Vertebrate Axial and Appendicular Patterning: The Early Development of Paired Appendages 1

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1 AMER. ZOOL., 39: (1999) Vertebrate Axial and Appendicular Patterning: The Early Development of Paired Appendages 1 MICHAEL I. COATES 2 * AND MARTIN J. COHN *Department of Biology, University College London, Gower Street, London WCJE 6BT UK f School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ UK SYNOPSIS. Determination of paired fin or limb number, identity and position are key issues in vertebrate development and evolution. Phytogenies including fossil data show that paired appendages are unique to jawed vertebrates and their immediate ancestry; that such fins evolved first as a single pair in an anterior location; that appendicular endoskeletons are primitively AP asymmetric; and that pectoral and pelvic fins primitively differ. It is conjectured that Hox gene expression patterns along the lateral plate mesoderm establish boundaries that contribute to localisation of AP levels at which signals initiate outgrowth from the body wall. Such regionalisation may be regulated independently of that in the paraxial mesoderm and axial skeleton. When combined with current hypotheses of Hox gene phylogenetic and functional diversity, these data suggest a new model of fin/limb developmental evolution. This coordinates body wall outgrowth regions with primitive boundaries established in the gut, and the fundamental non-equivalence of pectoral and pelvic structures. INTRODUCTION This article summarises (and updates) a recent synthesis of palaeontological and developmental perspectives on vertebrate postcranial skeletal patterning (Coates and Cohn, 1998), which re-examines major evolutionary changes in the axial and appendicular skeletons. Instead of returning to the fin-limb evolutionary transition, which has been the focus of a considerable body of work during the past decade or more (as a representative sample, see: Shubin and Alberch, 1986; Thorogood, 1991; Coates, 1994; Sordino et al., 1995), the present work addresses earlier events in vertebrate ontogeny and phylogeny. Key issues therefore concern the relation of paired fins to the axial skeleton, the position at which lateral fins first emerge from the body wall, and the origin of morphological differences between fore- and hindlimbs and fins. TETRAPOD LIMBS AND HOX GENES Tetrapod limbs have long been used as a model system for developmental research, ' From the Symposium Developmental and Evolutionary Perspectives on Major Transformations in Body Organization presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3-7 January 1998, at Boston. Massachusetts. 2 m.coates@ucl.ac.uk 676 but the nature of this work has changed significantly following the advent of techniques for investigating developmental control genes. Much of this research has concerned the Hox genes (Fig. 1), which are involved in the specification of positional identity during metazoan embryogenesis. Gain or loss of function mutations of these genes, as well as temporal changes in their expression patterns, can produce morphological transformations in which one body part or segment is substituted for another (homeotic mutations). Vertebrate Hox genes may be characterised by: (a) sequence similarity to the Drosophila Hom-C complex, (b) organisation in gene clusters, and (c) expression patterns along the embryonic axis matching their arrangement on the chromosome (Kappen, 1997). Genes located at the 3' end of the Hox cluster are expressed anteriorly, while genes located at the 5' end are expressed posteriorly. This rule of "spatial colinearity" (Duboule and Morata, 1994) between Hox gene position within each cluster and expression sequence within vertebrate embryos also correlates with expression timing, termed "temporal colinearity." Therefore, 3' Hox genes are activated both anterior and prior to expression of more 5' genes in posterior body regions.

2 VERTEBRATE AXES AND APPENDAGES D n-» Mouse Pufferfish Zebrafish Amphioxus FIG. 1 Hox gene cluster diversity and evolution. Simple tree shows evolutionary relationships of representative chordates in which more or less complete Hox clusters are known. Numbers identify paralogous gene groups between clusters; letters identify homologous clusters between taxa. Tetrapoda: mouse Hox network from Sharman and Holland (1996); Hoxbl3 from Zeltser et al. (1996). Teleostei: Pufferfish {Fugu) Hox network from Aparicio et al. (1997); zebrafish (Danio) after Prince et al. (1998). Cephalochordata: amphioxus Hox cluster from Sharman and Holland (1996). Interrelationships of anterior (open), medial (shaded), and posterior (black) Hox gene groups from Kappen (1996). No attempt is made to indicate inter-gene distances, except for mouse Hoxbl3, which is out of register with other group 13 genes (Zeltser et al., 1996). Outlined boxes in amphioxus cluster indicate incompletely characterised genes (Sharman and Holland 1996); dotted boxes indicate pseudogene remnants of former Hox gene cluster members (Aparicio et al., 1997). Discovery of at least six clusters in zebrafish illustrates increasingly complex pattern of Hox cluster evolution; complete contents of clusters X and Y and relation to clusters A-D as yet unknown (see Wittbrodt et al., 1998 for further discussion of gene families in teleost fish). This general rule of vertebrate Hox gene spatio-temporal colinearity is corroborated by studies of expression patterns within the paraxial mesoderm of several taxa (Kessel and Grass, 1991; Gaunt, 1994; Burke et al, 1995; Prince et al., 1998), although spatial and temporal colinearity can break down during development (Nelson et al., 1996). It is clear that some additional hypothesis is needed to explain the relation between axial and appendicular Hox gene expression patterns in tetrapods. This is because the Hox genes expressed in both fore- and hindlimbs are predominantly members of posterior paralogue groups 9 13 (Fig. 1; Nelson et al, 1996; Shubin et al., 1997). Posterior Hox genes, such as Hoxdl3, expressed in the pelvic limb buds therefore resemble those in the trunk at the same anteroposterior level, but expression of these posterior genes in pectoral limb buds is out of register with the axial expression pattern. A DEVELOPMENTAL SCENARIO OF FIN AND LIMB EVOLUTION Tabin and Laufer (1993) identified this inconsistency between axial and appendicular Hox gene expression, and explained it by means of a clever and heuristically valuable evolutionary transformation scenario (Fig. 2). This adapts Balfour's (1881) hypothesis of vertebrate paired fin origin from continuous lateral folds, and infers that mesoderm forming a primitive elongate fin bud expressed the same pattern of Hox genes as the adjacent body wall (Fig. 2B).

3 678 M. I. COATES AND M. J. COHN A. D. FIG. 2. Tabin and Laufer's (1993) model of vertebrate fin evolution. A, pre-paired fin condition; B, continuous lateral fin-fold; C and D, two fin pairs present: primitive and derived conditions respectively (see text for full explanation of model). According to their model, at a more advanced node in vertebrate evolution the mid-region of the lateral fin fold was suppressed, resulting in an anterior fin bud expressing anterior genes and a posterior bud expressing posterior genes (Fig. 2C). Consequently, the posterior fin expression pattern must have been recruited by, or imposed upon, the anterior fin or limb (Fig. 2D). In a more recent version of this scenario, posterior group Hox genes are simply co-opted (simultaneously) into pectoral and pelvic outgrowths (Shubin et al., 1997). TESTING THE MODEL AGAINST FOSSILS AND PHYLOGENY This development-based model of paired fin evolution can be tested against phylogenetic patterns of morphological change. Janvier's (1996a) analysis of primitive vertebrate interrelationships shows that anaspids (see Coates, 1994; Janvier, 1996Z?) are the most primitive fishes bearing paired fins. This places paired fin origins at a minimum of 450 Myr ago, and links the presence of all paired fins to the gnathostome "total-group" (Patterson 1993; i.e., the set of vertebrates related more closely to extant gnathostomes than to lamprey and/or hagfish lineages; this necessarily includes taxa exhibiting pre-jawed conditions). These simple fins lack an internal skeleton, and consist of dorsoventrally slender lateral keels with enlarged scales or spines. Such paired fins always extend from a pectorallike position; no primitive fish is known with only pelvic paired fins; and no paired fins extend from the flank to a point posterior to the anal level. The earliest evidence of paired fins with an internal skeleton derives from osteostracans, the gnathostome sister-group (Janvier, 1996a, b). These fins are lobate, implying body wall mesodermal input during ontogeny (Coates, 1994), and facets plus canals in the cranially integrated pectoral girdle indicate endoskeletal presence. The most primitive pelvic fin-like outgrowths are present in certain thelodont agnathans (Marss and Ritchie, 1998). Like primitive pectoral fins, these seem to be simple flaps with no evidence of endoskeletal support. Gnathostomes (chondrichthyans, osteichthyans, and the extinct placoderms plus acanthodians) appear between 435 and 450 Myr ago (Sansom et al., 1995; Janvier, 19966), and primitively possessed two finpairs. Primitively, gnathostome pectoral fin skeletons include several endoskeletal radials articulating with the girdle, the posteriormost of which articulates with secondary radials mostly along its anterior edge (Coates, 1994). This complex, asymmetrically patterned posterior radial may be described as a metapterygium (Shubin and Alberch, 1986; Coates, 1994). In contrast to pectoral fins, pelvic fins are primitively smaller and, usually, anatomically simpler. Pelvic fins therefore neither originate as simple copies, nor as identical serial homologues of the pectorals. Patterns of primitive fin phylogeny therefore provide little evidence of parallel (or concerted) evolution between pectoral and pelvic appendages {contra Shubin et al., 1997). Moreover, in male chondrichthyans and placoderms the pelvic metapterygial radial complex is modified into an intromittent clasper. Close similarity between pectoral and pelvic fins is therefore a specialised feature which is developed most clearly within sarcopterygian (lobe-finned) osteichthyans. But even in this taxon, which includes tetrapods and their closest fish-like relatives,

4 VERTEBRATE AXES AND APPENDAGES 679 pectoral and pelvic fins primitively differ (Andrews and Westoll, 1970; Coates, 1994). In summary, there is no evidence of primitive lateral folds subdividing into pectoral and pelvic fins. Pectoral and pelvic fins primitively differ; pectorals precede pelvic fin evolution; and anteroposterior asymmetry is a primitive and persistent characteristic of all vertebrate lateral appendages. Therefore, an alternative hypothesis is needed to explain the relation of axial to appendicular patterning systems. We propose a model for the origin of paired fins after the following review of postcranial developmental data. APPENDICULAR DEVELOPMENT: CELLULAR INTERACTIONS AND GENETIC PATHWAYS Limb and fin development involves a heirarchy of decisions including outgrowth level along the cranio-caudal axis, and appendage identity, i.e., the determination of pectoral or pelvic patterns. Vertebrate appendicular buds emerge from lateral plate mesoderm following localized, sustained cell proliferation. Limb bud outgrowth is maintained by thickened ectodermal epithelium at the bud apex, the apical ectodermal ridge (AER) (Saunders, 1948). AER signals maintain a high rate of cellular proliferation and an undifferentiated state in the subjacent progress zone, although it is here that cells acquire proximo-distal positional identities (Summerbell et al., 1973). The AER also maintains the zone of polarizing activity (ZPA) in the posterior mesenchyme, and this polarising region is involved in anterior to posterior patterning (Saunders and Gasseling, 1968; Tickle et al., 1975). Genetic pathways underlying these cellular interactions are now beginning to be unravelled. Members of the fibroblast growth factor (FGF) family control limb bud initiation from the lateral plate mesoderm, (Cohn et al., 1995; Crossley et al., 1996; Vogel et al., 1996; Ohuchi et al., 1997), and FGFs produced subsequently by the AER mediate ridge signalling activities (Niswander et al., 1993; Fallon et al., 1994). Antero-posterior polarizing activity is mediated by the Sonic hedgehog gene (Shh), which is expressed in the ZPA, and can be induced by retinoic acid (Riddle et al., 1993). Moreover, a positive feedback loop, coordinating limb outgrowth and patterning, involves reciprocal maintenance between Shh in the ZPA and FGF-4 in the AER (Laufer et al., 1994; Niswander et al., 1994). Shh expression is also maintained by the Wnt-7a signal from the dorsal ectoderm (Yang and Niswander, 1995), and this signal contributes to dorsoventral patterning by inducing expression of Lmx-1, a transcription factor, in the dorsal mesenchyme (Parr and McMahon, 1995; Riddle et al, 1995; Vogel et al., 1995). Radical Fringe (R-fng) is also expressed in the dorsal ectoderm, and appears to have an important role in positioning the AER (Laufer et al., 1997; Rodriguez-Esteban et al., 1997). Both R-fng and Wnt-7a are antagonised by the expression of Engrailed-1 in the ventral ectoderm (Loomis et al., 1996; Johnston et al., 1997). All three primary axes of limb bud outgrowth have therefore been linked to interacting gene networks. And a series of similar gene expression patterns and interactions have now been identified in teleost fin buds. Shh is expressed in the posterior fin bud mesoderm (Sordino et al., 1995). Fgf8 is also expressed in zebrafish fin buds, but in Fgf8 mutants (acerebellar), fin development is unaffected, suggesting that it is not required for normal fin morphogenesis (Reifers et al., 1998). Several FGF receptors have been identified, at least one of which is expressed in in the mesoderm beneath the apical ridge (Thisse et al., 1995). The ventral half of the pectoral fin expresses an Engrailed protein (Hatta et al., 1991), and Msx and Distal-less (Dlx) genes are expressed in the apical ridges of tetrapod limb buds and teleost fin buds (Akimenko et al., 1995). These likely homologies between tetrapod and teleost genes, the expression domains and interactions between such genes, and the primitive presence of anteroposterior asymmetry in gnathostome paired fins, strongly indicate that a plesiomorphic version of this signal network was active in the paired fins of the last common ancestor of osteichthyans, if not all gnathostomes.

5 680 M. I. COATES AND M. J. COHN AXIAL PATTERNS AND LEVELS OF OUTGROWTH Comparative analyses of expression patterns along the body axis are beginning to provide clues about the evolutionary relation of axial Hox gene expression to skeletal morphology and appendicular patterning. Paraxial mesoderm, which forms vertebrae, musculature and dermis, is a useful model for studying axial patterning because changes in somitic identity are detectable in vertebral morphologies. Axial Hox expression studies have linked "transposed" (Goodrich, 1930) vertebral landmarks to changes in Hox expression boundaries (Gaunt, 1994; Burke et al., 1995). The evolutionary polarity of these developmental changes can be inferred following the premise that members of each Hox gene paralogue group primitively shared identical expression boundaries. These conserved boundaries probably reflect an ancestral expression pattern present in a vertebrate ancestor prior to Hox cluster duplication. Therefore, the anteroposterior separation of Hox gene expression domains from a single paralogue group in an amniote tetrapod {e.g., Hoxa9, b9, c9 and d9 from an ancestral Hox9 gene) are derived, and this spread of expression boundaries may have allowed the evolution of anatomical divisions such as the lumbar-thoracic boundary and sacrum (Burke et al, 1995). The corollary of this argument is that simpler colinear expression patterns should be found in subjects with simpler, and arguably more primitive axial organisation, such as the zebrafish, Danio rerio. Preliminary results support this hypothesis. Zebrafish Hox genes of paralogue groups 6 to 8 share much closer anterior paraxial mesoderm expression boundaries than in tetrapods (Prince et al., 1998). Further clues about the developmental evolution of tetrapod axial skeletal conditions are provided by phylogenetic sequences of morphological change. Intriguingly, tetrapod-like characters replace fish-like (primitive) morphologies in a consistent, caudally directed trend spanning the fish-tetrapod evolutionary transition (Coates, 1996). Of perhaps greater significance is the fact that these changes occur without any related change of girdle and limb position. Transplantation studies show that decisions about pectoral versus pelvic appendage development are made long before budding is initiated (Kieny, 1971). Head to tail coordinates established early in development may provide the positional information needed to determine appendage type. Such early positional specification within the lateral plate mesoderm could therefore provide different sets of initial conditions at the levels of pectoral and pelvic bud outgrowth. Given that most genes involved in patterning are expressed in fore- and hindlimbs, these differences may exert a significant influence during early morphogenesis. On the basis of largely correlative data, we suggest that Hox genes establish the developmental markers responsible for positioning outgrowth and girdle development within lateral plate mesoderm (Coates and Cohn, 1998). In chicks, loss of HoxbS function causes the forelimb to shift anteriorly along the body axis (Rancourt et al., 1995). Furthermore, early in chick development, before bud outgrowth, Hoxb9, Hoxc9 and Hoxd9 genes are expressed throughout the lateral plate mesoderm, with staggered anterior boundaries within the prospective forelimb territory (Cohn et al., 1997). Then later, as bud outgrowth starts, expression levels change: for example, Hoxd9 flank expression is down-regulated and an anterior Hoxd9 expression patch remains stranded in the pectoral bud. This dynamic expression pattern results in different combinations of Hox gene expression characterising lateral plate mesoderm territories at wing, flank and hind limb levels. In addition to these observations, application of FGF to posterior flank, resulting in ectopic leg formation, reprograms Hox expression in the flank to reproduce expression pattern found at leg levels. Similar changes occur when FGF is applied to anterior flank regions where ectopic wings may be induced (Cohn et al., 1997). It is also noteworthy that in each case of ectopic limb development and transformed lateral plate expression patterns, vertebral morphologies and Hox gene expression in the somites are unaffected. Several studies

6 VERTEBRATE AXES AND APPENDAGES 681 have shown that the T-box genes, Tbx4 and Tbx5, are differentially expressed in forelimbs and hindlimbs, and experimental evidence suggests that they are involved with limb identity (Ohuchi et al., 1998; Isaac et al., 1998; Gibson-Brown et al., 1998). The Tbx gene family has undergone extensive expansion in vertebrate evolution, and duplication of these genes, like the Hox genes, may have provided the opportunity for cooption into the limb development pathway (Ruvinsky and Silver, 1998). These experimental results lead to several conclusions. First, like phylogenetic patterns of fin development, posterior group Hox gene expression in the anterior lateral plate mesoderm conflicts with Tabin and Laufer's (1993) model (Fig. 2). Second, FGF is probably central to the signal pathway involved in initiating limb and fin bud outgrowth. Third, paraxial and lateral plate mesoderm patterning appears to be mutually independent. This suggests that fin and limb diversity arose by similar, but independent, mechanisms to those that generate morphological diversity in the axial skeleton. Paired appendage evolution is therefore assumed to have included a mechanism which staggered Hox gene expression along the lateral plate mesoderm. This resembled patterns in the paraxial domain, and was probably achieved by modifying genes which regulate Hox gene expression, rather than the Hox genes themselves. Tissue-specific regulatory genes would permit such decoupling of anatomical systems and freedom for variation to occur without causing total anatomical transformations. AN ALTERNATIVE MODEL OF PAIRED FIN EVOLUTION The model illustrated in Figure 3 presents a new scenario of changes in Hox gene expression associated with paired fin evolution. The fish cartoons represent conjectures about developmental character states at specific nodes in vertebrate phylogeny. These are not intended as a series of hypothetical ancestors. Each cartoon includes an upper zone of Hox gene expression, which refers to paraxial mesoderm conditions, and a lower zone which refers to the lateral plate mesoderm. And each cartoon illustrates an equivalent stage in ontogeny at the point of fin bud outgrowth initiation. Group-9 Hox gene expression is shown extending anteriorly, but note that different combinations of these genes are expressed in cells proliferating into pectoral and pelvic buds. The anterior limit of group-9 Hox gene expression is probably crucial for subsequent 5' Hox gene activation, because Hoxd9 expression is a likely prerequisite for expression of more 5' genes within the outgrowing bud, such as Hoxdl3 (Sordino and Duboule, 1996; Zakany et al., 1997). This model assumes that staggered Hox gene expression boundaries in the lateral plate mesoderm of primitive vertebrates supplied differential outgrowth boundaries at the origin of paired appendages. However, this assumption requires a further explanation of how such lateral plate boundaries came to be created and stabilized in a pre-paired fin condition. We suggest that the evolution of gut regionalisation may hold the answer. Splanchnic mesoderm (producing the smooth muscle of the gut) and somatic mesoderm (producing body wall and paired appendages) are both derivatives of lateral plate mesoderm. Hox genes are expressed in both layers in early embryos (Roberts et al., 1995; Yokouchi et al., 1995); Hox gene mutations can have coordinated effects on limb and gut development (Kondo et al., 1996; Warot et al., 1997), and it is now clear that FGF application (to the lateral plate mesoderm) modulates gene expression and morphological patterning in the gut as well as the body wall (unpublished data, MJC and K. Patel). It therefore seems plausible that staggered Hox boundaries in the lateral plate mesoderm appeared concomitantly with gut regionalisation (see 'gut' cartoons in Figures 3A and B), given the likely co-regulation of hox genes in somatic and splanchnic mesoderm. This is not the first time that Hox gene expression has been linked to gut regionalisation as a primitive character relative to vertebrate evolution (Bienz, 1994; Kondo et al., 1996). Moreover, while extant agnathans lack a specialised foregut or stomach (Romer and Parsons, 1977), fossil data

7 682 M. I. COATES AND M. J. COHN Hox9 Hox13 FIG. 3. A model of evolving Hox gene expression and function in vertebrate axial and appendicular domains. Whole body cartoons of the fin buds and gut illustrate changing morphological and developmental conditions. Horizontal bars represent general features of Hox gene expression, rather than specific clusters or paralogous groups. Upper set of bars represent paraxial mesoderm (PM); lower bars represent body wall and lateral plate mesoderm (LPM). A. Agnathans and primtive stem-group gnathostomes. Primitively, Hox gene domains in PM and LPM share mostly coincident anterior expression boundaries; single posteriorly restricted Hox gene expression domain (block-shading) is associated with simple division of anterior and posterior gut regions. B. Advanced stem-group gnathostomes. Staggered Hox boundaries anteriorly in LPM coordinates specification of anterior (pectoral) fins and a specialised foregut/stomach. The Hox gene expression pattern in the LPM from which the fin bud emerges determines the initial profile of Hox gene expression in the bud. C. Gnathostomes. Emergence of new Hox boundaries in posterior LPM is associated with posterior (pelvic) fin specification. This posterior fin is initiated in a different, "posterior" context of Hox gene expression. Subsequent expression in the bud proceeds in conventional 5' sequence. The sum of subsequent expression of more 5' Hox genes resembles, but is not identical to, the total expression pattern in the anterior bud. Limited axial skeletal regionalisation reflects emerging Hox gene boundaries in PM. shows evidence of a stomach in thelodonts, indicating that a regionalised gut had evolved already in these advanced members of the gnathostome stem-group (Wilson and Caldwell, 1993, 1998; Janvier, 19966; note also first evidence of rudimentary pelvic fins in certain thelodonts: Marss and Ritchie, 1998). Consequently, once such (gut)

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