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MICHAEL . COATES LIMB EVOLUTION MICHAEL I. COATES LIMB EVOLUTION Fish fins or limbs - a simple twist of fate? Comparisons between expression patterns in fins and tetrapod limbs are revealing new insights into the developmental mechanisms underlying the evolutionary transition from fin to limb.

Phylogenetic trees represent nested patterns of develop- full breadth of the distal mesenchyme, coincident with mental diversification. Yet, until recently, attempts to link development of a 'digital arch' from which the digits will large-scale evolutionary morphological transformations form (see below, Fig 2c) [4]. Thus, HoxD expression to changes in ontogeny have yielded limited results. restriction is reoriented from an antero-posterior to a Explanations have usually characterized developmental proximo-distal axis [1,2]. HoxA expression, in contrast to evolution in terms of shifts in the relative timing of that of the HoxD members, shows no antero-posterior developmental events and alterations to gross embryonic restriction, and instead consists of a series of proximo- patterns, whether static or dynamic, instead of attempting distally nested bands spanning the entire bud (Fig.la). to identify changes affecting specific morphogenetic pro- cesses. But this situation is changing, largely because of The similarities and differences between the development rapid advances in the study of the genes that regulate of the tetrapod limb bud and that of the zebrafish fin bud development. Perhaps most significantly of all, after the are striking (Fig. lb). By comparison with limbs, fin bud initial interest that was inspired by the remarkably con- outgrowth ceases earlier; mesenchymal proliferation fin- served features of some of these developmental genes, ishes as the apical fin bud ectoderm transforms into a pro- research is now beginning to focus on their diversity. truding fold, enclosing the developing dermal rays [5]. In This renewed interest in comparative approaches to sub- the pectoral fin, proximo-distal subdivision of the meso- jects that are otherwise treated simply as 'experimental derm forms a series of four proximal radials, while model systems' is exemplified especially clearly by the peripheral foci form distal radials (Fig. ld) [1]. The com- recent paper from Sordino, van der Hoeven and Duboule position of the zebrafish HoxD gene complex' appears [1] on fin and limb development. Their to resemble that of , with posterior members research links developmental to morpho- expressed colinearly, although in a more restricted fash- logical variation, both within and between taxa; it illus- ion. HoxD members are barely expressed in the anterior trates the reciprocally informative nature of ontogenetic half of the pectoral fin bud, and there is no secondary, dis- and phylogenetic analyses, and suggests a series of impor- tal phase. Zebrafish HoxA expression is similarly simpler tant, new developmental and evolutionary hypotheses. than that of tetrapods. In the case of (at least) Hoxa-11, the expression domain remains distal and broad, extend- The zebrafish, Danio rerio, has become a well-established ing to the sub-apical fin bud mesenchyme. This is quite embryological experimental tool - a valuable vertebrate unlike amniote limbs, in which the expression domain is alternative to chicks, mice and Xenopus. As a non- restricted distally, resulting in a band which stops short of tetrapod osteichthyan (bony fish), it has several anatomi- the zone of digital arch development (Fig. la). Signifi- cal peculiarities which are usually regarded as primitive cantly, Sordino et al. [1] extended their analysis to the features, but these are rarely subjected to comparative pelvic fin. Teleost pelvic fins resemble abbreviated reitera- analysis. The description by Sordino et al. [1] of HoxA tions of pectoral patterns [6]. Zebrafish pelvic fin buds and HoxD gene expression patterns in paired fin buds, emerge 30 days after the pectorals, but outgrowth (and and the comparison with expression patterns in tetrapod mesenchymal proliferation) is attenuated, with earlier limbs, is therefore a significant departure from the usual development of an extensive fin fold. The reactivated Hox course of developmental research. The more 5' members gene expression domains are less elaborate (Fig. c), and of the HoxA and HoxD gene complexes are known to be the endoskeleton is correspondingly smaller (Fig. ld). expressed during tetrapod, and especially amniote, limb development (Fig. la) [2]. Limb buds enclose lateral plate Sordino, van der Hoeven and Duboule [1] clearly recog- mesenchyme, and their outgrowth is maintained by the nize the evolutionary implications of their research, and influence of an apical ectodermal ridge (AER) [3]. Skele- draw a series of conclusions and speculative hypotheses. tal pattern development within the mesenchyme occurs First, there is a causal correlation (and perhaps trade-off) in a proximo-distal direction, and follows conserved between AER or fin-fold initiation, endoskeletal devel- sequences of prechondrogenic focal condensation, seg- opment, and Hox gene expression. Second, the posteri- mentation and bifurcation [4]. Members of the HoxD orly restricted HoxD expression domains in limb buds complex (Hoxd-11-13) are expressed in a characteristi- and fin buds provide independent corroboration for cally nested, biphasic sequence [2]: initially, gene expres- long-established theories of homology between the sion is restricted towards the posterior edge, whereas in metapterygium - the posterior-most, asymmetrically the secondary phase HoxD expression extends across the branched fin support that provides a primary axis of the

844 © Current Biology 1995, Vol 5 No 8 DISPATCH 845 paired fins in jawed fish - and the main axis of the prox- imal part of tetrapod limbs (upper arm and forearm; see Fig. 2; reviewed in [4,6]). However, the morphological relationship between metapterygia and the distal (ankle/wrist plus digits) patterns of tetrapod limbs has only recently been resolved satisfactorily. Shubin and Alberch's analyses [4] of prechondrogenic limb patterns characterized metapterygia as continuous paths of seg- mentation and bifurcation, which in most tetrapod limbs (amniotes and anuran amphibians) turn antero-distally to produce distal carpals and digits. These twisted patterns of skeletal proliferation correspond closely to the bi- phasic, distally skewed HoxD expression domains in these species [1,7].

From this follows a third evolutionary hypothesis, namely that the reoriented distal phase of HoxD expression in limb buds is secondary, or 'derived', relative to the monophasic zebrafish pattern. Moreover, the correlation with digital arch development (derived relative to the straight metapterygial axes of finned stem-tetrapods, such as the extinct Eusthenopteron; see Fig. 2a) results from late distalization of the domain of expression of the Sonic hedgehog (shh) gene compared to zebrafish pelvic fin shh expression (Fig. lc), along with unequal postero-dis- tal cell proliferation. Finally, the proximally restricted Hoxa-11 expression band in tetrapod limb buds supports the hypothesis that the 'autopod' ('hand' or 'foot'), including distal carpals and digits, is neomorphic, or in some sense a morphological novelty, relative to fin buds.

These conclusions superficially resemble Holmgren's concept of a 'neopodium' to describe the hands/feet of tetrapods [8], but there are important differences. Holm- gren first identified the almost complete proximo-distal

Fig. 1. (a) Regions of Hoxa-I1 expression in the mouse forelimb (adapted from [1]) and Hoxd-11 expression in the chick hindlimb (adapted from [2]) are shown in dark blue; non-expressing regions of mesenchyme are shaded light blue. Note that by equivalent stages of development in the two species ('stage t2'), the Hoxa-11 distal expression boundary lies proximal to the 'autopodal' region (including distal carpals and digits), and that Hoxd- I 1 is now expressed antero-posteriorly, across the sub-api- cal mesenchyme (AER, apical ectodermal ridge; ec, ectoderm; mc, mesenchyme). (b) Hoxa-ll and Hoxd-12 expression (dark blue) in the zebrafish pectoral fin (adapted from [1]). In contrast to the amniote limb expression patterns, at the t stage, Hoxa-11 is expressed predominantly in the posterior region, and at t2 there is no significant distal zone of non-expression. Hoxd-12 expression remains in the posterior half of the developing fin bud; by 58 hours post-fertilization (hpf) the expression is weak- ening (aef, apical ectodermal fin fold). (c) Hoxd-12 and shh expression (dark blue) in budding pelvic fins (adapted from [1]). Both expression patches are confined to the posterior zone, and neither reaches the most distal mesenchymal cells. dpf, days post-fertilization. (d) Lateroventral views of near-adult pectoral and pelvic fin and girdle skeletons, with dermal fin rays omitted. Camera lucida drawing of a cleared and stained zebrafish speci- men (Danio rerio): blue, dermal bone; green, endoskeletal bone and cartilage (cl, cleithrum; pp, pelvic plate; sc, scapulocoracoid plate; R,proximal radials; r, distal radials). Distal isto the top and anterior to the left in all panels. 846 Current Biology 1995, Vol 5 No 8

discontinuity between digital arches and the rest of tetra- formulated to be consistent with a diphyletic theory of pod limbs (subsequently explained by Shubin and Alber- tetrapod evolution (urodeles arising from lungfish, the ch's scheme [4]). But although digits and carpels were rest from Eusthenopteron-like lobe-finned fishes). More- described as neopodial, so too was any distal structure over, simply stating that digits and carpals are neomor- consisting of'secondary rays' - that is, any developmen- phic is of limited use. This kind of description is tally late fin radials arising independently of an 'extremity biologically meaningful only if the morphological change stem' (which is not synonymous with a metapterygium). is related to a specific point on the phylogenetic tree. Holmgren also described lungfish fins (Fig. 2a) as neo- The results of Sordino et al. [1] need to be considered podial, and his definition applies equally well to zebrafish within the context of current phylogenetic hypotheses. distal radials (Fig. d). 'Neopodium' is an insufficiently precise term: it is based on out-dated morphological The zebrafish, a carp-like (cypriniform) teleost member descriptions of limb development; it bears the typologi- of the vast, ray-finned subdivision of bony fishes [9], has cal baggage of essential limb characteristics; and it was been evolving independently of lobe-finned bony fishes

Fig. 2. (a) Selected adult pectoral fin endoskeletal patterns plotted on a cladogram of major groups of bony fish (Osteichthyes; textinct taxon). The axes of the metapterygia are indicated with a red line; in the zebrafish, the metapterygial course is speculative, and differs from that suggested by Sordino et al. [1]. In fact, there may be a significant difference in the morphological assembly of metapterygia between lobe-finned fish (Sarcopterygii) and other jawed , including ray-finned fish (Actinopterygii). In the former, the metapterygia appear to consist of coalesced proximal radials; in the latter, metapterygia derive from a single proximal focal condensa- tion. Zebrafish skeleton redrawn from [1 ]; for others see [6] and references therein. A provisional hypothesis of paired fin developmen- tal characteristics is mapped onto the cladogram. (b) Prechondrogenic cell clusters (blue) segmenting to form endoskeleton in a chondrostean pelvic fin. The posterior-most clusters form a 1:2 branching pattern characteristic of the metapterygium, superimposed with the and bifurcation pattern (dark grey; note the close correspondence between the metaptarygial development zone in this fin bud and the posterior pelvic fin bud expression patch in Fig. 1c). Differentiation proceeds from posterior to anterior of the fin base (redrawn from [6]). (c) Shubin and Alberch's [41 scheme of tetrapod (amniote) limb skeletal pattern development. Blue shapes rep- resent prechondrogenic cell clusters, dotted lines indicate cellular connections. Development proceeds in a proximo-distal direction. In comparison with the short pelvic fin metapterygium, the branched and segmented limb axis extends antero-distally to form the digital arch (dark grey). The anterior edges of all fins and limbs are to the left. DISPATCH 847

(of which tetrapods are a highly evolved subgroup) for pre-axial (anteriorly facing) metapterygial surface (Fig. more than 410 million years. It can be inferred that their 2); the significance of this reorientation is uncertain. last common ancestor had two sets of paired fins with Thus, Sordino et al.'s 'neomorphic autopod' [1] is a mix- dermal rays, that the pelvic and pectoral fins were dissim- ture of old and new features: a new morphological zone ilar, and that at least the pectoral fin was metapterygial - wherein old features are sustained or replicated, but that is, it had a posterior, branched axis. These mor- deployed in an entirely new way. phologies indicate the presence of fin folds, differences between the relative timing of transitions from pectoral The transformations between fin and limb discussed and pelvic ectodermal ridge to fin-fold, and the imposi- above mostly concern the endoskeleton, but perhaps the tion of a fundamental asymmetry upon the endoskeletal clearest difference between fins and limbs is the presence pattern. Such asymmetry, and therefore proximal unequal or absence of rays. The changing structure and role of cellular proliferation, suggests that a network of signalling the apical ectoderm within which these originate appears factors - perhaps including members of the fibroblast to be crucial to our understanding of fin versus limb growth factor and Wnt families of signalling molecules development and evolution. Sordino et al. [1] suggest that interacting with shh and Hox genes [1,10,11] - may ray development within the fin fold may interrupt sig- have been established before the evolutionary split nalling and terminate endoskeletal proliferation and pat- between ray- and lobe-finned bony fishes (Fig. 2a). terning. Moreover, in contrast to the HoxA and HoxD genes, which appear to be confined to mesodermal The basal Hox gene complement of bony fishes remains expression, zebrafish fin-fold and dermal ray develop- uncertain in the absence of outgroup comparison - ment are known to involve at least four out of five mem- which in this case should be chondrichthyans: sharks, bers of the msx gene family [13], whereas only three have rays and chimaeroids - but it seems likely that at least been found in mouse limb buds. The evolutionary polar- Hoxd-10 to Hoxd-13 plus Hoxa-ll were present. Fur- ity and root of these differences is unknown. Available thermore, the comparison by Sordino et al. [1] between data hint that the Hox and msx gene families may be dis- pectoral and pelvic fin development sheds light on playing quite different rates or phases of evolutionary the absence of a discrete metapterygium in zebrafish change. Whereas Hox gene complements in bony fishes and other . The stepped, persistently asymmetric appear to be relatively conserved, with morphological arrangement of pectoral radials 2-4 suggests that this pat- transformations related primarily to shifting expression tern may result from an advanced fin-fold initiation, rela- boundaries [1,2,14], msx complements may still be tive to the non-teleost ray-finned condition. It would be changing, and so may be their roles in the development interesting to see if a broad-based metapterygium, like and regeneration of fin rays (and other structures). Fur- those of near-teleost actinopterygians (see, for example, thermore, the marked contrast between HoxA and HoxD the bow-fin Amia calva in Fig. 2a) could be induced by limb expression domains and those of HoxC [15] and the experimentally delaying the transition from pectoral msx genes begs questions about the relation between ectodermal ridge to fin fold. these gene families and the evolution of endochondral and dermal skeletogenic systems. Future research is likely In contrast to the paired fins of ray-finned fishes, those of to become increasingly concerned with these kinds of lobe-finned fishes are exclusively metapterygial, and dif- questions, and the establishment and regulation of many ferences between pectoral and pelvic morphologies are of the transformations described here (and plotted in Fig- significantly reduced. Thus, similarity between pectoral ure 2a). In order to tease out the derived from the primi- and pelvic ontogenies is not a unique feature of tetrapods, tively shared features, however, not only will this work and neither are distal mesenchymal proliferation or der- require an increasingly phylogenetically informed inter- mal ray loss (implying fin-fold reduction). Extant lungfish pretation of any results, it will also need a similarly species provide alternative examples of each of these informed and broadened choice of experimental subjects. characteristics [6]. The evolutionary uniqueness of tetra- pod limbs (relative to fins) appears to reside in the distal- Acknowledgements: Thanks to P. Ferretti and the Developmental ized shh expression domain, along with unequal cellular Biology Unit, Institute of Child Health, University of London, for proliferation related to the generation of an anteriorly access to zebrafish specimens, and to D. Duboule, C. Tickle, A.C. Burke and M. Cohn for helpful comments and discussion. twisted digital arch, and the secondary, similarly twisted phase of Hoxd expression. However, the detailed content References of this distalization process is unclear, and the distal effect l. Sordino P, van der Hoeven F, Duboule D: Hox gene expression in seems to be enhanced relative to the pattern of proximal teleost fins and the origin of vertebrate digits. Nature 1995, 375:678-681. symmetry. Conversely, the intrinsic composition of the 2. Morgan BA, Tabin C: Hox genes and growth: early and late roles in digital arch, rather than its orientation, can still most limb bud morphogenesis. Development 1994, Suppl:181-186. parsimoniously be interpreted as a conserved, although 3. Hinchliffe JR, Johnson DR: The Development of the Vertebrate Limb. Oxford: Oxford Science Publications, Clarendon; 1980. extended, metapterygium. It consists of no more than 4. Shubin NH, Alberch P: A morphogenetic approach to the origin reiterated skeletal structures which are serial homologues and basic organisation of the tetrapod limb. Evol Biol 1986, of those in the proximal limb [4,12]. Similarly, digits are 20:319-387. 5. Geraudie J: The fine structure of the early pelvic fin bud of the radial-like developments from the post-axial (posteriorly trouts Salmo gairdneri and S. trutta fario. Acta Zool 1978, 59: facing, relative to a straight metapterygial axis) instead of 85-96. 848 Current Biology 1995, Vol 5 No 8

6. Coates MI: The origin of vertebrate limbs. Development 1994, 12. Shubin N: The evolution of paired fins and the origin tetrapod Suppl:1 69-180. limbs. Phylogenetic and transformational approaches. Evol Biol 7. Coates MI: New palaeontological contributions to limb ontogeny 1995, 28:39-86. and phylogeny. In Developmental Patterning of the Vertebrate Limb. 13. Akimenko M-A, Johnson SL, Westerfield M, Ekker M: Differential Edited by Hinchliffe JR, Huerle JM, Summerbell D. New York: induction of four msx genes during fin development and regen- Plenum; 1991, 347-354. eration in zebrafish. Development 1995, 121:347-357. 8. Holmgren N: An embryological analysis of the mammalian carpus 14. Burke AC, Nelson CE, Morgan BA, Tabin C: Hox genes and the and its bearing upon the question of the origin of the tetrapod limb. evolution of vertebrate axial morphology. Development 1995, Acta Zool Stockholm 1952, 33:1-115. 121:333-346. 9. Meyer A, Biermann CH, Orti G: The phylogenetic position of the 15. Molven A, Wright CVE, Bremiller R, DeRobertis EM, Kimmel CB: zebrafish (Danio rerio), a model system in : Expression of a gene product in normal and mutant an invitation to the comparative method. Proc R Soc Lond [Biol] zebrafish embryos: evolution of the tetrapod body plan. Develop- 1993, 252:231-236. ment 1990, 109:279-288. 10. Niswander L, Jeffrey S, Martin G, Tickle C: A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 1994, 371:609-612. Michael I. Coates, Department of Biology, University 11. Martin G: Why thumbs are up. Nature 1995, 374:410-411. College London, Gower Street, London WC1E 6BT, UK.

THE AUGUST 1995 ISSUE (VOL. 5, NO. 4) OF CURRENT OPINION IN GENETICS AND DEVELOPMENT

will include the following reviews, edited by Robb Krumlauf and Clifford J. Tabin, on Pattern Formation and Developmental Mechanisms:

Signalling by hedgehog family proteins in and vertebrate development by Philip W. Ingham Homeotic genes and diversification of the insect body plan by Robert Warren and Sean Carroll The role of the notochord and floor plate in inductive interactions by Marysia Placzek Vertebrate limb development by Cheryll Tickle Early decisions in Drosophila eye morphogenesis by Nancy M. Bonini and Kwang-Wook Choi Plant development: pulled up by the roots by Liam Dolan and Keith Roberts Lateral specification of cell fate during vertebrate development by David W Raible and Judith S. Eisen Spatial patterning and information coding in the olfactory system by Susan L. Sullivan, Kerry J. Ressler and Linda B. Buck Morphogenesis in Dictyostelium: new twists to a not-so-old tale by JeffWilliams Chromatin complexes regulating gene expression in Drosophila by Vincenzo Pirrotta Fibroblast growth factors in mammalian development by Terry P. Yamaguchi and Janet Rossant Fate maps of the zebrafish embryo by Katherine Woo, John Shih and Scott E. Fraser Determination events in the nervous system of the vertebrate embryo by Laure Bally-Cuif and Marion Wassef The role of brahma and related proteins in transcription and development by John W. Tamkun