of a zoospore maintenance factor (Soli a n d Sonneborn, 1969, 1971). Note the two timing aspects of the system: temporal order, and the absolute times to r o u n d cell I, r o u n d cell II, and germling. BB, basal body; Nuc, nucleolus; R, ribosomes; NC, nuclear cap structure; GP, gamma particle; MVB, multivesicular body; V, vesicle; LG, lipid granule; SB, side body; M, mitochondrion; F, flagellum; CW, cell wall; FA, flagellar axoneme; VF, vesicular fusion; ER, endoplasmic reticulum; GT, germ tube (Soli a n d Sonneborn, 1971).

Regulation of Timing In Developing Systems

5

stimulation, or the differentiation of a myoblast to a muscle cell (Königsberg, 1963). It can also be applied to the genesis of multicellular form, and, in fact, multicellular morphogenesis in the slime mold Dictyostelium discoideum has been the prime experimental system for investigating the complexity and relationships of timing pathways. It will, therefore, serve as o u r major example in the timing discussions that follow.

What Don't We Mean by Developmental Timing? In the preceding section, I have attempted to provide a general notion of what will be considered developmental timing. Now let us distinguish between developmental timing and other timing processes in biological systems. T h e most important distinction must be made between developmental timing and circadian rhythms. This is best done by simply presenting a definition of circadian rhythms. "A circadian rhythm is an oscillation in a biochemical, physiological or behavioral function which u n d e r conditions in nature has a period of exactly 24 hours, in phase with the environmental light and darkness, but which continues to oscillate u n d e r constant but permissive conditions of light and temperature with a period of approximately but usually not exactly 24 hours" (Sweeney, 1975). Circadian rhythms have features of entrainment and temperature insensitivity (Takahashi and Zatz, 1982). They are f o u n d t h r o u g h o u t the eukaryotic world (Hastings and Schweizer, 1975) and can be affected by single-gene mutations (Gardner and Feldman, 1980; Yu et al., 1987). When we talk about developmental timing, we refer to the individual stages in an emerging, continuously changing developmental program. T i m i n g events in these systems may be entrainable, in some systems they may be 24 hours in extent, and in some cases they may have oscillatory characteristics, but they will not have the combined characteristics of circadian rhythms, and they will rarely be entrained by light-dark, 24-hour cycles. In most developing systems, especially in the lower eukaryotic systems examined in some detail, timing events are dramatically shorter than 24 hours and do not cycle (Varnum et al., 1983). This is not to say that developmental programs cannot be cued by circadian rhythms. It is, however, the timing characteristics of the stages in the cued program that we will consider in o u r discussion. A distinction must also be made between a hormonally induced event in a complex system and what we will consider to be developmental timing. In complex, multicellular eukaryotes, in order for tissues in one body location to signal tissues in another, the first tissue may release a h o r m o n e that travels through the bloodstream to the respon-

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David R. Soll

sive tissue (Snyder, 1985). Hormonal cues can be regulated in time (White and Nicoll, 1981; Granger and Bollenbacher, 1981), and their secretion can be the result of a program involving developmental timing. T h e hormone signal in turn stimulates in the responding tissue a developmental program with developmental timing at the level of cellular differentiation, proliferation, and tissue morphogenesis (Topper and Freeman, 1980; Kratochwil and Schwartz, 1976). In this case, our discussion will be limited to the secreting tissues' developmental program that leads to secretion, or to the developmental program in the responding tissue, but not to the dynamics of signaling between tissues in the complex organism. The Temporal Aspects of a D e v e l o p m e n t a l Program To develop methods that will allow us to dissect the order, complexity, relationships, and, eventually, molecular nature of timing pathways, we must carefully define the parameters of a hypothetical developmental system. In this system, there are three definable stages: A, B, and C. Stage A precedes stage Β and stages A and Β precede stage C. This relationship is shown in Representation 1. We must immediately scrutinize this representation for the elements that make it a graphic description. First, there is the necessary time element on the horizontal axis. We can detail Representation 2 with the scale in arbitrary units.

C'j

- (

Representation 1

Representation 2

Regulation of Timing In Developing Systems

7

Stage A, in this case, is the originating phenotype, stage Β is the phenotype at 5 time units, and stage C the phenotype at 10 time units. It should be noted that in the initial representation A, B, and C are ordered but not timed; in the second representation A, B, and C are both ordered and timed. In these representations, the arrows show the order of stages: A precedingß, Β preceding C. In turn, order usually suggests causality in the sense that Β cannot occur unless A has occurred, and that C cannot occur unless Β has occurred. It should be realized, however, that even if this causal relationship in a developmental system is true, it need not, and usually does not, reflect the complexity of the causal pathways involved in the genesis of each stage. In fact, the sequence of causally linked stages usually does not reflect the complexity of the timing pathways. In the representation of our developmental program, we have placed stages A, B, and C in discrete positions along the time axis. This representation, however, is ambiguous because there are several scenarios for the transitions from A to Β and from Β to C. First, A, B, and C may represent discrete stages and their rapid appearance may occur at the times noted on the time axis. Such a scenario would be described by Representation 3. In this case, A remains A between 0 and 5 time units. At 5 time units, there is a rapid transition to Β. Β then remains Β between 5 and 10 time units. At 10 time units, there is a rapid transition from Β to C. Alternatively, A, B, and C may represent identifiable moments in a continuum, in which case Representation 4 is applicable. In this case, the phenotype is continuously changing, A evolves to B, which in turn evolves to C. The phenotypes Β and C in this case have no permanency over a time interval, as in Representation 3. The distinction between the scenarios in Representations 3 and 4 is not trivial because it affects our consideration of timing regulation. In the continuum scheme in Representation 4, one would immediately

I 0

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Time (arbitrary

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Γ 10

units)

Representation 3

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David R. Soll

hypothesize that the periods between A and Β and between Β and C are dictated by the actual time necessary for the observed transitions. In contrast, the discrete steps observed in Representation 3 suggest that the periods between A and Β and between Β and C represent preparatory processes that precede rapid phenotypic transitions.

Causal Events and Timing Pathways In the preceding section, we have considered how to conceptualize the temporal aspects of a developmental program. Before we delve into the more f u n d a m e n t a l question of what time represents, however, we must discriminate between what is necessary for a transition f r o m stage A to B, or Β to C, and what dictates timing. In most developmental transitions in complex systems, a n u m b e r of gene products and specialized functions are necessary for a phenotypic transition f r o m A to B. Indeed, the estimates for the n u m b e r of genes involved in the first stage of slime mold development are well over 100 (Firtel, 1972; Loomis, 1978), yet this transition can occur in a few hours. Even the Saccharomyces cell cycle, which also can occur in a few hours, involves a large n u m b e r of gene functions (Pringle and Hartwell, 1981). T h e r e fore, it seems reasonable to suggest that in a developmental program, several essential pathways function in parallel. Recent observations not only on the cell cycle (Mitcheson, 1971; Hartwell et al., 1974; Pringle and Hartwell, 1981) but also on differentiation pathways (Varnum et al., 1983; Soli, 1986; Matsumoto et al., 1988; Peralta and Lodi, 1988) demonstrate that this is indeed the case. Let us consider the alternative possibilities for the relationships of essential events for the genesis of stage ß in a developmental program. In the following representations, the blunt arrow shows essential processes. T h e simplest model is a single process a between A and Β (Representation 5). Note that the single arrow represents a single process with uniform characteristics along its entire length. In this simple representation, the duration of a

0

5 Tlmt (arbitrary

10 unite)

Representation 4

Regulation of Timing In Developing Systems

9

is 5 arbitrary time units. In a m o r e complex model, a single d e p e n d e n t pathway includes a sequence of consecutive events, each d e p e n d e n t u p o n the preceding event (Representation 6). Note that each arrow represents a d i f f e r e n t essential event with a u n i q u e set of characteristics; deletion of any event results in pathway termination and, therefore, n o genesis of stage B. In a n even m o r e complex situation, parallel d e p e n d e n t pathways may e x t e n d f r o m A to Β (Representation 7). In this representation, t h e r e are two parallel d e p e n d e n t pathways, 1 a n d 2. Pathway 1 includes t h r e e events a n d pathway 2 includes two events. Deletion of any event leads to the termination of that pathway, b u t n o t to t h e termination of the parallel pathway. Deletion of any o n e essential event t h e r e f o r e leads to either n o genesis o r a b e r r a n t genesis of stage B. Convergence a n d divergence of pathways may also occur. In the f o r m e r case, two o r m o r e pathways may converge into a single pathway

ι

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Representation 5

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David R.Soll

(Representation 8). In the latter case, one pathway may diverge into multiple pathways (Representation 9). In all of the preceding models, each essential event occurs over a time period. Does this mean that each event is involved in developmental timing? The best way to answer this question is to consider how dependent events are involved in the timing of stage B. Let us consider a situation in which there are three parallel pathways essential for the genesis of stage Β (Representation 10). In order to generate stage B, one must complete pathways 1, 2, and 3. Termination time, however, varies for the three pathways. Pathway 1 is completed first, pathway 2 next, and pathway 3 last. Β cannot be generated until pathway 3 is complete, making pathway 3 the determining timing pathway. Therefore, although all three pathways are essential, only pathway 3 dictates the timing of stage B. This simple consideration of timing regulation leads

T

T

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Time (arbitrary units) Representation 7

ΊΓ

T

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Time (arbitrary units) Representation 8

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to a useful definition of a timing pathway, or developmental timer (Soli, 1983): "the pathway that dictates the timing of a developmental stage under a single set of environmental conditions." This definition must be considered to be rigid in order to relieve it of any unnecessary assumptions. In its simple form, it does not address the complexity of a timer pathway (i.e., the number of components in sequence that constitutes the pathway); it does not preclude the possibility that the identity of the timing pathway may change when one or more environmental variables are changed; and it does not enumerate the number of stages regulated by a single timer (Soli, 1983). The only limitation on the definition is that it is best applied to a developmental situation in which the genesis of stage Β is a discrete event, as in Representation 3.

Representation 9

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Time (arbitrary units) Representation 10

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David R.Soll

Developmental Timers Is a developmental timer simply that essential pathway that dictates when a stage is generated under a particular set of environmental conditions, as in Representation 10, or have developmental timers evolved as timing cues? The answer to these questions is relevant both in our understanding of timing regulation and in consideration of the evolutionary theory of heterochrony (Gould, 1977). Let us begin with timing regulation. If a developmental timer simply represents the slowest or last-tobe-completed essential parallel pathway under one set of environmental conditions, then it is possible that, as a result of differential sensitivity, the identity of the developmental timer for that stage could

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A. Environmental conditions X

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Representation 11

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change under different environmental conditions (Representation 11). In the situation in Representation 11(A), pathway 3 dictates the time to Β u n d e r the set of environmental conditions labeled X. When the conditions are changed to Y, the time to stage Β expands from 5 to 8 time units, but the three essential pathways do not expand proportionately. Pathway 2 is far more temperature sensitive than pathway 3 under conditions Y and the last-to-be-completed pathway essential for stage B. Therefore, the identity of the timing pathway changes from 3 to 2 with a change from X to Y conditions. This scenario would suggest that there is no unique timing pathway for stage Β and that a timer is simply the slowest or last-to-be-completed essential pathway u n d e r a particular set of environmental conditions. In contrast, we may find that under all sets of conditions in which Β is generated, one pathway acts as a cue and therefore has evolved as a specific developmental timer. In this case, if the system included a

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Timer)

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5 Time (arbitrary units) A. Environmental conditions X

2

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ο

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B. Environmental conditions Y Representation 12

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David R.Soll

number of essential parallel pathways for the genesis of stage B, the timing pathway would function as a cue under all conditions. In such a situation, a set of conditions that would expand an essential parallel pathway beyond the length of the timing pathway would lead to an aberrant stage B, since the cue would precede the completion of all essential pathways (Representation 12). If this was the case, then it would be possible for a subset of essential processes to evolve as temporal cues in a developmental program and, therefore, to represent an underlying regulatory program that temporally organizes the diverse molecular events essential for each phenotypic or morphogenetic transition. Timing Models for Consecutive Stages in a Developmental Program In the previous sections, we have considered timing regulation of a single stage B, but we have not considered relationships between timers for consecutive stages in a developing system. There are four major models that may function in the temporal regulation of consecutive stages (Soil and Finney, 1987): the single timer model (Figure 1-2A), the sequential timer model (Figure 1-2B), the parallel timer model (Figure 1-2C), and the branched timer model (Figure 1-2D). The single timer model The most discussed model of timing regulation in biological systems, especially for the cell cycle (Wille et al., 1977; Lloyd et al., 1982), is the single timer model, which includes clock models. In the single timer model, a single, continuous process, with uniform characteristics along its entire length, cues sequential stages in the developmental program and is represented in Figure 1-2 as a single horizontal timing pathway. Vertical arrows in this case simply represent the times at which consecutive stages are cued but do not represent additional timing components. The single timing pathway cues multiple stages and is a single, continuous molecular process. The sequential timer model The most assumed mechanism for timing regulation in developing systems is the sequential timer model. As noted in the Introduction, because the stages of a developmental program are in sequence, there is an unstated assumption that essential processes and, therefore, timing processes are also in the same sequence. In the sequential timer model (Figure 1-2B), the timing pathway for Β precedes the timing pathway for C. The two pathways are in a dependent sequence. Com-

Regulation of Timing in Developing Systems

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pletion of the initial pathway results in the genesis of B, and completion of the second pathway results in the genesis of C. Although in sequence, the two pathways represent different molecular processes. If they did not, they would in fact represent the single timer model (Figure 1-2A). The parallel timer model The least considered model is the parallel timer model (Soil, 1979) (Figure 1-2C), in which two, independent, and parallel timing path-

A. Single timer model

B. Sequential timer model

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Figure 1-2. The major models of timing regulation for consecutive stages Β and C in a developmental program. The horizontal arrows represent temporal progress along a timing pathway.

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ways dictate the timing of stage Β and stage C, respectively. In this situation, the genesis of stage C may be dependent on the genesis of stage B, but the time to C is not dependent upon the timing of B, except that Β must occur before C. The timer pathway to C is, therefore, progressing prior to the genesis of B, and represents a molecular process distinct from the timing pathway for B. The branched timer model The final timing model is a composite and includes a point of pathway divergence prior to the genesis of stage Β (Figure 1-2D). In this model,

Figure 1-3. T h e expansion of single timing processes occurs linearly in time. In the first case, a linear reaction cues stages Β and C under short (panel A) and long (panel B) conditions; in the second case, an exponential reaction cues stages Β and C under short (panel C) and long (panel D) conditions. Dashed lines indicate the product concentrations that cue stages Β and C.

Regulation of Timing In Developing Systems

17

a single pathway initiates the timer program, then cues two parallel timing pathways that cue Β and C. Alternatively, a single timer pathway may cue a subpathway for either Β or C. In the models in Figure 1-2, the arrows represent single molecular processes and should exhibit uniform properties along their entire lengths. Therefore, if these timing processes do represent single molecular reactions or molecular interactions, it does not matter whether the reactions are linear, exponential, or logarithmic. In all cases, progress in time dictated by these processes will be linear. This point is perhaps best demonstrated by comparing a single timer based on a reaction in which the concentration of product, which acts as a cue, increases linearly (Figure 1-3A), with a single timer based on a reaction in which the concentration of product increases exponentially (Figure 1-3C). In both cases, stage Β is cued at 5 hours and stage C at 10 hours. In the case of the linear reaction, 5 units of product cue stage Β and 10 units cue stage C (Figure 1 -3A). In the case of the exponential reaction, 2 units of product cue stage Β and 6 units cue stage C (Figure 1-3C). If the rate of the linear reaction is halved by a change in an environmental parameter, it takes twice as long to accumulate 3 and 6 units of product, and the times to stages Β and C, therefore, increase to 10 and 20 hours, respectively (Figure 1-3B). If the rate of the exponential reaction is halved by a change in an environmental parameter, it again takes twice as long to accumulate 2 and 6 units of product, and the times to stages Β and C again increase to 10 and 20 hours, respectively (Figure 1-3D). Therefore, regardless of the reaction kinetics, the changes in the time to the sequential stages are affected proportionally. In other words, doubling the time to C doubles the time to Β regardless of the reaction kinetics, as long as both stages are cued by the same reaction. The First Questions to Be Asked about Timing Regulation In the preceding sections, I have attempted to develop a simplified view of timing regulation that includes (1) a distinction between causal events and timing events, (2) a definition of timing pathways, or developmental timers, (3) a consideration of timing pathways as cues that have evolved for that purpose, and (4) the development of the simplest alternative models for the temporal regulation of sequential stages in a developing system. I will next consider the questions to be asked in an initial investigation of timing regulation in a developmental program. To begin with, we must know if the genesis of multiple stages of a

18

David R. Soll

developmental program represents discrete events at the end o f interphase periods (Representation 3) or whether it represents highlights in an observable continuum (Representation 4). Next, we must know which timer model or combination o f timer models applies to the program. Third, we must know the minimum complexity o f the timing pathway for each developmental stage. Fourth, we must know if there are changes in timer identities when the developmental program is contracted o r expanded, and if any o f these changes result in aberrant phenotypes. Finally, we must elucidate the molecular nature o f timing pathways or components. T h e general approach o f dissecting an entire system has been most successful in elucidating the dependent pathways involved in the genesis o f the T 4 virus (Hood et al., 1968), the dependent pathways involved in the yeast cell cycle (Hartwell et al., 1974), and, to a lesser extent, the essential events in simple developmental programs (Loomis et al., 1976). In these cases, combined use o f developmental mutants and biochemical or ultrastructural measurements, or developmental mutants and inhibitors, have played paramount roles in dissecting both the complexity and the interactions o f dependent pathways, and have led to models that include sequences o f dependent events, parallel pathways that converge, and parallel pathways that diverge. In these classic studies, however, methods were developed solely to distinguish between dependent and independent events, and to order these events in dependent pathways (Jarvik and Botstein, 1973; Pringle, 1978). T h e s e methods were not developed to identify those pathways or processes that dictate timing (Soil, 1979, 1983).

Conditional Methods for Examining the Complexity and Relationships of Developmental Timers T h e first formal set o f methods for examining the relationship of timing pathways for consecutive stages in a developmental program was quite simple (Soil, 1979) and could be applied to any developing system that included well-defined, consecutive stages with relatively reproducible timing between experiments. O n e o f the assumptions upon which it was based, however, was an oversimplification. Testing this assumption (Soil, 1983) provided a new method for examining not only timer relationships, but also minimum timer complexity and identity changes. This new method is relatively assumption-free.

The Original M e t h o d T h e original methods for distinguishing among timing models (Figure 1-2) and defining the minimum complexity o f timing pathways were

Regulation of Timing In Developing Systems

19

based o n differences in sensitivity to a general environmental p a r a m eter, such as t e m p e r a t u r e . If two o r m o r e consecutive stages in a developing system are timed by a single timer, as in Figure 1-2A, t h e n a c h a n g e in an environmental p a r a m e t e r that e x p a n d s t h e timer will increase proportionally the time to both stages. Conversely, if two o r m o r e consecutive stages are timed by d i f f e r e n t timers with different sensitivities to the environmental parameter, t h e n a c h a n g e in that p a r a m e t e r will not result in proportional changes in the timing of these stages. This m e t h o d includes two c o m p o n e n t s : the timing of consecutive stages over a r a n g e of conditions (for instance, a t e m p e r a t u r e range) a n d a simple shift e x p e r i m e n t between the extremes of the range. For simplicity, we will use t e m p e r a t u r e as the m a n i p u l a t e d environmental parameter, but o t h e r p a r a m e t e r s could work equally well. Timing of consecutive stages over a temperature range In the first p r o c e d u r e , the time to each stage is m e a s u r e d over a short t e m p e r a t u r e r a n g e in which o n e obtains fast timing at the u p p e r e n d of the r a n g e a n d slow timing at the lower e n d . T w o indexes of these data a r e t h e n calculated. T h e first is At, which simply represents the difference in the time between the t e m p e r a t u r e extremes (At = tl-t2, w h e r e tl is the time to t h e stage at t h e lower t e m p e r a t u r e a n d t2, the time at t h e h i g h e r t e m p e r a t u r e ) . T h e second index is %At, the percent difference in time between the t e m p e r a t u r e extremes (%At = (tl — t2) x 100). T h e Ats f o r consecutive stages provide a m e a s u r e of the increase in time with decrease in t e m p e r a t u r e , a n d the %Ats provide a m e a s u r e of uniformity. If %Ats are equal f o r two or m o r e stages, the effect of a decrease in t e m p e r a t u r e is equal. In Figure 1-4, hypothetical data f o r the timing of sequential stages Β a n d C are presented with At a n d %At relationships, a n d the most likely, as well as excluded, timer-model interpretations. It is clear that a l t h o u g h these two indexes allow o n e to exclude single timer models, they rarely allow discrimination between parallel a n d sequential timer models. The simple shift experiment T o discriminate between parallel a n d sequential timer models, in the original m e t h o d , a simple shift e x p e r i m e n t was developed in which the predicted o u t c o m e would be quite d i f f e r e n t d e p e n d i n g u p o n w h e t h e r the timers f o r consecutive stages were in sequence o r in parallel. In the shift e x p e r i m e n t , the system is allowed to progress to stage Β at the low t e m p e r a t u r e extreme, t h e n shifted to t h e high t e m p e r a t u r e e x t r e m e a n d allowed to progress to stage C. Conversely, the system is allowed to progress to stage Β at the high t e m p e r a t u r e extreme, t h e n shifted to

Α.

Β.

ί tlB·))t|B2] Τ, Τ2 Temperature ( T W At(B)At (c); %ΔΙ ( Β ) > %At ( C ) (cross-over) Parallel (single and s e q u e n t i a l excluded)

Figure 1-4. Hypothetical data for the timing of consecutive stages Β and C in a limited temperature range. The formulas for At and %At are developed in the text. The At and %At relationships for the two stages as well as the most likely and excluded model interpretations are presented for each data set (Soli,

1979).

Regulation of Timing In Developing Systems

21

the low temperature extreme and allowed to progress to stage C. A shift f r o m high to low temperature is diagrammed in Figure 1-5. If the timing pathways to Β and C are in sequence, the predicted value for the interval f r o m Β to C after a shift f r o m high to low temperature is the normal interval at low temperature, and the predicted value for the interval after a shift f r o m low to high temperature is the normal interval at high temperature. If, however, the timing pathways for Β and C are in parallel, the predicted value for the interval after a shift f r o m high to low temperature reflects the time it will take to complete the parallel timer at the lower temperature. This predicted value t(BC) is obtained by first calculating the proportion of the parallel timer to stage C, which must still be completed at high temperature. This proportion is t(C2) -

t(B2)

t(C2)

where t(B2) and t(C2) are the times to stages Β and C, respectively, at the higher t e m p e r a t u r e (Figure 1-5). This proportion is then multiplied by

A

Temperature

( T ) — •

Figure 1-5. T h e temperature shift experiment of the original method for analyzing time regulation. In this case, the shift is from high to low temperature (down). T h e system is allowed to develop to stage Β at high temperature and then shifted to low temperature. B2 and C2 represent the time points of stage Β and C at high temperature for cultures maintained continuously at high temperature, and B , and C, represent the time points at low temperature for cultures maintained continuously at low temperature. T h e dashed line represents the switch to low temperature. T h e question mark represents the test times to Β and C after a shift (Soil, 1979).

22

David R. Soll

the time it takes to stage C at t h e lower t e m p e r a t u r e , t(C,), to obtain the predicted time interval, t(BC), if the timers to Β a n d C are in parallel t(BC) = t(C])

t(C2)2) - t(B2) t(L,2)

=

t(B2)

T h e same logic is used to calculate the predicted interval time f o r a shift f r o m low to high t e m p e r a t u r e if timers are in parallel. T h e final formula in this case is t(BC) = t(C2) 1 -

j-r-

T h e usefulness of the simple shift e x p e r i m e n t in discriminating between timer models is best d e m o n s t r a t e d by considering t h e predicted interval t(BC) f o r the hypothetical data in Figure 1-4 if the timers are sequential or if the timers are parallel f o r consecutive stages Β a n d C. If sequential, the predicted interval value a f t e r a shift f r o m high to low t e m p e r a t u r e would be 0.5 arbitrary time units, a n d the predicted interval value a f t e r a shift f r o m low to high t e m p e r a t u r e would be 2.0 arbitrary time units. If parallel, the predicted interval value f o r a shift f r o m high to low t e m p e r a t u r e would be 2.8 time units a n d f o r a shift f r o m low to high t e m p e r a t u r e 0.36 time units. T h e r e f o r e , if t h e timers f o r stages Β a n d C are in sequence, t h e predicted interval a f t e r a shift f r o m high to low t e m p e r a t u r e (shift down) would be o n e - f o u r t h the predicted interval a f t e r a shift f r o m low to high t e m p e r a t u r e (shift up). In m a r k e d contrast, if the timers were in parallel, t h e predicted interval a f t e r a shift d o w n would be 7.8 times t h e predicted interval a f t e r a shift up.

The application of the original methods to Dictyostelium morphogenesis T h e m e t h o d s originally developed f o r analyzing timer relationships were first applied in detail to t h e sequence of morphological stages in the slime mold Dictyostelium discoideum (Soli, 1979). In this system, a m e b a e aggregate after 7 h o u r s of starvation (the preaggregative period), t h e n proceed t h r o u g h a constant a n d temporally reproducible sequence of multicellular, m o r p h o g e n e t i c stages (Figure 1-5), leading to t h e genesis of a f r u i t i n g body. T h e s e stages include ripple (B), loose aggregate (C), tight aggregate (D), finger (E), early culminate I (F), maxifinger (G), early culminate II (H), late culminate (I), and f r u i t i n g

Regulation of Timing In Developing Systems

23

body (J). T h e timing of these stages t h r o u g h a limited t e m p e r a t u r e r a n g e (18 to 24°C) is p r e s e n t e d in Figure 1-7. It is immediately clear that m a n y of the hypothetical relationships described in Figure 1-4 can be observed in the actual timing data in Figure 1-7. Note that stages Β a n d C relate to subsequent stages by the model depicted in Figure 1-4B; thus a single timer model can be excluded f o r these two stages, b u t the parallel a n d sequential timer models cannot be distinguished. Stage G relates to stage Η by the model depicted in Figures 1-4E a n d Figure 1-5, with At(G) > At(H) a n d %M(G) > %At(H). As previously noted, direct timing results are informative b u t limited because they rarely distinguish between parallel a n d sequential timer models (Figu r e 1-4). T o accomplish this distinction, t h e simple shift e x p e r i m e n t was applied, a n d the results were indeed revealing f o r some relationships. For instance, t h e predicted p r o p o r t i o n of shift-down to shift-up f o r t h e tight aggregate-finger interval would be 3.05 if the timers were sequential a n d 0.75 if parallel. T h e observed p r o p o r t i o n was 0.88, suggesting that the timers f o r the two stages were in parallel. T h i s result held t r u e f o r a n u m b e r of o t h e r timer relationships f o r consecutive stages in the timing p r o g r a m of Dictyostelium, leading to a very u n e x p e c t e d model in which m a n y of the stages were controlled by

SMOOTH CARPET

RIPPLE

0

LOOSE AGGREGATE

6-9 (t4)

AAAA _ FINGER

aaax EARLY CULMINATE Π « *

C».e)

MAXI-FINGER

13.1 (t .e)

_

AGGREGATE

10.3 (· 4)

AAAA EARLY CULMINATE I

1« (* 5)

TIGHT

8.9 (! 4;

m t LATE CULMINATE 19.8

i t 9)

14.4

_

{! 7)

H H FRUITING BODY 25.7

(S

6

)

Figure 1-6. Morphogenesis in the cellular slime mold Dictyostelium discoideum. The mean time in hours (± standard deviation) of each stage for six independent experiments is presented beneath each stage. This timing is specific for midlog phase cells of strain Ax-3, clone RC-3, grown in axenic nutrient medium at 25°C (Soli, 1979).

I

18

1

20

1

22

Temp.(°c)

1

24

Figure 1-7. T h e timing of consecutive stages of Dictyostelium, morphogenesis in the temperature range 18°C to 24°C. Each point represents the mean of seven separate experiments. B, ripple; C, loose aggregate; D, tight aggregate; E, finger; F, early culminate I; G, maxifinger; H, early culminate II; I, late culminate; J, fruiting body (Soli, 1979).

Regulation of Timing In Developing Systems

25

parallel timers beginning close to the time the entire program was initiated. The major assumption of the original method The major assumption on which the original method was based (Soil, 1979) was that timer pathways are uniformly affected by the discriminating condition, in this case temperature, and therefore represent uniform biochemical processes. In spite of this assumption, which turned out to be invalid for several timers, application of the original method to Dictyostelium morphogenesis demonstrated two things. First, a single timer, or clock model, was not applicable to timing regulation in this system. Second, the use of conditional methods for dissecting the relationships of timing pathways was a useful initial approach to the problem. The N e w Method To test whether timers represent single processes or whether they are composed of subprocesses in sequence, a reciprocal shift experiment was developed (Soli, 1983). This method determines, for each timer, its complexity, defined as the number of individual processes functioning in sequence along the timer pathway for one stage under one set of environmental conditions. The reciprocal shift experiment The method is again relatively simple (Figure 1-8). Two sets of conditions are first defined in which the time to a developmental stage (DS) is altered by changing one environmental parameter (e.g., low and high temperature). One set of conditions (e.g., high temperature) results in short timing and the alternative set (e.g., low temperature) in long timing (Figure 1-8A). In the reciprocal shift experiment, development is initiated in one set of parallel cultures under short conditions and another set of parallel cultures under long conditions. At short time intervals prior to the genesis of the developmental stage, cultures under short conditions are shifted to long conditions and cultures under long conditions are shifted to short conditions (Figure 1-8B). The total time to the stage (the time under the first condition plus the time under the second condition) is measured for each shift experiment and plotted as a function of the time of shift. The reciprocal shift results (from short to long and from long to short) are plotted separately (Figure 1-8C). Finally, each plot is analyzed for the number of components, the magnitude and sign of the slopes of components, absolute times of origins and termini of components, and discontinu-

A.

Two conditions are developed for short and long timing to a developmental stage (D.S.)T

SHORT CONDITION IS1 TIMER i

I

1

3

1

• D.S.

tONG CONDITION HI LONG h — t TIMER Ο 1 1 Β.

Μ

1 1

i

D.S.

Α Ι

κ,

Shifts are performed at time intervals from short To long and long to short conditions. The total tTme to the stage (time under first condition pTüs time under second condition) 1s then scored"!

LONG TO SHORT SHIFTS (t-s)

S H O R T T O L O N G SHIFTS (s-Q

ο

ι S > — ( ? ) — » · D.&

l U ( ? ) — D.S.

Ο

τ—ι—r r-r 1 2 3 4 TIME—•

ι

2

r—τ I I 1 2 3 TIME—•

I ' 4

Total developmental time is plotted as a function of time of snift for shifts from short to long conditions (S-i-L) and long to short condition~s(L->S). . S-»l

. l-S

h ft βBil d

I I I I I • 0 1 2 3 4 5 SHORT TIMER

TIME O F SHIFT D.

1 I I I I I 2 3 4 5 6 7 8 9 10 LONG TIMER

Each plot is then analysed for number of components, slopes of components, absolute times of origins and termini of components, and discontinuities between components. Timer interpretations are then made according to the combinations of these characteristics for the two plots.

Figure 1-8. The reciprocal shift experiment for analyzing timer complexity (Soli, 1983).

Regulation of Timing In Developing Systems

27

ities between components. It is imperative that the intervals between shifts are as short as possible and that shifts are performed in both directions. Interpretations of timer complexity are then made according to the combinations of the characteristics of the two plots. Interpretations of reciprocal shift data

A detailed treatment of possible data sets and interpretations was presented in the original publications of the reciprocal shift experiment (Soil, 1983; Varnum et al., 1983), so only a few examples will be considered in the discussion that follows. The single component timer. If a timer consists of a single component, and is therefore uniformly affected along its entire length by a change in temperature, one obtains a straight line with negative slope for short to long shifts and a straight line with positive slope for long to short shifts. In the example in Figure 1-9A, the timer is 5 arbitrary time units under short conditions and 10 arbitrary time units under long conditions. In the model in the upper portion of Figure 1-9A, one observes the uniform expansion of the timer in the short to long direction. The multiple component timer. If a timer consists of two components in sequence exhibiting different sensitivities to temperature, short to long and long to short curves are composed of two components with different slopes. In the example in Figure 1-9B, the first component is temperature insensitive and is 2.5 time units long under short and long conditions. The second component is 2.5 time units long under short conditions and 7.5 time units long under long conditions. Shifts from short to long conditions yield plots in which the first component exhibits a slope of zero and the second component a negative slope. Note the very clear transition between the first and second components. Shifts from long to short conditions yield plots in which the first component again exhibits a slope of zero and the second component a positive slope. Multiple component timers may also include two timers that are sensitive in the same direction—both expanding in the short to long direction—or in a less likely situation, one expanding and one contracting in the same direction. In either case, the reciprocal shift data are easily interpreted (Soil, 1983). Reversibility. If progress along a timing component is reversed by shifting in one or both directions, then total developmental times are usually greater than undisturbed times. An example of a twocomponent timer in which the first component is reversed by shifts from short to long or long to short conditions, and the second component temperature insensitive, is presented in Figure 1-9C. For shifts from the short to long condition, the plot is composed of two discon-

28

David R. Soll

tinuous lines; the discontinuity is depicted by a dashed line. T h e first component of the plot has a positive slope, beginning at 10 time units and terminating at 12.5 time units. In this case, the terminus is 2.5 hours longer than the time to the stage under unperturbed long timing conditions. T h e second component of the plot is horizontal at 5 time units. For shifts f r o m the long to short condition, the plot is again composed of two components. T h e first component has a positive

A. Single

0

S^L ΙΛ

2

8·

component

1

2

3

4

2

3

4

timer

Two

5

5

0 6

7

8

9

10

0 1

component

1

2

3

4

timer

5

2 3 4 5 8

7 8 9

10

L—S

\ ί »

c.

Two component timer with reversible first component

D.

A d d i t i o n of new c o m p o n e n t under long c o n d i t i o n s

TIME Of SHIFT

Figure 1-9. Reciprocal shift data for a single component timer (A), a two component timer (B), a two component timer with a reversible first component (C), and a timer in which a new component is added under long conditions (D). Equivalency maps of the timing component, or components, are presented in the upper portion of each panel for the timers under short (S) and long (L) conditions. The numbers along the components represent arbitrary time units. The lower graphs in each panel represent hypothetical data for shifts from the short to long (S —> L) and long to short (L —* S) conditions. The vertical axes represent total time to the development stage (time under first condition plus time under second condition) (Soil, 1983).

Regulation of Timing In Developing Systems

29

slope, b e g i n n i n g at 5 time units a n d t e r m i n a t i n g at 12.5 time units. T h i s is followed by a discontinuity in the initiation of t h e second c o m p o n e n t . T h e second c o m p o n e n t is horizontal at 10 time units. Reversibility is readily distinguished by c o m p a r i n g the plots in Figure 1-9B f o r a s t a n d a r d two-component timer, with the plots in Figure 1-9C f o r a two-component timer with a reversible first c o m p o n e n t . A l t h o u g h I have presented only o n e example of a timer with a reversible c o m p o n e n t , f o r two-component timers t h e r e are 123 combinations that d e p e n d u p o n which c o m p o n e n t or c o m p o n e n t s are reversible, the direction or directions of shifts that cause reversibility, a n d the sensitivity of the c o m p o n e n t s (Soil, 1983). Addition of a component under long conditions. T h e difference in the length of a timer u n d e r short a n d long conditions may also be the result of the addition of a c o m p o n e n t at the b e g i n n i n g o r e n d u n d e r long conditions. Figure 1-9D shows a n addition at the origin u n d e r long conditions. In this case, t h e timer u n d e r short conditions is composed of a single insensitive c o m p o n e n t equivalent to the terminal 5 time units of the long timer. T h e first 5 time units of the long timer r e p r e s e n t a new c o m p o n e n t . T h e plot f o r shifts f r o m short to long conditions is composed of o n e point at the origin at 10 time units, a n immediate discontinuity, t h e n a horizontal plot at 5 time units. T h e plot f o r shifts f r o m long to short conditions includes an initial plot with a positive slope, followed by a horizontal plot. A n u m b e r of additional models have been developed f o r addition at the t e r m i n u s o r insertion along the length of t h e short timer model b u t d o not w a r r a n t detailed analysis in this discussion. T h e details of these model a n d data interpretations have been discussed at length (Soli, 1983). Identity change. Finally, the difference in time between short a n d long conditions can be d u e to an identity change. For a system in which a single c o m p o n e n t timer dictates the time to a stage in both directions, a n d a shift initiates t h e alternative timer at its origin (Figure 1-10A), the plots g e n e r a t e d are indistinguishable f r o m a system in which t h e timers are reversed by shifts in both directions. A m o r e plausible situation f o r a n identity c h a n g e is described in Figure 1-10B. In this case, pathways a a n d b a r e both necessary f o r the genesis of the developmental stage, b u t a is the last to be completed u n d e r short conditions a n d b is t h e last to be completed u n d e r long conditions. T h e r e fore, a is the timer u n d e r short conditions a n d b the timer u n d e r long conditions. Shifts f r o m short to long condition yield a plot composed of two a d j o i n i n g lines, t h e first with a very negative slope a n d the second with a less negative slope. Shifts f r o m long to short condition yield a plot with two a d j o i n i n g lines, the first with a positive slope a n d the second with a m o r e positive slope.

30

David R. Soll

A far more complex and interesting set of plots is obtained in the identity change outlined in Figure 1-1OC. In this situation, both pathways a and b are essential for the genesis of the developmental stage. Stage a is the last to be completed and therefore the timer under both unperturbed short and long conditions, but b is reversible for shifts in either direction. For shifts from short to long and long to short condi-

Figure 1-10. Reciprocal shift d a t a f o r a single c o m p o n e n t , reversible timer that u n d e r g o e s a n identity c h a n g e a f t e r shifts in either direction (A); a single c o m p o n e n t , irreversible timer that u n d e r g o e s a n identity c h a n g e a f t e r shifts in either direction (B); a n d a single c o m p o n e n t timer that u n d e r g o e s a n identity c h a n g e a f t e r shifts at i n t e r m e d i a t e times because of t h e reversibility of c o m p o n e n t b (C). Vertical d a s h e d lines r e p r e s e n t discontinuities in the plots. N o t e that in p a n e l A the timers u n d e r short a n d l o n g conditions r e p r e s e n t d i f f e r e n t pathways, that in panel B, a is rate-limiting u n d e r short conditions, but b is ratelimiting u n d e r long conditions, a n d in p a n e l C, a is rate-limiting u n d e r u n d i s t u r b e d short a n d long conditions, b u t since b is reversible a f t e r shifts in either direction it becomes rate-limiting a f t e r shifts at i n t e r m e d i a t e times. S, short conditions; L, long conditions (Soil, 1983).

Regulation of Timing In Developing Systems

31

tions early or late in the pathway, pathway a is rate-limiting. For shifts midway in the interval, however, b is rate-limiting. This leads to plots with three lines for short to long and long to short shifts. In both cases, a discontinuity can be observed between the second and third line. Interpreting data sets in terms of timer complexity and timer characteristics. In the preceding discussion, I presented a brief summary of the different types of timer complexity and timer characteristics that may exist in developing systems, and the data sets they would generate when the reciprocal shift experiment is applied. In the application of the reciprocal shift experiment, however, one first obtains the data, then generates the best model, or models, for describing the results. In a few cases, timers with very different characteristics generate similar data, but for the most part, detailed data are easily interpreted. T h e method is only effective if shifts are performed in both directions. An example of application of the methodfor determining timer complexity of a single stage. T h e reciprocal shift experiment is not a theory, but rather a quantitative method for determining the minimum complexity of the timing pathway to a developmental stage. Application of the method to the morphogenetic program of Dictyostelium discoideum demonstrates that the method indeed works. T h e results for the tight aggregate stage (Figure 1-11) demonstrate just how detailed the data can be in application of the reciprocal shift experiment. T h e average time to the tight aggregate stage of Dictyostelium morphogenesis under the short condition (24°C) is 9.9 hours (standard deviation, SD, ± 0.8; number of experiments, N, 6), and the average time under the long condition (18°C) is 13.2 hours (SD, ± 0.5; N, 6). I f the timer for the tight aggregate stage was composed of a single component 9.9 hours long under the short condition, expanding to 13.2 hours under the long condition, then one would expect short to long and long to short plots of a single line with a negative slope, in the former case, and a positive slope in the latter case, depicted as a dashed line in Figures 1-11A and B, respectively. T h e actual results were far more complex. T h e results (n = 6) are presented as filled squares in Figures 1-11A and Β for shifts from short to long and long to short conditions, respectively. T w o individual data sets (filled and unfilled) circles are presented in Figures 1-11C and D. T h e plots for shifts from short to long conditions are composed of components with distinct transition points at roughly 1.75 and 6 hours. T h e first component has a negative slope, the second a slightly positive slope, and the third a strongly negative slope. T h e plots for shifts from long to short conditions are also composed of three components, with a discontinuity between the first and second component at roughly 3.0 hours and a transition point at roughly 6.8 hours. T h e first component

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Figure 1-11. T h e reciprocal shift e x p e r i m e n t (Figure 1-8) applied to t h e tight a g g r e g a t e stage of Dictyostelium m o r p h o g e n e s i s . T h e averaged d a t a f o r six i n d e p e n d e n t e x p e r i m e n t s f o r shifts in the short to long direction ( • ) are p r e s e n t e d in panel A a n d in t h e l o n g to short direction ( • ) in panel B. T h e data sets of two i n d e p e n d e n t e x p e r i m e n t s f o r shifts in the short to long direction ( · , O) a r e p r e s e n t e d in panel C a n d in t h e long to short direction (Α, Δ ) in panel D. T h e d a s h e d lines in panels A a n d Β r e p r e s e n t t h e hypothetical data expected f o r a single c o m p o n e n t timer. T h e m o d e l of t i m e r complexity that best fits b o t h the averaged d a t a a n d the i n d e p e n d e n t d a t a sets is p r e s e n t e d f o r shifts in the short to long direction (panel E) a n d f o r shifts in the long to short direction (panel F). In t h e model, t h e solid, horizontal arrows indicate individual c o m p o n e n t s exhibiting d i f f e r e n t t e m p e r a t u r e sensitivities. Dashed arrows r e p r e s e n t equivalence a f t e r shifts. T h e n u m b e r at t h e e n d of each solid arrow r e p r e s e n t s the time at which the c o m p o n e n t terminates u n d e r short (S) a n d long (L) conditions. T h e bold n u m b e r s u n d e r each c o m p o n e n t r e p r e s e n t the short to long equivalences in panel Ε a n d t h e long to short equivalences in p a n e l F. REV r e p r e s e n t s reversibility. T h e hypothetical data g e n e r a t e d f r o m this m o d e l (X) are p r e s e n t e d in panels A a n d C f o r shifts in the short to long direction a n d panels Β a n d D f o r shifts in the long to short direction (Varnum et al., 1983).

Regulation of Timing In Developing Systems

33

has a positive slope of 1.0, the second component a slightly negative slope, and the third component a strongly positive slope. The following model (diagrammed in Figures 1-1 IE and F) appears to be the best description of the results. The first component is sensitive to a shift from short to long conditions, exhibiting a short to long equivalence (the ratio of short to long length) of 0.58. The first component is completely reversible for shifts in the long to short direction because the slope of the plot (Figure 1-1 IB and D) is 1.0. The total time to the tight aggregate stage for shifts from the long to short condition during the first 3.0 hours of development exceeds the time to tight aggregate under the unperturbed long condition, and a discontinuity occurs between the first and second component. The second component is relatively insensitive to temperature changes, and the third component is extremely sensitive, expanding in the short to long direction and contracting in the long to short direction. The third component has a short to long equivalence of 0.61. Using the results from Figures 1-1 IE and F, hypothetical plots were generated in Figures 1-11A to D (lines with x's) and the shapes of the plots compared with actual data. The agreement is high, suggesting that the models are indeed applicable. Therefore, by simple shifts between two temperatures we have delineated and partly characterized three sequential timing components. We have elucidated a unique characteristic of the first component (reversibility for shifts in one direction), which eventually will be useful in its identification, and we have demarcated transition points between components along the developmental program. Employing the new method to distinguish between timer models for the regulation of consecutive developmental stages The reciprocal shift experiment is useful not only for characterizing the length of a single timer pathway for a single developmental stage, but also for assessing timer relationships and thus distinguishing between timer models (Figure 1-2) for consecutive stages in a developmental program. To do this, one simply extends the reciprocal shift experiment for a single stage (Figure 1-8). Shifts are performed from time zero to the time of the last stage of interest (Figure 1-12). From this single set of data, the complexity of the timing pathway to each stage is dissected, and the sensitivity of each timer component characterized in a manner similar to that described for a single developmental stage (Figure 1-8). Timer relationships are then assessed according to the similarities or dissimilarities of parallel components (Figure 1-13). If the timing pathways for consecutive stages Β and C are each composed of a single component, and if both show the same sensitivity to

34

David R. Soll

shifts from short to long or long to short conditions (Figure 1-13A), then the best interpretation is that they are regulated by a single timer (Figure 1-2A). Other models, however, cannot be excluded since the condition for generating timer complexity (e.g., temperature) may not have discriminated components or differences among parallel timers. If the pathways for Β and C are each composed of a single component, but the timers have different sensitivities (Figure 1-13B), the best interpretation is the parallel timer model (Figure 1-2C); single, sequential, and branched models can be excluded in this case. This method of comparison can discriminate among models for timers composed of multiple components (Figures 1-13C to I). The important point is that the reciprocal shift experiment is an improvement over earlier methods because it does not assume that timers are uniformly affected by the environmental change employed for discrimination.

A. TIMING

SHORT CONDITION (S) Β C D I—I—I—I—I—I—I—I—I—I—I

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•

t

t

I

t S

t

t

»

•

t

Figure 1-12. Using the reciprocal shift experiment to assess timer relationships for consecutive stages B, C, and D of a developmental program. The vertical arrows in panel Β represent times of shift. The data obtained from this experiment are plotted according to the method in Figure 1-8 and analyzed according to the rules in Figure 1-13.

A

E a c h timer one c o m p o n e n t , similar c h a r a c t e r i s t i c s

Β

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*

* B e s t interp. parallel E x c l u d e d : single, sequential, branched.

B e s t interp single E x c l u d e d : nothing

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The Β timer one c o m p o n e n t , the C timer two c o m p o n e n t s . The β timer and first component of C timer similar.

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The Β timer one c o m p o n e n t , the C timer two c o m p o n e n t s . The Β timer a n d first component of C timer dissimilar.

* B e s t interp.: parallel E x c l u d e d : single, sequential, branched

B e s t interp.: sequential Excluded: single

E.

The Β timer two c o m p o n e n t s , the C timer one c o m p o n e n t . T h e first c o m p o n e n t of Β timer similar to C timer, s e c o n d component dissimilar.

*

F.

*

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G.

The Β timer a n d C timer b o t h t w o c o m p o n e n t s ; first a n d s e c o n d c o m p o n e n t s similar.

*

H.

T h e Β timer a n d C timer b o t h two c o m p o n e n t s ; first similar, s e c o n d dissimilar.

*

*

*

B e s t interp.: b r a n c h e d E x c l u d e d : nothing.

B e s t interp.: s e q u e n t i a l E x c l u d e d : nothing.

1

The Β timer two c o m p o n e n t s , the C timer one c o m p o n e n t . T h e first c o m p o n e n t of Β timer d i s s i m i l a r to C timer, s e c o n d c o m p o n e n t similar or d i s s i m i l a r .

T h e Β timer a n d C timer b o t h t w o c o m p o n e n t s ; first d i s s i m i l a r , s e c o n d similar or dissimilar. -

Β • c

B e s t interp parallel E x c l u d e d : single, sequential, branched.

Figure 1-13. T h e rules for assessing timer relationships for consecutive stages Β and C in a developing system. Each horizontal arrow represents a timing component. Angled equivalence symbols (\\) indicate that parallel components for stages Β and C exhibit similar sensitivities to environmental change. Dashed equivalence symbols QK) indicate that parallel components exhibit dissimilar sensitivities (Soli, 1983).

36

David R. Soll

Application of the new method to Dictyostelium morphogenesis T h e reciprocal shift experiment was applied to the morphogenetic program of Dictyostelium discoideum to obtain not only a picture of minimum timer complexity for each of the morphological stages but also a picture of timer relationships (Varnum et al., 1983). T h e model that has evolved for timing regulation in this system is presented in Figure 1-14. It has at least one example of every possible timer relationship originally considered (Figure 1-2): single, sequential, parallel, and branched. To begin with, the first component, a, of the timing program is common to all subsequent stages. This component has the unique feature of reversibility (in the long to short direction for temperature shifts). An identity change appears to occur for the ripple stage timer for shifts in the long-to-short direction after 1.25 hours (dotted arrow to R), but, u n d e r nonshift conditions, no identity change occurs. T h e second component of the timing program, b, is also common to all stages and follows α in a d e p e n d e n t fashion. C o m p o n e n t b terminates at a branch point f r o m which a n u m b e r of pathways emerge. T h e last component of the timers for the ripple and loose aggregate stages, c and d respectively, a p p e a r to be in parallel with the common timing pathway e for the tight aggregate, finger, early culminate I, and maxifinger stages. It is clear that at about 6 hours u n d e r short conditions and 7 hours u n d e r long conditions, a n u m b e r of parallel pathways emerge f r o m a branch point, and it is probably not fortuitous that this breakpoint coincides temporally with the onset of multicellularity in this system (Varnum et al., 1983). A single timer component, e, appears to time four late stages (TA, F, ECI, and MF), but short components preceding some of the stages may indeed exist (Varnum et al., 1983). A timer component for the early culminate II stage, / , branches f r o m component e, and the data supporting the independence of this branch are indeed unambiguous. Shifts in the short to long direction after 6 hours, however, indicate an identity change for the timer pathway to ECII (dotted arrow to ECU) (Varnum et al., 1983). It should be clear f r o m the diagram of minimum timer complexity in Figure 1-14 that particular developmental periods normally considered interphases can be dissected into timing components, with clearly defined transition points that must eventually correlate with molecular transitions. T h e best case is the transition between the first and second timing component in the preaggregative period. It is worth considering in detail how the two major timing components and the transition point relate to the molecular changes accompanying the preaggregative period of Dictyostelium.

A. SHORT CONDITION (24°C)

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Figure 1-14. A model of timer complexity and timer relationships under short and long conditions for morphogenesis in Dictyostelium discoideum. T h e horizontal, solid arrows represent clearly distinguished, independent components that are rate-limiting under short and long nonshift conditions. The horizontal, dashed arrows represent components that we have tentatively concluded are independent of and in parallel with the more clearly defined components (solid arrows). T h e dotted lines represent pathways that become rate-limiting after shifts from the short to long direction only. The horizontal termini of arrows indicate the end of a rate-limiting component. Vertical arrows indicate the genesis of a particular stage. The termini of the two pathways exposed by shifts in the short to long direction (dotted arrows) have not been defined, but they must occur just prior to the termini of the pathways rate-limiting under nonshift conditions. T h e lower-case letters refer to independent components rate-limiting under nonshift conditions. R, ripple; LA, loose aggregate; TA, tight aggregate; F, finger; ECI, early culminate I; MF, maxifinger; ECU, early culminate II.

38

David lt. Soll

To begin with, it is clear that the preaggregative period terminates with the onset of aggregation, represented by the ripple stage (Figure 1-6). In the aggregation process, precocious cells in the population first emit a cAMP signal that is relayed outwardly by cells in the aggregation territory (Robertson et al., 1972; Gerisch and Hess, 1974; Tomchik and Devreotes, 1981). These precocious cells then function as aggregation centers, and cells in the territory assess the direction of the outwardly moving cAMP wave (Varnum-Finney et al., 1987a, b), moving in a directed fashion toward the aggregation center. Once in the aggregation center, they adhere to each other to maintain a multicellular aggregate (Barondes et al., 1982). T o accomplish aggregation, cells must acquire all of the necessary chemotactic and adhesion machinery. T h e first question to be asked is whether the new molecular components of the machinery accumulate t h r o u g h o u t the preaggregative period, in which case the time to the ripple stage would simply reflect the time necessary for synthesis and assemble, or whether the new molecular components accumulate only at the end of the preaggregative period, in which case the time to the ripple stage would reflect preparatory processes preceding synthesis and assembly. By monitoring several developmentally acquired functions and cell surface components (cell motility, Varnum et al., 1986; chemotactic responsiveness, Varnum and Soil, 1981; cAMP binding sites, Green and Newell, 1975; and contact sites A, Beug et al., 1973), the latter alternative was demonstrated (Figure 1-15 A). All of these aggregation associated characteristics appeared in the final h o u r or two of the preaggregative period, suggesting that the rate-limiting components a and b that make u p the major portion of the preaggregative period represent preparatory processes leading to the acquisition of morphogenetic machinery and do not represent the slow, continuous accumulation of these characteristics (Soli and Finney, 1987). In addition, the onset of component a and the onset of component b correlated with two major points in gene regulation (Finney et al., 1986b; Soli and Finney, 1987). By monitoring the synthesis of 778 polypeptides during growth and early development, it was demonstrated that there were significant decreases in the rate of synthesis of 93 major polypeptides normally synthesized at high rates d u r i n g growth, and significant increases in the rate of synthesis of 74 major polypeptides either undetectable or synthesized at insignificant rates during growth. In terms of timing, there were two peaks of activity for both decreases in synthesis of vegetative polypeptides (Figure 1-15B) and increases in synthesis of developmental polypeptides (Figure 1-15C). T h e peaks of decreases correlated tightly with the onset of timing components a and b, and the peaks of increases occurred roughly 1.5 hours after the onset of the timing components.

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Ο ο

l· 0

1

2

3

4

,5

6

7

8

9

10

D E V E L O P M E N T A L TIME (HR)

ATE LIMITING COMP

Β.

C.

Γ

ΓΉ

DEVELOPMENTAL TIME (HR)

Figure 1-15. T h e relationship between gene expression and the two major ratelimiting components of the preaggregative period of Dictyostelium morphogenesis. A, the temporal kinetics for the acquisition of aggregation-associated functions during the preaggregative and aggregative periods. B, histogram of the number of polypeptides synthesized at high levels during growth that decrease dramatically in rate of synthesis during consecutive 1.5-hour periods throughout the initial 10.5 hours of Dictyostelium morphogenesis. The height of each vertical bar (hatched plus unfilled) represents the number of polypeptides that exhibits a moderate to major decrease in synthetic rate; the height of the hatched bars alone represent the number that exhibits a major decrease. C, histogram of the number of polypeptides synthesized at negligible levels during growth and increase dramatically in rate of synthesis. T h e height of each vertical bar (hatched plus unfilled) represents the number of polypeptides that exhibits a moderate to major increase; the height of the hatched bars alone represents the number that exhibits a major increase. At the top of panels Β and C, the first and second timing components (C, and C 2 , respectively), as well as the periods of the ripple (R) and loose aggregate (LA) stages, are diagrammed (Finney et al., 1986b; Soil and Finney, 1987).

40

David R. Soll

In the latter case, the delays probably represented the time necessary for transcription, processing, and transport of messages. Indeed, a careful analysis of the level of the transcript for the cohesion molecule gp80 demonstrated that accumulation begins between 4 and 6 hours (Kraft et al., 1989), the approximate time of the onset of timing component b. It is clear that the dissection of timing components provides the molecular biologist with a temporal framework for analyzing coordinate gene regulation during development (Finney et al., 1986b; Soil and Finney, 1987). Application of the methods for timing analysis to two other developing systems: Blsstocladiella sporulation and early development of sea urchin embryos Peralta and Lodi (1988) have applied the reciprocal shift experiment to sporulation of the water mold Blastocladiella emersonii. During sporulation of B. emersonii, the sporangial syncytium stops growing, and the multinucleate cytoplasm is carved into zoospores of equal size and organellar composition (see Figure 1-1 for diagram of zoospore). T h e landmark events monitored in this developmental program were SZ, the septate zoosporangium; PZ, the papillate zoosporangium; CZ, the cleavage zoosporangium; and EZ, the empty zoosporangium. T h e short condition was 22°C and the long condition 27°C. T h e results of reciprocal shifts suggest a basic model that includes three parallel timer pathways apparently originating at time zero of induction, one cuing SZ, one CZ, and one PZ and EZ. In an alternative model, two parallel pathways are initiated at time zero, one branching to cue SZ and CZ, and one cuing PZ and EZ. T h e results of the Peralta and Lodi (1988) study are at least as good as the original Varnum et al. (1983) study, and the reciprocal shift plots have discrete transition points between components. Matsumoto et al. (1988) have applied both temperature dependency and shift experiments to the early program of sea urchin development. T h e monitored landmark events in this system were first cleavage; second cleavage; third cleavage; fourth cleavage; cil, initiation of ciliary beat; mes, mesenchyme cell ingression; and gas, onset of gastrulation. T h e range used to examine differences in temperature sensitivity was 11°C to 20°C. Reciprocal shift experiments were performed between 11 to 17°C and 17 to 11°C. T h e results of this study suggest a basic model in which the first to fourth cleavage and cil are cued by a single timer, and mes and gas by a timer that branches from the cleavage-cil timer. In addition, there is an initial variable component at the beginning of the entire timing program. Matsumoto et al. (1988) have used slightly different calculations for assessing differen-

Regulation of Timing In Developing Systems

41

tial sensitivities to temperature including a Q 1 0 value, and have introduced a modification of the original equation for predicting parallel versus sequential interval times (Soil, 1983). The modification allows calculation of the fraction of the interval between two stages (a) representing the initiation point for a new timing pathway. What does the information obtained from the reciprocal shift experiment really tell us? The value of characterizing timer pathways in a developmental program resembles that of analogous studies in which the number and dependencies of gene products essential in the assembly of bacteriophage T4 (Hood et al., 1968) and progression of the yeast cell cycle (Pringle and Hartwell, 1981) were assessed. In the case of T4, simply identifying the components that make up the final virus tells very little about the developmental, or dynamic, program that leads to phage assembly, or to the causal relationships between independent processes. A combination of mutational, biochemical, and microscopic analyses led to a model of tail, tail fiber, and head development in which parallel but independent pathways converge to generate the completely assembled bacteriophage. The description of the complexity of pathways and their relationships is an important component of the holistic view of T4 development, even if the processes or components comprising the pathways are not completely identified or their functions understood. This point is even more poignant in the case of the dissection of the yeast cell cycle. Employing cell cycle mutations, Hartwell and coworkers initially demonstrated that parallel pathways diverged and converged, and that within pathways one could order dependent events (Hartwell et al., 1974). These researchers dissected the complexity of these pathways and their relationships, thereby presenting an overview of the entire cell cycle. This overview has expanded from two parallel pathways (Hartwell et al., 1974) to a model including at least five parallel pathways with ordered dependent events (Pringle and Hartwell, 1981). Most of the events in these pathways were identified by mutations in individual genes more than 10 years ago, but the gene products and their functions are only now being elucidated (Reed et al., 1985; Haarer and Pringle, 1987). Again, the importance of the functional sequence map developed by Pringle and Hartwell (1981) lies not only in the eventual identification of the gene products or their function, and no one questions the importance of this information, but also in the detailed view of how the entire program operates. As I argued earlier in this chapter, the first questions that must be answered concerning timing regulation in a developmental system

42

David R. Soll

concern the complexity o f the timing program. Unlike studies o f dependent events (Hood et al., 1968; Jarvik and Botstein, 1973; Hartwell et al., 1974; Pringle, 1978), the initial methods developed to dissect timing events (Soli, 1979, 1983) have been conditional rather than mutational. Nevertheless, they serve to obtain a picture, such as the one presented in Figure 1-14 for Dictyostelium, o f the minimum complexity o f the timing program.

Combining a Mutational Approach with the Already Established Conditional Approach to Further Dissect Timing Regulation In the set o f conditional methods described, timing components are discriminated by differential sensitivity to the particular environmental parameter employed (e.g., temperature or pH). T i m i n g mutations can be used in a similar fashion, although they must be quite different in character from standard developmental mutations that normally block the entire program or a portion o f the program (Soli, 1987; Soll et al., 1987b). T o be useful, timing mutations must selectively alter the length o f one or more components in a program without blocking the subsequent program. Again, their usefulness is in dissecting the timing program as in the case o f the reciprocal shift experiment. Also, a timing mutation may not be a timer mutation, even though it is selective. In many cases it may affect a physiological process that in turn selectively affects the length o f a timer component o f a timing pathway. T h i s point does not diminish the usefulness of timing mutants.

Combining mutational and conditional methods Let us consider a hypothetical timing mutant in which the time to stage Β is halved under short conditions (Representation 13). We already

ο

s Time (arb. units)

0

2.5 Time (arb. units)

A. Wild-type

B. Timing mutant Representation 13

Regulation of Timing In Developing Systems

43

know from the reciprocal shift experiment employing the wild-type parent that the timer from A to Β is composed of two components: the first is 4 arbitrary time units long and the second component 1 arbitrary time unit long under short conditions. To test whether the timing mutation affects both components or is selective for a particular component, the reciprocal shift experiment (Figure 1-8) is performed on the mutant. If the mutation affects timing in general and causes a uniform reduction in all timing components, then both components discriminated by the reciprocal shift should be half as long as their counterparts in the wild-type strain: the first component should now be 2 arbitrary units long and the second component 0.5 arbitrary units long. If the mutation is selective for the first component, then the first component should be 1.5 arbitrary time units long and the second component still 1 time unit long in the mutant. A mutational approach may also be useful in the further dissection of timer components already distinguished by the conditional approach. Some timing components may have quite different identities but similar sensitivities to the environmental parameter being manipulated in the reciprocal shift experiment, and distinction may be effected in this case only by mutation. Application of mutational and conditional methods to determine timer complexity in a morphogenetic program A combined mutational and conditional approach was recently applied to the preaggregative period of Dictyostelium, discoideum. To isolate putative timing mutants, a procedure was employed (Soil, 1987; Soll et al., 1987b; Soli and Finney, 1987) that took advantage of colony morphology (Figure 1-16). During Dictyostelium morphogenesis, starving amebae progress through a morphogenetically uneventful preaggregative period, and then begin to aggregate. Aggregation initiates a morphogenetic program that includes a sequence of defined stages (Bonner, 1967; Loomis, 1975; Soli, 1979). Dictyostelium morphogenesis is best visualized on buffer-saturated filters, but it can also be observed in clonal colonies growing in bacterial lawns. To generate such a colony, a single ameba in a bacterial lawn feeds and divides, generating a clonal colony that appears as a plaque in the lawn. As the colony spreads, amebae on the colony periphery feed and divide and are, therefore, responsible for colony expansion. Amebae in the colony interior, however, begin to starve and enter the developmental program. Cells at the center of the colony have starved the longest and have, therefore, progressed through the greatest portion of the morphogenetic program. Cells at greater distances from the center have progressed through proportionately less of the program. After a set number of days under

44

David R. Soll

rigidly defined conditions (Soll, 1987), rings of the different stages are apparent in a normal colony, with the outer ring consisting of nonaggregating cells, followed by a ring of aggregating cells, then rings of intermediate stages, and finally fruiting bodies in the colony center (Figure 1-16A).

WILD TYPE COLONY

P U T A T I V E TIMER C O L O N Y ( E X P A N D E D PRE A G G R E G A T I V E PERIOD)

Colony

Colony

Figure 1-16. A method for isolating putative timing mutants according to colony morphology. A, wild-type colony and putative timer mutant colony after 7 days on agar. Note the expanded preaggregative ring in the putative timing mutant (Soll et al., 1987b; Soil, 1987). B, example of a young wild-type colony, with average-size preaggregative band (PB); C, example of a young fast timing mutant colony, with extremely narrow preaggregative band; D, example of a young slow timing mutant colony, with extremely broad preaggregative band. FA, feeding amebae; PB, preaggregative band; AA, aggregating amebae; BL, bacterial lawn (Soli, 1987).

Regulation of Timing In Developing Systems

45

Putative timing mutants are screened by differences in ring diameters. Examples of a young 6-day colony of the wild-type parent strain are shown in Figure 1-16B, of a fast mutant with a reduced preaggregative band in Figure 1-16C, and of a slow mutant with an expanded preaggregative band (Figure 1-16D). In a screening experiment, cells are grown to midlog phase, mutagenized, and clonally plated, and colony morphologies are examined (Soli, 1987). Putative mutants are then characterized carefully for the timing of morphogenesis and compared to the timing of parallel wild-type cultures. A comparison of the timing mutant FM1 (Soll et al., 1987b) with wild-type (WT) is presented in Figure 1-17A for the following stages: ripple, R; loose aggregate, LA; tight aggregate, TA; finger, F; early culminate I, ECI; maxifinger, MF; early culminate II, ECU; midculminate, MC; and late culminate, LC. It is immediately clear that FM1 progresses through all of the stages in the developmental program but that there are decreases in two timing intervals: the period between initiation and ripple, and between maxifinger and early culminate II. T h e preaggregative period of FM1 is roughly 3.5 hours and that of the parallel wild-type culture 7.0 hours. Since the preaggregative period is composed of two major timing components, the results of reciprocal shift experiments for FM1 and wild-type were compared (Figure 1-17B). T h e first component of the timing program, which expanded in the short to long direction and was reversed after shifts from the long to short condition in wild-type, was absent in FM1. Therefore, FM1, which is relatively normal in all aspects of growth and in the morphogenetic sequence, has a preaggregative period composed solely of component b, or C2 in the presentation in Figure 1 -17. In other timing mutants, the first component is reduced or expanded in length. An example of the former type, FM2, has a 4.5 hour preaggregative period, which is the result of a dramatic and selective reduction in the length of the first timing component. An example of the latter type, SM3, has a 10-hour preaggregative period, which results from a dramatic and selective expansion of the first component of the preaggregative period. Although dissecting timer complexity by a combination of timing mutants and the reciprocal shift experiment has so far been applied only to the first component of the preaggregative period, it is applicable to the timers for all stages in the morphogenetic program of Dictyostelium. A complete study of this type would provide us with a more detailed description of timing regulation in a single developmental system, and the mutants might also be useful in the eventual elucidation of timing components if they represented mutations in the structural genes of timer components.

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214

Stephen Roth

the same method when the complete variable regions of λ and κ chains are compared, and scores of 3.5 and 0.6 when a heavy chain constant region is compared to two light chain variable regions. The probability of obtaining a score of 3.4 or greater, by chance, is less than 0.001. Figure 5-2 shows a short fragment of the bovine galactosyltransferase compared with a fragment of HLA DR. The characteristic, immunoglobulin superfamily motif Y-C-V is apparent. None of these comparisons is alone strong evidence of an evolutionary relationship between the transferases and the immunoglobulins. On the other hand, these relationships might become much stronger as more, and perhaps more relevant, glycosyltransferase sequences become available. In a recent review, Williams and Barclay (1988) have divided the immunoglobulin superfamily proteins into three classes, based on their primary structures. They find that the proteins tend to resemble the variable region (V-like), the first constant region (Cl-like), or the second constant region (C2-like) of the immunoglobulins. C2-like proteins tend, further, to have cell surface recognition functions. Fragments of ten such C2-like proteins were listed by Williams and Barclay (Figure 5-3a). When a corresponding fragment of the murine galactosyltransferase is added to these ten C2-like proteins, the primary structure of the transferase allows it to be included at least as well as some of the others. Figure 5-3b shows that NCAM and the murine galactosyltransferase fragments share ten identical residues, whereas NCAM and CD2 share only nine. Immunoglobulin-specific glycosyltransferases

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