JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 285:307–325 (1999)

Modularity in Animal Development and Evolution: Elements of a Conceptual Framework for EvoDevo

GEORGE VON DASSOW*AND ED MUNRO Department of Zoology, University of Washington, Seattle, Washington 98195-1800

ABSTRACT For at least a century biologists have been talking, mostly in a black-box sense, about developmental mechanisms. Only recently have biologists succeeded broadly in fishing out the contents of these black boxes. Unfortunately the view from inside the black box is almost as obscure as that from without, and developmental biologists increasingly confront the need to synthesize known facts about developmental phenomena into mechanistic descriptions of com- plex systems. To evolutionary biologists, the emerging understanding of developmental mecha- nisms is an opportunity to understand the origins of variation not just in the selective milieu but also in the variability of the developmental process, the substrate for morphological change. Ultimately, evolutionary developmental biology (EvoDevo) expects to articulate how the diversity of organic form results from adaptive variation in development. This ambition demands a shift in the way biologists describe causality, and the central problem of EvoDevo is to understand how the architecture of development confers evolvability. We argue in this essay that it makes little sense to think of this question in terms of individual gene function or isolated morphometrics, but rather in terms of higher-order modules such as gene networks and homologous characters. We outline the conceptual challenges raised by this shift in perspective, then present a selection of case studies we believe to be paradigmatic for how biologists think about modularity in devel- opment and evolution. J. Exp. Zool. (Mol. Dev. Evol.) 285:307–325, 1999. © 1999 Wiley-Liss, Inc.

Biologists have long recognized that the evolv- for granted that logical separability reflects under- ability of development is the key to morphological lying modularity: the experimental study of de- evolution. However, only recently has molecular velopment assumes that one may meaningfully genetics produced substantial characterizations isolate (physically or conceptually) and study in- of developmental regulatory processes and thus dividual processes independent from one another. made it possible to analyze them comparatively. Functional decomposability is thus a necessary This has fueled resurgent interest in the evolu- presumption to considering developmental mecha- tion of development, but evolutionary develop- nisms either as units of explanation within de- mental biology (EvoDevo) lacks a clear conceptual framework. Blithe attempts to apply classical comparative concepts to development lead more 1This essay arose from a workshop on modularity in development often than not to confusing or contradictory re- and evolution organized by the authors and Dr. Evelyn Fox Keller, held in September 1997 at the Friday Harbor Laboratories. In addi- sults. Furthermore the idea that one should fo- tion to the authors, the participants were Drs. Jessica Bolker, Richard cus on developmental mechanisms as units of Burian, Marie-Anne Félix, Scott Gilbert, Jason Hodin, Evelyn Fox Keller, Eli Meir, Jay Mittenthal, Lisa Nagy, Garrett Odell, Jarmila evolutionary change begs the question, what con- Kukalova-Peck, Louise Roth, Günter Wagner, and William Wimsatt. stitutes a developmental mechanism?1 To the workshop proceedings and participants we owe much of the development of our thinking on the topics discussed in this essay Classical accounts of animal form concentrate on and several of the examples discussed, and we are forever grateful to identifying morphological characters and on trac- the participants for sharing their thoughts and collegiality both dur- ing the workshop and after. We express our appreciation to the Fri- ing their phylogenetic modification. The dichoto- day Harbor Laboratories, its staff, and director for a wonderful mous notions of homology and analogy are implicitly environment for a meeting. We are also especially grateful to Dr. Evelyn Fox Keller for encouraging us in these directions, for the idea predicated on a modular view of animal design; their of a workshop on modularity, and for providing the bulk of the fund- ease of use is proportional to the ease with which ing for that workshop out of her own pocket. Grant sponsor: National Science Foundation: Grant number: MCB- one can identify the modules. Embryogenesis, too, 9732702. consists of logically separable processes—gastrula- *Correspondence to: George von Dassow, Department of Zoology, Box 351800, University of Washington, Seattle, WA 98195-1800. tion, establishment of body axes or morphogenesis E-mail: [email protected] of individual organs or appendages—and we take Received 17 March 1999; Accepted 3 September 1999 © 1999 WILEY-LISS, INC. 308 G. VON DASSOW AND E. MUNRO velopment or as units of evolutionary change. De- THE EMERGENCE OF MECHANISM spite the pervasiveness of this implicit assump- IN DEVELOPMENTAL AND tion, and despite its importance as a pre-requisite EVOLUTIONARY BIOLOGY to mechanistic explanations, there exist only ru- A century ago Wilhelm Roux introduced the no- diments of a developmental modularity concept. tion of developmental mechanics (Roux, 1894), a Those rudiments are the many intuitive notions mechanistic analysis of development by experi- about modularity that have attracted so much at- ment. Embryology before Roux’s time was largely tention recently (Raff, ’96, esp. chapter 10), in- descriptive and comparative. By focusing on analy- cluding morphogenetic fields (Gilbert et al., ’96), sis and experiment, Roux and less polemically in- gene networks (Bonner, ’88; Arnone and Davidson, clined colleagues effected a profound shift in ’97), and the several notions of homologues (Hall, biological thought, a conceptual thread to which ’92). Existing ideas about modularity are based most modern studies of development can be traced on good biological intuitions about the nature of both (Maienschein, ’91). To this lineage we owe such the developmental and the evolutionary process, and concepts as regulative development, induction, therefore the challenge is to develop generally use- morphogenetic fields, and more. However, Judson ful criteria that handle diverse phenomena within (’79) recounts in “The Eighth Day of Creation” in a common explanatory framework. In this essay, the chapter “Morgan’s Deviation” how, shortly af- we discuss why this is not a trivial problem. Our ter Roux’s declaration, T.H. Morgan abandoned em- existing concepts in developmental and evolution- bryology for fruit fly genetics, opining that no one ary biology shape the ways in which we analyze could make any progress in developmental mechan- both development and form, so we start by trac- ics until biology had made sense of heredity. In- ing historically where present ideas about mecha- deed, despite the conceptual success of experimental nism are coming from and where they converge. embryology, it hit a very rough patch until it could Next, we consider two problems with modularity reconcile its differences with genetics. as a conceptual tool, both of which biologists might Experimental embryology ultimately stumbled be stuck with. The way we conceptualize module on its own rocks, the morphogenetic field and the and mechanism depends both on the reference pro- organizer (see Gilbert et al., ’96 for review). Need- cess (on whether we are interested in morphogen- ham and others, inspired on the one hand by the esis or epigenesis, or in the evolutionary process success of Spemann, Harrison, and other embry- itself) and on whether we are thinking analyti- ologists in defining specific, causally relevant cally or synthetically. The first arises because we events in embryos and on the other by the emer- are talking about disjunct types of process in gence of metabolic and enzymatic biochemistry, which biological entities participate differently; sought to develop “chemical embryology” as an ex- the second selects our operational criteria. The perimental study that would reveal mechanisms practical consequence of both is merely that bi- underlying the various tissue-level phenomena ologists must be careful not to assume any par- defined by earlier embryological studies. Need- ticular correspondence between units that emerge ham’s three-volume Chemical Embryology (’31) from each perspective. The map between them is testifies to both the theoretical richness and the itself an architectural problem that merits atten- empirical drought that this movement experi- tion. In the third and fourth sections we consider enced. The organizer, in particular, led straight how modules of animal form and function (mor- to confusion, as a frenzy set in to find the sub- phological characters) relate to modules of devel- stance responsible for its remarkable properties. opmental process. Whatever the nature of that relationship in either general or specific terms, No such agent was found, or rather too many such individual modules must possess a history through agents were found, and embryologists ultimately phylogeny of functional adaptation. Biologists set aside chemical embryology (see Nieuwkoop et have become quite comfortable with the idea that al., ’85 for review). morphological characters are units of phenotypic In hindsight we might say today that the chemi- adaptation. If we say the same is true of develop- cal embryologists failed only because they sought mental mechanisms, then we encounter a problem answers in the wrong place; urea metabolism and of correspondence. Thus, we need to understand the other favorites of Needham and his colleagues interface between developmental and evolutionary don’t seem like developmental mechanisms as we mechanics, and shape our ideas about character think of them today. Things might have gone dif- identity to match. ferently for chemical embryologists had they MODULES IN DEVELOPMENT AND EVOLUTION 309 known enough about genes and their products. the 1960s, 1970s, and since (for example Garcia- Actually, embryologists of that era, as a group, Bellido, ’98 relates the history of interest in dismissed genetics as irrelevant because no one engrailed). There’s plenty more to learn about he- could explain how genes continually present from redity, about the organization, transformation and egg through adult could have anything to do with transmission of genetic information, but we cer- epigenesis (Gilbert et al., ’96). Jacob and Monod tainly know enough about it to explore develop- (’61) provided the first widely noticed answer with mental mechanism in terms of gene function. the demonstration of regulated gene expression Unequivocally, we know something meaningful in bacterial cells. The lac operon laid the ground- about development from the vast literature of de- work for the emergence of developmental genet- velopmental genetics. As a case in point, we know ics as the modern version of chemical embryology not just that without the bicoid gene flies don’t by showing how inherited genes could directly in- form an anterior-posterior axis correctly; we also fluence physiology in an epigenetic manner.2 know bicoid is transcribed in nurse cells, the But is it really just a matter of choosing, say, mRNA is deposited in the egg, anchored at the transcriptional control over urea metabolism? The anterior end, then translated early in develop- last decade has seen a replay of the “organizer ment; the Bicoid protein forms a gradient in the substance” frenzy with mRNAs and proteins in egg; Bicoid is a transcriptional regulator that place of alcoholic tissue extracts, and the pros- binds to target genes (like hunchback), which in pects seem only slightly improved. We now know turn have target genes, and so forth (Driever and of a variety of macromolecules that do interest- Nusslein-Volhard, ’88a,b, ’89; Macdonald and ing things to frog embryos, and this time around Struhl, ’88; Struhl et al., ’89, ’92; Hulskamp et there is good evidence that some of them are ac- al., ’90; Pokrywka and Stephenson, ’91; reviewed tually involved in the developmental process (see in Lawrence, ’92). The point is, developmental ge- Lemaire and Kodjabachian, ’96; Harland and netics reveals more than just a catalog of required Gerhart, ’97 for reviews). However, despite the parts, and begins to reveal something about what emerging body of literature on secreted signals the parts do, which ones interact, and why things and localized gene expression, there is still no ex- go wrong without them. planation for certain fundamental properties of What’s missing is a way to get from this kind the organizer and the primary embryonic field. of knowledge to a formal understanding of devel- For example, Cooke (’81) demonstrated that when opmental phenomena. Mechanism, per se, is an an exogenous organizer is implanted ventrally in explanatory mode in which we describe what are a host embryo, despite the famous twinning phe- the parts, how they behave intrinsically, and how nomenon the relative proportions of tissue types those intrinsic behaviors of parts are coupled to remain constant throughout the embryo. How does each other to produce the behavior of the whole. this phenomenon emerge from what is presently This common sense definition of mechanism im- known about neural inducers, organizer-specific plies an inherently hierarchical decomposition; gene expression, ventralizing signals, and so on? having identified a part with its own intrinsic be- Is something missing? havior, that part may in turn be treated as a whole Morgan’s deviation is finished; Morgan would to be explained. Historically, developmental biolo- have returned to developmental mechanics de- gists haven’t been able to take this approach, pri- cades ago, as did many Drosophila geneticists in marily because of insufficient knowledge of the parts; you can’t explain how the whole skeleton

2Whether or not their paper was in fact a historic stimulant to works if you’ve only got a few bones. Instead for developmental genetics is beside the point; before Jacob and Monod, explanation we rely primarily on a sort of local the relation between inherited information and dynamic regulated processes in a living animal—epigenesis—was obscure. Thus the elu- causation. The operational approach of develop- cidation of the lac operon, and later studies of gene expression in mental genetics, for instance, takes for granted prokaryotes and their viruses, should perhaps be thought of as the greatest theoretical advance of developmental mechanics in the cen- the organism as a working whole, with no gen- tury since Roux. Almost all other broad-brush concepts in use today eral assumptions about the nature of developmen- by developmental biologists date back to the age of Roux, August Weissman, and Hans Driesch. One notable exception is the morpho- tal mechanisms beyond the empirical fact that genetic field, which was developed by Needham, Hans Spemann, and some fraction of mutations have discrete, visible others. Another is Paul Weiss’s notion of “molecular ecology” which surfaces in a number of his essays; Weiss’s idea of how to think of effects. Developmental genetics begins with an in- gene regulation never seems to have made it into common parlance, duced anomaly (a mutation) and a (hopefully dis- but in this essay we make the argument that its time has come. The reader interested in the conceptual lineage of developmental biology crete) consequence, then proceeds to decipher a is encouraged to turn to an excellent volume compiled by Gilbert (’91). perturbation-to-consequence chain. As above with 310 G. VON DASSOW AND E. MUNRO bicoid, one begins with a mutation and a pheno- of developmental mechanism and solid examples type at the ends of the chain and fills in the links to consider, evolutionary biologists have had to of cause and effect until it may be said that muta- take for granted the existence of developmental tions in bicoid lead to some phenotype because mechanisms that are responsive to selective pres- Bicoid normally binds to the promoter of hunch- sure. The field has historically focused on the na- back, causing hunchback transcription, etc. The per- ture of that pressure and on interpreting scenarios turbation-to-consequence chain between mutant to account for the existence of specific forms, which gene and abnormal phene is then taken as an ex- themselves are presumed to be more or less planation for the role of part in process. We owe to “adapted” in aggregate. Thus evolutionary biology this habit the prevalence of metaphors like “genetic employs among its explanatory modes something program” and “developmental pathway”; implicit in akin to final causation. Gould and Lewontin (’79) this discussion is a reminder that we oughtn’t take point out in their criticism of the “adaptationist such metaphors too seriously as literally descrip- program” that natural selection (Dawkins’ “blind tive of mechanistic architecture. Rather, they are watchmaker”) is a nearly teleological explanation summarily descriptive of a chain of causation that for phenotypic adaptation, in which possible sce- we have been able to tease out analytically from an narios for adaptive change are limited only by as-yet-undescribed architecture. one’s ability to imagine how a surviving outcome While this kind of explanation allows one some can be achieved through a series of selectively fa- description of what parts (genes and their products) vorable intermediates.3 On the other hand, evolu- do to each other, it doesn’t articulate any sense of tionary biologists are equally at home with the the mapping from genotype to phenotype, which is encapsulation of natural selection in population what we ultimately want. But as the body of indi- genetic theory (the watchmaker’s apprentice, if vidual perturbation-consequence chains grows, they you will), which forms the basis for a formal overlap and interweave, and the whole sooner or mechanistic mode of explanation. But it’s hard to later must emerge from the sum of the parts make the leap; without some concrete idea about (shouldn’t it?). The opportunity arises to integrate how genotypes determine morphological pheno- this information within a mechanistic explanatory types it becomes difficult to say how allele spread mode, an opportunity literally unavailable to biolo- in populations is related to patterns of morpho- gists until very recently, and we expect this to lead logical change. to a fundamental transition in biological thought. One of the persistent problems is how we are Developmental biologists have worked for nearly a to explain patterns of adaptive morphological century in the perturbation-consequence mode, in change manifest in phylogeny, such as, among which explanation resembles Aristotelian efficient other things, missing phenotypes (e.g., eight- causation. We are on the verge of a shift toward legged insects) or parallelisms (e.g., independent something much more like formal causation; that evolution of saber teeth in “cats”). More gener- is, explanation in terms of dynamical formulation ally, one would like to understand why the adap- and behavior. The first step is to assess where we tive process follows the particular course it does are with respect to this opportunity. Where in the in each instance, rather than another. Most bi- study of development is mechanism within reach? What do developmental mechanisms look like, 3We wish to be clear that nothing pejorative is intended in this and what are the problems associated with iden- passage. On the contrary, it is only because evolutionary theorists chose tifying or characterizing them? Do we understand to think in terms of selective optima, goals, and adaptationism that evolutionary problems could be spoken about without reference to the any developmental mechanisms well enough to nature of developmental mechanism. Certainly during the construc- use them as a basis for hypotheses about mor- tion of the Neo-Darwinian synthesis there existed too much debate about the nature of the developmental process for evolutionary theory phological evolution? The case studies sketched to consider it explicitly. Moreover, the vibrance and utility of evolu- at the end of this essay make us think the situa- tionary theory testify to the validity, if not the completeness, of final causation as an explanatory mode in this context. Finally, despite the tion is quite promising. critique of adaptationism, the critics do not shift away from a funda- Evolutionary biology has come via a different mentally teleological viewpoint; introducing terms like exaptation (due originally to Gould and Vrba, ’82) or pre-adaptation, does not alter the route to much the same point. Evolutionary explanatory model. The anonymous reviewers also drew our attention change in morphology is, literally, heritable varia- to two additional points. First, finalistic language may result as much from the prevalence of artificial selection experiments (in which there tion in development. The developmental mecha- really is a goal) as from any underlying philosophical dilemma. Sec- nism is the fundamental entity whose behavior is ond, developmental mechanics, to the extent that it is described by dynamical systems theory, itself requires a good deal of finalistic ter- altered by mutation, and is thus the substrate of minology because of the prominence of stable states, attractors, and phenotypic variation. Lacking a coherent concept limit cycles in the analysis of deterministic systems. MODULES IN DEVELOPMENT AND EVOLUTION 311 ologists concur that natural selection can’t achieve ’92). Conceivably, whatever mechanism makes in- every conceivable goal and that some adaptive sects bear legs can only make six; maybe the routes may be more likely than others. But one en- mechanism that makes mammalian teeth has counters difficulty in saying, in a definite sense, some innate propensity to increase the length of whether there is anything more to explain about canines. The debate has to do with the difficulty evolutionary patterns beyond the blind watchmaker. of saying, in any particular case, whether an ob- There are bound to be missing phenotypes, because served pattern is due to constraints or to corre- we creatures of the earth can’t have had a chance sponding selective patterns; in effect, whether a to explore all the possibilities yet, and anyway, what pattern is due to intrinsic or extrinsic factors. We would insects need another two legs for? Further- feel this argument misses the point, for it is the more, evolution is a branching process; daughter case that without even being able to describe de- variants tend to inhabit the same neighborhood of velopmental mechanics, one can say for certain phenotypic space as their parents, so parallelisms that any mechanism (developmental, electronic, may reflect the fact that similar adaptive pressures economic, telepathic, whatever) must have describ- lead (unsurprisingly) to similar adaptive responses. able variational tendencies. That is, for any So it can’t be completely clear whether patterns of mechanism in the rational universe there must adaptive change reflect patterns in the selective mi- be some set (which could be empty!) of available, lieu or reveal constraint. functional modifications. This is as true of the The word “constraint” reveals the perspective most sophisticated mechanisms as it is of the sim- in which this dialectic originated: constrained with plest. To make an account of such a set in any respect to what? Clearly, with respect to the abil- specific case, one would need detailed knowledge ity of selection to accomplish some goal. “Goal” is of the mechanism in question that is well beyond not far from “purpose,” and purpose is final cau- present reach. But we do know some developmen- sation. We aren’t casting aspersions on evolution- tal mechanisms as rudiments, well enough to for- ary biologists. Every biologist knows that adapting mulate hypotheses about how they could be organisms do not have goals in this sense, that transformed by mutation. In these cases we can members of an adapting population have no view make educated guesses as to how these specific of the adaptive landscape, and that even if they mechanisms might be the substrate for evolution- did they would have no control over the muta- ary change. From this perspective the term “con- tional motor that propels them across it. We straint” isn’t appropriate. Instead, when we think merely talk about adaptation in this way because about morphological evolution in terms of devel- it reflects our notion that there exists some ideal opmental mechanisms, we are really thinking response to each selective pressure—the goal.4 Se- about variational tendencies, of which develop- lection, however slowly, should pull an adapting mental constraints per se are a special case. population quite near such a goal (i.e., to a fit- Considering this, we see that this question mark ness peak) within the adaptive landscape. There that has traveled so long under the label “devel- has been a minor rumbling of doubts about the opmental constraints” is really the plane of inter- general validity of this assumption. Selection may section between developmental mechanics and not, for example, hold a population at an adap- evolutionary mechanics. This question mark is tive peak (without incurring some burdensome central to the evolution of development. To turn cost of selection) in the face of high mutation rate. our focus here requires developmental and evolu- Other doubts arise when one considers the adap- tionary biologists to construct an explicit concept tive landscape metaphor: populations could be- of developmental mechanism and how it functions come trapped on sub-optimal peaks, unable to as the substrate for morphological evolution. reach the “best” solution without traversing val- leys of critically low fitness (Kauffman, ’93, chap- MODULARITY, MECHANISM, AND THE ters 2 and 3). DECOMPOSITION PROBLEM However, the most debate has been sparked by Whether we start with a decomposition of de- the notion of developmental constraints, the hy- velopment into analytic units or with a construc- pothesis that patterns of phenotypic change may tion of synthetic units from lower-level entities, sometimes result from the nature of underlying we hope to arrive at a conceptualization of build- developmental mechanisms (reviewed by Hall, ing blocks from which could be assembled models of higher-order behavior. There are many ways 4See footnote 3. to identify modules with respect to any particu- 312 G. VON DASSOW AND E. MUNRO lar whole, and in general each different concep- higher-order groups below the scale of the entire tual decomposition of that whole engenders a dif- genome, grouped perhaps by the density of inter- ferent set of experiments, a different mode of reactivity among gene products. We expect fur- explanation, and ultimately a different insight. thermore that any decomposition of the genome For example, developmental biologists studying in functional terms will be explicitly hierarchical. the establishment of primary axes within verte- In this case, however, it remains an operational brate limb buds recognize at least two different matter how one chooses to individuate epigenetic decompositions of the developing limb. One, based modules. One arrives at different criteria analyti- on classical embryology, identifies regions of tis- cally and synthetically. Imagine being presented sue within the rudiment, such as the progress with a map of the epigenetic interactions between zone, the zone of polarizing activity (ZPA), and all an organism’s genes. It should be possible to the apical ectodermal ridge (AER), and ascribes individuate modules (if they exist) within such a to them experimentally defined developmental map by connectivity criteria alone: one might functions and interactions (Saunders, ’48; Saunders adopt the operational definition that a module is et al., ’57; Tickle et al., ’75; Summerbell, ’79). An- any subset with a high internal density of inter- other, based on genes as abstract units of develop- actions and sparse connection to the rest. This mental function, has led to the study of a variety of criterion, however, while it suits some applica- gene products, including sonic hedgehog (Riddle et tions, doesn’t help us integrate molecular genetic al., ’93), FGF family members (Martin, ’98), and the data into formalized mechanisms simply because Hox complex (Nelson et al., ’96), that are involved too small a fraction of the total map is yet known. in the establishment of axes within the limb bud. For that problem we need criteria that help us to Each of these modes of decomposition has become tell, as we build from the ground up, when we’ve associated with an accepted type of explanation achieved some degree of “wholeness”; one might within mainstream biology, and when we put each adopt module-individuation criteria based on the class of unit into the pot we hope to get out an ex- constitution of an intrinsic behavior. In either case planation of limb development. The more we know one picks out intermediate entities that are ex- within either decomposition, the more comfortable pected usually to fall into a nested hierarchy. we are taking one as proxy for the other. It is such intermediate entities that pose the Usually, as in this example, units of decompo- most difficulty, and with which we would like to sition are chosen prior to analysis of a given phe- associate the term module (as opposed to “unit,” nomenon or object, for historical reasons, or based which connotes a much more discrete entity than on the available tools, or because they are appro- we have in mind). Modules may be more difficult priated from existing disciplines. Furthermore, to define in abstract terms and to identify or dis- many of the entities familiar in developmental and tinguish from one another in practice than obvi- evolutionary biology (molecules, cells, individual ous things like proteins and cells, but they are by organisms, species, etc.) are obvious in that we no means less real as a consequence. Perhaps if readily distinguish them and could articulate why. we take as a starting point the obviousness of mol- These obvious entities, defined in more or less con- ecules, genomes, cells, and individuals, we can say crete terms, constitute the framework of an that we would like to think about intermediate equally evident hierarchy of biological organiza- entities that are either composed of or that com- tion. Yet there are clearly more elusive entities that pose the obvious ones. But here we would run into inhabit intermediate levels within the framework difficulties as we approach the boundaries. After defined by the obvious ones. For example, body parts all, there are well-known problems in trying to are decompositional units of animal form, adapta- define the gene or even the individual in certain tion, and development. Clearly body parts are not contexts (colonial organisms, notably). Paradoxi- as concretely defined as the individual bodies they cally, our intuition recognizes at least certain mod- are parts of or the cells they are composed of. More- ules (body parts, for instance) much more readily over, anatomy is explicitly hierarchical unto itself, than our reason allows; one is tempted to wonder unlike either the cell or the individual; thus we what intuition knows that reason does not, and naturally consider a knuckle part of a finger, which thus one pragmatic aspect to the decomposition is part of a hand, etc. problem involves a search for rational criteria that Consider genes, which are clearly organized into accomplish in general what our intuition provides genomes. Empirical experience tells us that there in specific cases. must be some functional organization of genes into There is an intuitive relationship between mod- MODULES IN DEVELOPMENT AND EVOLUTION 313 ules as defined above and the developmental nation, etc.) and the morphogenetic process (cell mechanism: a developmental module is a collec- shape change, the emergence of physical form). tion of elements whose intrinsic behaviors and Furthermore, we also have the evolutionary pro- functional interactions yield a mechanistic expla- cess to consider, and moreover it is often difficult nation of an identifiable developmental process or to draw any line between the epigenetic process transformation.5 Having identified a module with and plain old metabolism. For each of these ref- its own intrinsic behaviors, one may subject it to erence processes we might expect to identify dif- further decomposition. Similarly, we expect that ferent compositional units and to find that they modules we treat operationally as isolated enti- enter in different ways into mechanistic explana- ties are coupled to other concurrent modules to tions. Alternatively we may find that the same effect higher-order entities. Thus it is clear why compositional units take on different significance we should be concerned with the existence and when viewed with respect to some alternative pro- extent of modularity in development: because de- cess or organization. Thus it is important to in- composability is prerequisite to mechanism, the vestigate how decompositions with respect to characterization of modularity in development ought different reference processes are related. to make developmental mechanics more accessible. Furthermore, while it is surely too difficult ever to THE INTERFACE BETWEEN apprehend the entire logic of development all at DEVELOPMENT AND EVOLUTION once, it may be possible to build such an apprehen- If morphological adaptation necessarily means sion through a hierarchical decomposition. As good heritable change in developmental mechanisms, reductionists, we biologists all hope this to be so or how are developmental modules related to units of we would never make it to the bench; the extent phenotypic adaptation? One way to operationally to which reduction succeeds is related to the de- define developmental modules is that any sub- gree of identifiable modularity. The point is not system manifesting some quasi-autonomous behav- to conceptualize the organism or the developmen- ior qualifies. Applying this to the evolutionary tal process as a bag of modules, any more than as process, one decomposition readily presents itself: a bag of genes, and certainly it will be true that given a series of ancestrally related forms (i.e., some aspects of development, or some organisms, primitive and derived forms plus intermediates), are more or less modular than others. Neverthe- we expect to be able to identify subsets of the less, the better one can identify logically separable shared body plan elements that manifest adap- modules, the better paved the road to mechanics. tive change more or less autonomously. We might So far in this essay we have evoked consistently hope that subsets so characterized would corre- “developmental process” even though it is far from spond to anatomical elements that we would iden- clear that it makes sense to speak of developmen- tify within the individual forms, and indeed this tal process as if it were a single domain of phe- expectation is generally borne out. Instances nomena in which the modules we want to look for abound: tetrapod limbs, insect wings, arthropod participate. Encompassed under the umbrella of limbs and segments, mammalian teeth, vertebrate “developmental process” are the epigenetic pro- eyes; all are clearly distinct anatomical elements cess (meaning regulated gene expression, dynamic that exhibit specialization autonomous to the body interactions among gene products, cell determi- plan of which each is part. Thus, “unit of pheno- typic adaptation” means the continuum of homolo- 5An anonymous reviewer questioned why we focus on the role of gous body parts. modules in explaining things rather than on their role in process per se. We do not know how better to deal with the difficult issue of Given such an adaptive unit, we expect an un- whether we see modules because they are a useful metaphor or be- derlying cause in the developmental mechanisms cause they are really there, other than to say that it seems useful to think about modules in the context of present knowledge. We don’t associated with that unit to account for its coher- deny the possibility that once more data are available, modularity ence. We might naively anticipate a one-to-one (as we describe it) will no longer be the most useful way to think about things. The point here is to say “it looks like it’s turning out map between logically separable modules of the this way, so let’s see where it can get us,” so we are not mere ideal- developmental process and autonomous body plan ists concerned only with how we conceptualize things. Still, there is a deep connection between the available data and the metaphors that elements, remembering that both decompositions might be useful to describe those data. Thus, the notion of the gene are richly nested and hierarchical. One could ex- as determinant flourished when single genes unconnected to each other constituted the available data, and the metaphor of the devel- tend this argument beyond body plan elements to opmental pathway has been useful as long as most of the available any feature that might conceivably be homologized data fit into linear chains. To a certain extent the issue becomes moot because our saving grace, as scientists, is that sooner or later among a group of related forms, including some- we junk explanations and metaphors if they don’t fit the facts. thing as abstract as the dorsal-ventral axis of an 314 G. VON DASSOW AND E. MUNRO animal or a particular arrangement of cell types ferent were it possible to repeat it even under the in a tissue. Indeed, when we ask if there is such same external conditions. One can distinguish con- a one-to-one relationship, we tend to find that ceptually between processes that determine specific there is: thus the arthropod segment has its seg- patterns of phenotypic change within natural popu- ment polarity genes, the vertebrate axial complex lations given the range of available heritable phe- has its Hox genes, and the tetrapod limb has its notypic variation, and processes internal to embryos panoply of hedgehog, patched, BMPs, FGFs, wnts, governing the production of that variation, (i.e., that and (again) Hox genes. determine the structure of the genotype-phenotype Since it seems clear that within an individual map [sensu Wagner and Altenberg, ’96]). The species we might expect the modules of develop- present discussion is concerned mainly with the mental regulation to correspond, at least often, to latter type of process, the internal production of modules of phenotypic adaptation, we must ques- heritable phenotypic variation and the ways in tion the extent to which these are phylogeneti- which this defines possibilities for adaptive evo- cally stable associations. The easy answer is to lutionary change. The same decomposition de- cite examples, of which there are many, that prove scribed above seems natural, based on logically that such relationships are not (always) stable. separable parts or processes and their associated Instead we would like to explore why this is likely developmental mechanisms. But in this case we to be generally true, because it has important con- are not interested in specific instances of mecha- sequences for how we study the evolution of de- nism with respect to a specific part or process velopment and how we interpret data. To frame within an individual organism, but rather in the this problem more clearly, it is useful to contrast variational properties of developmental mecha- development as a process with evolution; there are nisms with respect to genetic mutation. That is, at least these two reference processes of interest. we are interested in the range of variation that Biologists view development as a collection of dy- genetic mutation could produce in the properties namic processes arranged hierarchically in space of a given developmental module or mechanism and time that collectively produce from some well- given both its current structure and the ways in defined initial state (the fertilized egg) an adult which coordination with other modules determine organism (i.e., an equally well-defined specific col- which variants might “work”. Given a mechanism lection and arrangement of anatomical parts). We at work in some context, in what sense is it a tend to associate with each subprocess some land- substrate for evolutionary change? What can it mark end state (e.g., establishment of primary axes, be made to do? allocation of an organ rudiment, etc.) or, more gen- Now we must face the fact that the criteria de- erally, a transformation of embryonic state. While termining which variants persist have, in all like- we can study such individual processes in concep- lihood, little to do with the preservation of any tual isolation, they are coordinated in space and particular relationship between mechanism and time to produce a harmonious whole. For each such function. The evolutionary process doesn’t neces- process we seek to identify a set of parts—a mod- sarily care so much about exactly how the dorsal- ule—whose intrinsic properties and interactions col- ventral axis is set up, the tooth anlage is specified, lectively determine, given appropriate initial and or the neural tube formed, as long as the end state boundary conditions (which represent coordination is equivalent. Consider a trivial example: imag- with other subprocesses), the mechanism underly- ine a signal transduction pathway in which a cell- ing that dynamic transformation. Here the focus is surface receptor activates a kinase, which then primarily on identifying the specific properties and activates a target. Now imagine introducing an- arrangements of parts plus the specific initial and other protein kinase, and that this kinase can be boundary conditions that get the job done—within activated by the receptor and in turn activate the an individual organism. appropriate target, and moreover do a better job, By contrast, evolution is an interplay between somehow, than the original. Couldn’t the evolu- natural selection (Dawkins’ “blind watchmaker,” tionary process junk the original in favor of the who cares little for design as long as “it works”) newcomer? and the processes by which variants arise and dis- Surely if we can imagine this scenario with a tribute (what we called the “watchmaker’s appren- single gene product, we can also imagine an en- tice” earlier in this article). Phenotypic variation tire module, an entire mechanism, being sub- is filtered by natural selection into a specific his- sumed should something better arise to take its tory of change that would likely be somewhat dif- place. Moreover, what if we begin with a very so- MODULES IN DEVELOPMENT AND EVOLUTION 315 phisticated mechanism, say one that patterns an degree of persistence over evolutionary history, but entire organ rudiment; such a sophisticated mecha- how could we trace such a scenario as described nism might readily be broken down into many above? We have developed a hypothetical night- modular parts: perhaps a few signal transduction mare in which a putative phylogenetic unit un- cascades, several gene regulatory switches, etc. dergoes continual xenoreplacement at the Might we not imagine that each of these parts could molecular level; of course the corollary is that the be successively replaced, even several times over, replaced parts are probably not being junked, but in a genetic version of continual xenoreplacement? rather are recycled into another context, to replace This may sound far-fetched, but consider one no- another module in turn. The more dissociable (i.e., table example: there can be little doubt that the the more modular) a mechanism, the more obscure segmentation of long-germ and short-germ insects will be its phylogenetic origins and its correspon- is homologous in a rather deep sense. In Drosophila dence to body plan elements. If we take modular- we know how segments are laid out, and this ity at all seriously, then any attempt to use mechanism depends on the fact that it takes place developmental mechanisms as phylogenetic tools in a syncytium (reviewed in Lawrence, ’92). No is doomed: how could one hope to distinguish be- such mechanism could possibly make segments tween bona fide conservation (a stable history be- in short-germ insects, where all but the first few tween mechanism and character) and re-use or segments are patterned as they proliferate from (worse yet) re-invention? a growth zone. A massive replacement of devel- An inversion of perspective makes the situation opmental mechanism has taken place, and the look much more promising, and a colleague of ours evolutionary intermediates may be obscure. has put it best: This roundabout approach is intended to expose “...the character [identity] problem and the a challenge that arises at the interface between homology problem have been primarily dis- developmental and evolutionary mechanics. The cussed over the generations from a taxonomic implication is that we will only rarely, if ever, be and comparative point of view; but I think this able to pin down any stable complex of develop- is completely wrong-headed and most of the mental mechanism and phenotypic characters that baggage we are dealing with here is coming behaves as a discrete unit with respect to both from this history. Characters do not exist to the evolutionary and developmental process. De- please taxonomists, [just as] genes do not ex- spite a few attention-grabbing examples, there is ist to please geneticists; rather the fact that no a priori reason to believe that the same in- we can identify and work with genes tells us stantiation of a developmental mechanism under- something about the functional and physical lies a conserved developmental process in even organization of the genetic material. In the closely related organisms. Indeed, empirical obser- same sense, the fact that we can identify ho- vation confirms that this need not be so (see Félix, mologous body parts tells us something about ’99; see examples in Roth, ’88; also Table 1 in how the phenotype is organized. I would like Wagner and Misof, ’93). These considerations re- to look at characters and homologues from veal grave difficulties for applying any of our clas- this biological point of view, rather than the sical comparative tools, particularly our classical perspective of how to recognize them and how notions of character identity, to the evolution of to use them to reconstruct phylogeny, because development. One principle goal in evolutionary that’s not the reason they exist; that’s not the biology is to reconstruct the actual history of phylo- mechanistic problem that we have to solve.” genetic change, and a central problem of phylogen- — G. Wagner, speaking at FHL modularity etics is the identification of homologous characters workshop, ’97 and determination of polarity of evolutionary change in those characters. Many current workers, inspired Rather than view dissociability as a problem for by the apparent conservation of a few better-known comparative biologists, let us recognize it as an ar- developmental mechanisms, are eager to use de- chitectural feature of evolvable developmental sys- velopmental mechanisms (or at least spatial regimes tems, a feature whose origins and consequences of gene expression) as quasi-phylogenetic tools, es- deserve attention. Over the years, authors too nu- pecially as diagnostics for homology (for example merous to cite have noticed that, indeed, it is diffi- see DeRobertis and Sasai, ’96; Arendt and Nübler- cult to imagine evolution of morphological diversity Jung, ’99). Clearly one property that a unit of phy- as we know it without dissociability. Furthermore, logenetic change must possess is at least some the litany of well-known evolutionary transforma- 316 G. VON DASSOW AND E. MUNRO tions, including homeosis, all types of heterochrony, character through the adaptive process. We cease adoption of direct development, and so on, literally (or should cease) to identify homologues when we imply dissociability either among body plan ele- can no longer imagine transformation by naturally- ments, among developmental mechanisms, or both. arising, heritable phenotypic variation. Moreover, if we ask how phenotypic characters participate in CHARACTER IDENTITY, HOMOLOGUES, the evolutionary process, it is through the variety of AND VARIATIONAL TENDENCIES possible forms determined by the variational ten- Nevertheless, we need ways to assess charac- dencies of each character. ter identity. In the case of body parts, for example, Whence variational tendencies? Obviously, when we need rational criteria that will generalize for we are talking about morphological characters, us what our intuition does so well in particular variational tendencies come largely from devel- cases. To our knowledge the most useful account opmental mechanism, whatever it is. This is para- of character identity for EvoDevo is the “biologi- doxical: there is no inherent stability to the cal homology concept” developed by Roth (’88, ’91) genotype-phenotype map, no one-to-one gene- or and Wagner (’89a,b, ’95), and our gloss is based developmental process-to-character relationship, on their work. Let us begin by asking, what does yet we confront the widespread existence of stable our intuition do? Wagner (’95, citing McKitrick, patterns whose coherence must be embodied caus- ’94) notes that we recognize homologues based on ally within a shifting sand of developmental pro- parsimony: given a character and a choice between cess beneath it. Roth (’88, ’91) and Wagner (’95) candidate homologues, we choose the correspon- conclude that the intrinsic stability of homologues dence that requires the least transformation. To amidst the evolutionary process is an emergent borrow Wagner’s illustrative style, it is easier to property that transcends the (possibly rapid) evo- think of the bird’s wing “becoming” a dinosaur’s lutionary transformation of the processes which forelimb than to think of the wing becoming, say, form them. 6 the tail. There is a naive way out of this conundrum. If Wagner (’95) notes that this parsimony approach most of the time organisms experience stabiliz- makes an implicit statement about the variational ing selection with respect to most characters, then tendencies of the characters in question. We can- if a variant were to arise in which one module of not imagine how natural variation could trans- a particular developmental mechanism were to be form a bird wing to a dinosaur tail, but we can replaced by another that performs equivalently, see how natural variation could transform it to a this replacement could be selectively acceptable. dinosaur forelimb, therefore we conclude that the So we get a little bit of change in the develop- bird wing and the dinosaur forelimb are manifes- mental mechanism, and none in the character it tations of the same homologue. We have there- fore already assumed not only the existence but produces. Functional constraints impose conser- also the evolutionary relevance of what are classi- vatism at the level of phenotypic units, while al- cally called developmental constraints! Now we lowing some flexibility in the developmental have come full circle: we began the previous sec- process. This explanation, while plausible, is so tion with an intuitive decomposition of animal mundane as to merit no further attention. Also, form into units of phenotypic adaptation; we it assumes an extreme degree of modularity equated a continuum of homologues with such among developmental processes that is unlikely units; the unit, in reference to the adaptive pro- to be general enough to explain the origins of char- cess, is manifest within the continuum of variant acter stability in more than a few instances. forms; and now we see the unit must in fact be Wagner and Misof (’93), inspired by experiments defined by its variational tendencies, for it is only on regeneration in blennie fin rays, suggest an those tendencies that maintain the identity of a interesting possibility. They distinguish between generative mechanisms (like rudiment specifica-

6A flip side to this, though perhaps obvious, deserves emphasis: it is tion, pattern formation, and morphogenesis) and an important criterion that it be easier to draw a correspondence than morphostatic mechanisms, which include any it is to postulate novelty. Thus, we have to convince ourselves that it is easier to transform the reptilian forelimb to a bird wing than it is to mechanism involved with maintenance of the or- first dispose of the forelimb and then invent a wing. There is little gan or structure in question during ontogeny. A danger of forgetting this criterion in the case of complex body parts such as limbs, but there is much danger in the case of simpler “char- trivial example would be a mechanism that main- acters” such as differentiated cell types or developmental mechanisms, tains the relative distribution of cell types in a to say nothing of attempts to use the expression of individual genes such as Distal-less (for example, Panganiban et al., ’95, ’97) or growing organ. Regenerative processes certainly Brachyury/T (Kispert et al., ’94) as homology markers. reflect the existence of morphostatic mechanisms, MODULES IN DEVELOPMENT AND EVOLUTION 317 because to account for regeneration there must (six-and seven-toed cats, the occasional polydac- be some regulatory mechanism that senses incom- tylous human); some sort of mechanism evidently pleteness and activates wound healing, blastema is able to convert the extra rudiments into us- formation, etc. and then turns it all off again when able digits. Somewhat more extreme is the re- completeness is again achieved. cent finding that metamorphic brittle star pluteus Clearly, morphostatic mechanisms could buffer larvae can drop off fully-formed juveniles, swim against variation in generative processes. There back into the plankton, regenerate, and form a may be a wide array of functional forms that new juvenile (Balser, ’98). Starfish larvae also variation in generative processes could manifest, have remarkable regenerative capacity (Minako but a morphostatic mechanism could attenuate Vickery, personal communication), and Jaeckle that variability. On the other hand, a character (’94) demonstrated that starfish larvae can repro- could not be buffered in the same sense against duce asexually by budding. Thus, morphostatic changes in morphostatic mechanisms. Unless the mechanisms (of which regenerative powers are a variability of generative mechanisms is insuffi- manifestation) can open up entire life history cient to fill the space of possibilities allowed by variations. They do not always impose the outer morphostatic variability, variation in morphostatic bound on the space of possibilities, but may even mechanisms should correspond to variation in the facilitate hops through that space. phenotype. Both classes of mechanism therefore We do not pretend herein to solve everything determine variational tendencies, but Wagner and by applying conceptual distinctions and imagin- Misof argue that because morphostatic mecha- ing the consequences. Rather, the point we wish nisms can impose an outer limit in the space of to make is that the problem of correspondence be- possible variation, they may be primary determi- tween natural units of developmental process and nants of the continuity of homologues in the face natural units of evolutionary process is neither of changing generative processes. Thus, pheno- trivial nor presently soluble. The existence of the typic characters may be endowed with a certain problem tells us first, to turn our attention to ex- degree of conservatism by developmental con- ploring the nature and origins of variational ten- straints, again while allowing some variation in dencies in developmental mechanisms; second, to generative mechanisms. ask what systems-level properties emerge from The conceptual division of developmental pro- conspiracies of lower-level entities; third, what can cess into morphostatic and generative modes has we learn about what makes developmental archi- other interesting implications. First, the notion tecture evolvable; finally, how did we get such an of morphostatic mechanisms may help explain architecture in the first place? cases of “bottlenecking” in the evolution of devel- opment. For instance, the generative processes A FEW INSTRUCTIVE EXAMPLES that lead to segmentation differ radically between We present a selection of case studies that illus- short- and long-germ insects, yet both types of de- trate some of the concepts to which we want to draw velopment lead to a primitively-segmented embryo attention here. These examples are not presented with similar neurogenic and appendage-making to prove any particular point, but rather because patterns in corresponding segments (Patel et al., each of these cases is an exceptional opportunity to ’92). The segment polarity genes, which likely study the evolution of developmental mechanisms function similarly in short- and long-germ insects in relation to phenotypic adaptation, using molecu- (Nagy and Carroll, ’94), may be among the morpho- lar and comparative approaches together. static mechanisms that maintain the identity of seg- ments in insects. Our own efforts to simulate the Hedgehog, patched, and company: dynamic behavior of segment polarity genes have an epigenetic module shown that even a small subset of known interac- First we discuss a simple example of a tiny epi- tions can stably maintain an asymmetric segmen- genetic network that appears to behave evolution- tal pattern in a field of cells (von Dassow, Meir, arily as a module. This example is afforded by Munro, and Odell, in preparation). recent studies of the regulatory interaction be- Second, under certain conditions morphostatic tween the Drosophila segment polarity genes mechanisms might channel otherwise aberrant patched (ptc) and hedgehog (hh) along the ante- generative variation into useful functional varia- rior-posterior compartment boundary in Droso- tion. The autopodium of the amniote limb can gen- phila imaginal discs. In the posterior, engrailed erate more than the usual five condensations (en) promotes hh transcription (Tabata et al., ’92). 318 G. VON DASSOW AND E. MUNRO ptc is active in the anterior compartment and is Nusse, ’97). In the row of cells posterior to the repressed in the posterior compartment by en parasegment border, as in imaginal discs, En pro- (Hidalgo and Ingham, ’90). However, ptc is tran- motes hh transcription (Tabata et al., ’92). Hh scribed only at very low levels in most cells of the tirates away Ptc, consequently altering the rela- anterior compartment because the Ptc protein, tive abundance of repressor and activator forms when active, leads via a subtle interaction with of Ci, such that the activator accumulates in cells the transcription factor encoded by cubitus inter- just anterior to the boundary (Aza-Blanc et al., ruptus (ci) to the repression of ptc transcription ’97). One Ci target is wg; full-length Ci activates (Aza-Blanc et al., ’97). The secreted protein en- wg anterior to the boundary (Von Ohlen and coded by hh binds to and titrates away Ptc, thus Hooper, ’97). Wg is required for en expression in relieving transcriptional repression of ptc; this al- cells posterior to the boundary (Vincent and lows accumulation of Ptc adjacent to the poste- O’Farrell, ’92), and thus there is a closed loop of rior compartment, where hh is expressed (Marigo interactions that, it has been hypothesized, sta- et al., ’96). Wherever hh is expressed, neighbor- bly maintains the boundary throughout develop- ing cells express ptc unless something else (such ment. This hypothesis implies that the hh/ptc as Engrailed) prevents ptc transcription. module has been incorporated, in this instance, into hh, ptc, ci and their associates interact in such a higher-order network that exhibits a more com- a way as to constitute a tiny, re-usable epigenetic plicated intrinsic behavior, namely the stable spa- module. The re-usability of this module has been tial regime of segment polarity gene expression. demonstrated strikingly in vertebrates. In the As mentioned previously, the segmental organi- mouse, hedgehog-like genes are expressed in a va- zation of insect embryos seems conserved through- riety of tissues including notochord and floorplate, out insect evolution, including the manifestation of limb bud, gut epithelium, whisker buds, nasal pla- parasegments (for example, Nagy et al., ’91), the code, and more; in every case the mouse patched mechanism of limb specification (Panganiban et is expressed in cells immediately neighboring al., ’94; Warren et al., ’95), and the function of at hedgehog-expressing cells (Goodrich et al., ’96). least some of the segment polarity genes in main- Since many of these structures are evolutionary taining the compartment boundary (Nagy and novelties, one cannot help but consider this a Carroll, ’94). Yet segment specification differs radi- manifold example of reuse. Whatever property cally among insects, and it is impossible to imag- makes this tiny module useful, it is abundantly ine that the same molecular mechanisms are at clear that it is not common ancestry but common work in every case (Patel et al., ’92; Patel, ’94; biochemistry that unites the long list of embry- Grbic et al., ’96; Ho et al., ’97). These observa- onic structures in which hh and ptc are expressed tions led us to hypothesize that the segment po- in complementary patterns.7 larity genes must be a module according to the One of the best-studied roles of the hh/ptc mod- following strong definition: a module has a char- ule is in the formation of parasegmental bound- acteristic intrinsic behavior in the absence of any aries and polarization of segments in Drosophila specific, persistent, exogenous influences on its embryogenesis. They were originally identified as components, and it may be triggered to express segment polarity genes, a class that includes genes this behavior through a small number of (one or involved in Hh signaling (fused, smoothened, cos- a few) generic inputs. We set out to test whether tal-2, and others; for review see Kalderon, ’97), or not this is the case by building a dynamical en, wingless (wg), and genes like porcupine, ar- simulation of the segment polarity network. In madillo, and disheveled, whose products partici- this case, the requisite behavior seems to be the pate in Wg signaling (reviewed in Cadigan and asymmetric, segmentally reiterated co-expression pattern of wg, en, and hh, all of which function as 7Indeed we can’t even be sure we’re talking about re-use or rein- “outputs” of the segment polarity gene network. vention here. It is no more “homologous” that hh and ptc are coexpressed in two different tissues than it is that serine, histidine, We found that a surprisingly small subset of and aspartate are found at the active site of both subtilisin and chy- known segment polarity gene products and their motrypsin family proteases. Serine, histidine, and aspartate are not the only way to cleave a peptide bond (many other proteases exist), known interactions (as characterized in Droso- but it does not nowadays surprise us much that two otherwise unre- phila) can satisfy these criteria for modularity lated proteins should have come upon the same three-dimensional arrangement of amino acids, and that, given such an arrangement, (von Dassow, Meir, Munro, and Odell, in prepa- both proteins act as proteases. By analogy, hh and ptc are probably ration). That is, the skeleton of the segment po- not the only way to do whatever it is that they do, but we should not be surprised if two otherwise unrelated tissues have employed them larity gene network can stably maintain the for that function. appropriate expression patterns for wg, en, and MODULES IN DEVELOPMENT AND EVOLUTION 319 hh in a two-dimensional field of cells without any The anchor cell participates, at least in some exogenous influences given a suitable choice of ki- nematodes, in the induction of the vulva. In C. netic parameters. Moreover, our simulation is sur- elegans, six cells (P[3–8].p) arrayed along the AP prisingly insensitive to both initial conditions and axis of the animal may participate in vulva for- the choice of kinetic parameters. Although there mation. The anchor cell is normally closest to P6.p; are about 50 free parameters, each of which might it secretes a signal that is received in highest con- vary over several orders of magnitude, an astound- centration by P6.p, and in lower concentration by ing one in 200 randomly chosen parameter sets the two neighbors. P6.p thus adopts the central leads to the proper behavior, and it is typical to vulval fate (1°), its neighbors adopt the outer vul- find that the model is insensitive to variation in val fate (2°), and the remaining three cells do individual parameters over a range of 2- to 100- something else entirely (reviewed in Félix, ’99). fold or more! Thus, we believe we have shown that There are at least two other regulatory processes the inherent modularity of the segment polarity in C. elegans that influence vulval cell fate deci- network could explain how insect embryos pos- sions in this population of cells. First, the cell sess homologous segments despite radically dif- (P6.p, normally) that receives the most signal from ferent means of segment specification. the anchor cell inhibits its neighbors from adopt- ing the 1° fate. According to Félix (personal com- The evolutionary lability of munication) it is not clear in C. elegans whether nematode vulval development this lateral inhibitory mechanism or the anchor Recent studies of the evolution of vulval devel- cell signal predominates in normal development. opment in worms reveal a surprisingly rapid evo- Second, the six vulval precursor cells are not uni- lution of developmental process despite overall formly competent; the expression of the Hox com- conservatism in adult morphology; Félix (’99) re- plex gene mab-5 makes P7.p and P8.p somewhat views these examples in detail. In C. elegans, two refractory to induction. Sommer and Sternberg cells (Z1.ppp and Z4.aaa) interact symmetrically (’96) and Félix and Sternberg (’96, ’97) have char- to decide which will become the anchor cell and acterized development of the vulva in six nema- which the ventral uterine precursor. This inter- tode species, and found surprising variation in the action is mediated by a feedback loop involving relative role of these different regulatory interac- the Notch-like receptor lin-12 and its Delta-like tions. In some species the primary mechanism for ligand, lag-2. In C. elegans Z1.ppp and Z4.ppp vulval fate specification seems to be restriction of form an equivalence group because in half the ani- competence; other species have varied the timing mals one cell forms the anchor cell, and in half of inductive events relative to division of the pre- the animals the other cell does so. In other nema- cursor cells; in one the induction from the anchor tode species, these two cells are not equivalent cell seems to have been dispensed with entirely. but still constitute a competence group; both cells Yet apparently only in the last instance is the can form the anchor cell, but usually Z4.aaa does change in the mechanism of fate specification as- so. In still other species, Z4.aaa always forms the sociated with a morphological alteration, and in anchor cell but Z1.ppp can do so in the absence of only one other species is there any change in the the normal precursor. Finally, there are nematode contribution of precursor cells to each vulval fate. species in which the anchor cell and the ventral In the case of cell fate specification among the uterine precursor are each specified autonomously P(3–8).p cells, the evolutionary variability in de- (reviewed in Félix, ’99). Based on causally associ- velopmental mechanism is explained by the fact ated changes in gonad morphology, Félix (personal that nematodes have a kit of at least three mecha- communication) regards the equivalent condition, nistically distinct but functionally overlapping pro- as in C. elegans, as primitive. Thus we have a con- cesses that contribute to the specification of vulval tinuous series in which the differentiated charac- cell fates in this population of cells. The evolu- ter is preserved (namely, the presence of the anchor tionary series characterized by Sternberg, Félix, cell and the ventral uterine precursor); in all likeli- and their colleagues is based on altering the rela- hood the molecular mechanism is to a certain ex- tive weighting of these mechanisms. In the case tent conserved (otherwise we might not see such a of anchor cell determination, there is as yet no continuous series), and yet the nature of the devel- reason, as far as we are aware, to think that there opmental process has completely changed. Indeed, is more than one basic mechanism at work, though this case illustrates conversion of a regulative to a this is a possibility. Instead, one hypothesis is that mosaic process. this one mechanism, based on the lin-12 signal- 320 G. VON DASSOW AND E. MUNRO ing pathway, experiences different contexts in each Ubx selects between wing on the mesothorax and species; perhaps the initial conditions differ or per- haltere on the metathorax. Yet Ubx expression has haps exogenous inputs modulate the response of barely changed since the dawn of the insects: not one or the other cell. Strikingly, exactly such varia- only in four-winged insects (Kelsh et al., ’94) but tion in the function of lin-12-dependent fate speci- even in apterygotes (Carroll et al., ’95), a Ubx- fication mechanisms exists within C. elegans: like gene is expressed in the metathorax. Thus, while this molecular mechanism mediates a sym- changing boundaries of Ubx expression can’t be metric interaction between the anchor cell pre- responsible for reduction of the hindwing in cursors, at other times and places in development Diptera. The expression domains of abd-A and Scr it mediates biased interactions, inductions, and also seem to be similarly stable (Tear et al., ’90; autonomous specifications (Félix, ’99). Rogers et al., ’97). These two Hox genes repress In both cases the conservatism of the end state wing primordium formation in Drosophila. Yet could come from functional constraints alone, with there is good reason to believe that wings initially the evolutionary process oblivious to these sorts arose on all trunk segments (Kukalova-Peck, ’78), of changes in developmental process as long as despite expression of Scr-like genes in the dorsal “it” (the anchor cell or the vulva) still works. In base of the prothoracic limb (Rogers et al., ’97) neither case is it clear whether changes in the and abd-A-like genes in the abdomen (Tear et al., developmental process are selected according to ’90). Thus it must be the case that the wing-form- the changes in morphology they are associated ing mechanism acquired an input from each of with, or if they are completely independent of it. these Hox complex genes at some point in evolu- As Félix (’99) observes, it’s possible that the ob- tion, definitely after the invention of wings. Nor served evolutionary changes in developmental can Ubx- or abd-A-like genes have an ancient role mechanism are nearly neutral. If so, it is facilitated in limiting limbs to the thorax, since Averof and by the modularity of the developmental process. Akam (’95) have shown that in crustaceans both are expressed throughout the leg-bearing region. Adaptation of insect segments In Drosophila and butterflies these genes repress So far, we have discussed dissociability between Distal-less, which is part of the trigger for limb morphological characters and developmental pro- primordium formation (Warren et al., ’95). Again, cesses from the perspective of the character, mak- the limb-forming mechanism must have acquired ing it seem as if characters are stable and this input after the origin of arthropods. Looked developmental processes come and go. Of course at the other way around, the Hox genes are part characters come and go as well, and some devel- of an evolutionarily stable mapping mechanism in opmental processes may be remarkably stable. insects, and as a ready source of positional infor- The role of the insect Hox cluster in segment iden- mation they have progressively insinuated them- tity is an important paradigm. Early on, there was selves into a position of control over morphogenetic the notion that insect segmental diversity was due modules like wings, limbs, and other organs. to the invention of new homeotic genes (Akam et The Hox complex does not have quite the same al., ’88). Starting with a myriapod-like animal, a evolutionary role in other notable groups, like the new gene might arise to suppress appendage for- crustaceans and the vertebrates. Hox gene expres- mation in the abdomen. Perhaps another new sion boundaries do shift relative to each other and gene distinguished legs from antennae; and more relative to the major body regions in the crusta- recently, perhaps the origin of two-winged from ceans, and these shifts may be involved in append- four-winged insects involved invention of a wing age diversification (Averof and Patel, ’97). A suppressor gene active in the metathorax. With similar phenomenon has been demonstrated in the the discovery that genic diversity of the Hox com- vertebral column of amniotes, although in this plex has been essentially conserved since well be- case the Hox complex appears to retain a causal fore the origin of insects (reviewed by Carroll, ’95), link to the distinctions between body regions; the the relevance of this hypothesis in insects is mini- number of segments in the major body regions and mal at best. the detailed morphology of segments seems to Instead, perhaps the expression domain of Hox change along with changing Hox expression genes changed. This turns out not to be the rule boundaries (Burke et al. ’95; reviewed by Carroll, either; numerous reports demonstrate that expres- ’95; and by Müller and Wagner, ’96). The deriva- sion domains of Hox complex genes are rather tion of the tetrapod limb from the fins of fishes stable evolutionarily. For instance, in Drosophila provides yet another example of a complex rela- MODULES IN DEVELOPMENT AND EVOLUTION 321 tionship between Hox genes and morphological This is a terribly simplified sketch of the devel- evolution, in which an anchored Hox expression opmental mechanism at work in the limb bud, but domain may have been elaborated into the novel we can expect that a fuller account would reveal autopod (reviewed by Müller and Wagner, ’96). many more interdependencies of this sort (some of which are indicated in Martin, ’98). Such inter- The vertebrate limb I: an epigenetic trap dependencies may be commonplace. The vertebrate limb bud affords an example of a developmental constraint—a systems-level prop- II: urodele versus anuran erty that limits variability (reviewed in Martin, and amniote limbs ’98). In the limb bud there are three classically We continue with another vertebrate limb ex- defined territories: the apical ectodermal ridge ample that illustrates how variational tendencies (AER), the zone of polarizing activity (ZPA), and serve as homology criteria. The tetrapod limb con- the progress zone. The progress zone consists of sists of three units, of which only the hand is unique proliferating cells that fuel the growth of the limb; to tetrapods. Although anatomically urodele and the ZPA consists of mesodermal cells that produce anuran limbs are fairly similar, developmentally the a signal that specifies the anterior-posterior (AP) urodele hand differs from the anuran or the am- axis of the limb; and the cells of the AER signal niote hand (reviewed by Wagner et al., ’99). The to the progress zone to continue proliferating urodele limb has no morphologically distinct AER. (Saunders, ’48; Saunders et al., ’57; Tickle et al., Urodele fingers grow out as a bud from the devel- ’75; Summerbell, ’79). The most important signals oping hand, whereas the anuran and amniote hand produced by the AER and ZPA are FGF-4 (Nis- is formed by remodeling a paddle-shaped rudiment. wander et al., ’93) and sonic hedgehog (Riddle et Anuran and amniote digital condensations form in al., ’93), respectively, and the combined action of a different order from urodele digits; the anuran/ these two signals maintains the activity of the amniote hand begins with the fourth digit, then progress zone. Also, sonic hedgehog triggers pat- three, two, and one, concomitant with the fifth, tern formation along the AP axis. However, to re- whereas urodele fingers develop one through five. spond to sonic hedgehog, cells in the limb bud need Wagner et al. (’99) found that Hox expression do- to see FGF-4 from the AER. Furthermore, activ- mains in the urodele limb are significantly differ- ity of the AER depends on sonic hedgehog. Thus, ent from the anuran/amniote limb. The urodele and there is a positive feedback loop between the AER anuran/amniote hands also have different varia- and the ZPA that is causally relevant to the tional tendencies: the anuran/amniote hand tends growth of the limb bud, and there is also an to retain primarily the fourth finger when it loses equally important requirement for both signals in digits in either phylogeny or under experimental the axial patterning of the organ (Laufer et al., conditions, whereas the urodele hand tends to re- ’94; Niswander et al., ’94). This double dependence tain digits one and two. imposes a constraint; we quote Wagner, who ex- If we take seriously the notion that homologous plained it to us: characters are united during adaptive processes by their innate capacity to produce characteristic “[in this instance] pattern formation and heritable phenotypic variation, then urodele and (thereby) constraints on variation are mecha- anuran/amniote hands are different things and we nistically linked to the very existence of [the oughtn’t consider these two limbs strictly homolo- limb]... So constraints [arise] because you can- gous. After all, one criterion for homology is that not fiddle around with the existence of ante- we must convince ourselves that the hypothesis rior-posterior polarity without endangering of independent origin is less parsimonious than the very existence of the whole limb. In this the most reasonable hypothesis of sameness. Each type of system a constraint comes into play type of hand has distinct properties that unite when the same genes are involved in some instances of it and distinguish it from the other, aspect of pattern formation and in the very and it is impossible to see how the variational existence of the organ... the evolutionary pro- tendencies of one type of hand could encompass cess has gotten into an epigenetic trap; it’s the other type. Are urodele hands a separate in- trapped into producing something that is po- vention from anuran hands, which happened to larized or not producing it at all.” — G. co-opt the mechanism used to determine the mor- Wagner, speaking at FHL modularity work- phological characteristics of digits? That seems shop, ’97 unlikely since the living amphibia are monophyl- 322 G. VON DASSOW AND E. MUNRO etic. Is this a case in which the generative process mechanisms that shape the limb. Anatomically the differs but morphostatic processes preserve form? forelimb digits of birds correspond to digits I, II, This would be surprising since the developmental and III of the reptilian hand; this derivation is processes responsible for the anuran/amniote limb evident in the lineage of theropod dinosaurs from don’t seem intrinsically labile; from what little we which birds arose. Developmentally, the digits of know of these processes at the molecular level, they birds ought to be II, III, and IV. As noted previ- have been conserved since before the divergence of ously, when amniotes (which ancestrally possess amniotes from the amphibia. five digits on each limb) undergo evolutionary re- Wagner and colleagues suggest two more plau- ductions in digit number, they “invariably” (Wag- sible scenarios. In the first, they note that fossil ner and Gauthier, citing Morse for “Morse’s law”) forms at the base of the tetrapods have eight-fin- lose the first, then the fifth. During amniote limb gered hands. Perhaps the urodele hand is derived development, the fourth primordial finger forms from the posterior five digits of the ancestral eight first, then three more condensations, in a line with and the anuran hand was derived from the ante- and connected to the fourth, form successively rior five. This would mean that the most stable more anterior (Alberch and Gale, ’85). The thumb digit (the fourth-most-anterior in anurans and is the last to form on this “metapterygial” axis. amniotes, the anterior-most in urodeles) is actu- The fifth condensation appears posterior to the ally the same in each group, corresponding to the fourth concomitant with the elaboration of the fourth digit of the ancestral eight-fingered hand. metapterygial axis. In birds, the forelimb devel- This doesn’t explain differences in digit morpho- ops four digital condensations in the autopodium, genesis, but it explains the difference in the or- three of them along the metapterygial axis. These der of development and in which digits tend to be are therefore identified embryologically as digits lost. Second, Wagner et al. (’99) point out that II, III, and IV. The fourth condensation in bird among urodeles are a number of groups with a forelimbs is identified with digit V, and is resorbed strong tendency to drastically reduce digit num- before differentiation of the hand. In addition, ex- ber. Perhaps the urodele hand is not a separate periments with mitosis inhibitors show that the invention, but a re-invention after reduction; the thumb is most sensitive. Thus the paradox is: how urodeles re-invented additional digits out of func- can birds have an anatomical thumb when they tional necessity but there was no reason to place have no embryological thumb? them anteriorly or posteriorly, or develop them To resolve the paradox, Wagner and Gauthier by budding or not. Neither scenario accounts for propose that the developmental process that the fact that on the basis of adult morphology com- makes the digital condensations is causally inde- parative anatomists have been comfortable iden- pendent from the “ensuing developmental indi- tifying the five digits of the anuran, amniote, and vidualization of those repeated elements as they urodele hands as I, II, III, IV, and V in each group. become the functional fingers in the mature In the first, we would have to posit that the hand.” If this is so, then they suggest it is pos- urodele hand, once the posterior five digits had sible that in the evolution of what eventually be- been culled from an eight-fingered ancestor, un- came the bird wing a frame shift (their term) took derwent a homeosis so that the original fourth place that associated the condensation originally digit (the new first digit) adopted adult morphol- destined to make digit IV with the morphological ogy of the first digit. In the second, we would have fate of digit III, condensation III to morphology to expect that the mechanism responsible for II, and condensation II to the thumb. They sup- specifying digit identity could be anchored by the port their hypothesis on the basis of Hox gene one or two ancestral digits, then re-adopted by expression patterns in the fore- and hindlimb of newly reinvented ones. chick embryos, which reveal the requisite frame shift. Thus, Wagner and Gauthier propose a phy- III: dissociability within the limb logenetic scenario in which the ancestors of birds, In a related phenomenon, the tetrapod limb pro- the theropod dinosaurs, faced a conflict between vides an excellent example of dissociability among a developmental constraint that favors loss of par- developmental mechanisms. One long-standing ticular condensations and a functional require- paradox has been the homology of the forelimb ment for a thumb, and that the evolutionary route digits of birds, which may have been resolved by through this conflict was facilitated by the dis- Wagner and Gauthier (’99); their solution assumes sociability of the differentiative process that con- the dissociability of some of the morphogenetic fers adult functional morphology on the digits, MODULES IN DEVELOPMENT AND EVOLUTION 323 and the morphogenetic process that provides the Arendt D, Nubler-Jung K. 1999. Comparison of early nerve primordia. cord development in insects and vertebrates. Development 126:2309–2325. The reason we choose this example to close our Arnone MI, Davidson EH. 1997. The hardwiring of develop- essay is that there seems to be no way to resolve ment: organization and function of genomic regulatory sys- the paradoxes that the tetrapod limb presents tems. 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