The Origin of Digits: Expression Patterns versus Regulatory Mechanisms
2010; Elsevier BV; Volume: 18; Issue: 4 Linguagem: Inglês
10.1016/j.devcel.2010.04.002
ISSN1878-1551
AutoresJoost M. Woltering, Denis Duboule,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoIn the emerging discipline of Evo-Devo, the analysis of gene expression patterns can be deceptive without a clear understanding of the underlying regulatory strategies. Here, we use the paradigm of hand and foot evolution to argue that the consideration of the regulatory mechanisms controlling developmental gene expression is essential to resolve comparative conundrums. In this context, we discuss the adaptive relevance of evolving stepwise, distinct developmental regulatory mechanisms to build an arm, i.e., a composite structure with functional coherence. In the emerging discipline of Evo-Devo, the analysis of gene expression patterns can be deceptive without a clear understanding of the underlying regulatory strategies. Here, we use the paradigm of hand and foot evolution to argue that the consideration of the regulatory mechanisms controlling developmental gene expression is essential to resolve comparative conundrums. In this context, we discuss the adaptive relevance of evolving stepwise, distinct developmental regulatory mechanisms to build an arm, i.e., a composite structure with functional coherence. Along their proximal to distal axes, limbs can be broadly divided into three parts: the most proximal part or stylopod contains the humerus (the femur in hindlimbs), an intermediate part or zeugopod contains the ulna and radius (tibia and fibula in hindlimbs), and a distal part or autopod contains the bones of the hand (foot in himdlimbs) including both the carpals (or tarsals) and digits (Figure 1; see Tabin and Wolpert, 2007Tabin C. Wolpert L. Rethinking the proximodistal axis of the vertebrate limb in the molecular era.Genes Dev. 2007; 21: 1433-1442Crossref PubMed Scopus (180) Google Scholar). When and how did digits appear? And how well do we understand the evolutionary relationships between the different kinds and numbers of digits found in various animals? Over the past 25 years, the discovery of molecular markers has greatly helped to address these questions. Among these markers, Hox genes belonging to the "posterior" halves of the HoxA and HoxD clusters are critical for the development of the proximo-distal organization, as illustrated by multiple series of gene disruption experiments (Davis et al., 1995Davis A.P. Witte D.P. Hsieh-Li H.M. Potter S.S. Capecchi M.R. Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11.Nature. 1995; 375: 791-795Crossref PubMed Scopus (492) Google Scholar, Davis and Capecchi, 1996Davis A.P. Capecchi M.R. A mutational analysis of the 5′ HoxD genes: dissection of genetic interactions during limb development in the mouse.Development. 1996; 122: 1175-1185Crossref PubMed Google Scholar, Dollé et al., 1993Dollé P. Dierich A. LeMeur M. Schimmang T. Schuhbaur B. Chambon P. Duboule D. Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs.Cell. 1993; 75: 431-441Abstract Full Text PDF PubMed Scopus (390) Google Scholar, Zákány et al., 1997Zákány J. Fromental-Ramain C. Warot X. Duboule D. Regulation of number and size of digits by posterior Hox genes: a dose-dependent mechanism with potential evolutionary implications.Proc. Natl. Acad. Sci. USA. 1997; 94: 13695-13700Crossref PubMed Scopus (192) Google Scholar, Zákány and Duboule, 1996Zákány J. Duboule D. Synpolydactyly in mice with a targeted deficiency in the HoxD complex.Nature. 1996; 384: 69-71Crossref PubMed Scopus (174) Google Scholar, Kmita et al., 2005Kmita M. Tarchini B. Zàkàny J. Logan M. Tabin C.J. Duboule D. Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function.Nature. 2005; 435: 1113-1116Crossref PubMed Scopus (196) Google Scholar). These analyses have uncovered functions for these genes during both the patterning and the subsequent growth of limb elements; while mutant specimens can indeed be polydactylous, oligodactylous, or even adactylous, the relative sizes and shapes of particular skeletal elements are generally affected too. Both expression and functional analyses have established distinct spatial and temporal signatures for Hoxa and Hoxd genes, which clearly distinguish proximal from distal limb regions. In the HoxA cluster, Hoxa11 functions in the zeugopod whereas Hoxa13 labels the autopod (Yokouchi et al., 1991Yokouchi Y. Sasaki H. Kuroiwa A. Homeobox gene expression correlated with the bifurcation process of limb cartilage development.Nature. 1991; 353: 443-445Crossref PubMed Scopus (274) Google Scholar, Nelson et al., 1996Nelson C.E. Morgan B.A. Burke A.C. Laufer E. DiMambro E. Murtaugh L.C. Gonzales E. Tessarollo L. Parada L.F. Tabin C. Analysis of Hox gene expression in the chick limb bud.Development. 1996; 122: 1449-1466PubMed Google Scholar, Tamura et al., 2008Tamura K. Yonei-Tamura S. Yano T. Yokoyama H. Ide H. The autopod: its formation during limb development.Dev. Growth Differ. 2008; 50: S177-S187Crossref PubMed Scopus (30) Google Scholar). Likewise, posterior Hoxd9 to Hoxd13 are coordinately expressed in two subsequent phases (Figure 1). In the early phase, Hoxd9 to Hoxd12 are transcribed in proximal regions, up to the boundary between the zeugopod and the autopod, i.e., in those cells that will ultimately build the humerus, radius, and ulna. Subsequently, a second phase of expression develops into a clearly distinct, more distal domain covering most of the autopod (Figure 1; Dollé et al., 1989Dollé P. Izpisúa-Belmonte J.C. Falkenstein H. Renucci A. Duboule D. Coordinate expression of the murine Hox-5 complex homoeobox-containing genes during limb pattern formation.Nature. 1989; 342: 767-772Crossref PubMed Scopus (460) Google Scholar, Nelson et al., 1996Nelson C.E. Morgan B.A. Burke A.C. Laufer E. DiMambro E. Murtaugh L.C. Gonzales E. Tessarollo L. Parada L.F. Tabin C. Analysis of Hox gene expression in the chick limb bud.Development. 1996; 122: 1449-1466PubMed Google Scholar). During this second phase, Hoxd10 to Hoxd13 are expressed concomitantly, in the same domain yet with decreasing transcriptional efficiencies, such that Hoxd13 is transcribed at the highest level (Montavon et al., 2008Montavon T. Le Garrec J.F. Kerszberg M. Duboule D. Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness.Genes Dev. 2008; 22: 346-359Crossref PubMed Scopus (118) Google Scholar). As a consequence of this robust transcription, this latter gene is expressed throughout the five digit primordia found in amniotes, whereas Hoxd12, Hoxd11, and Hoxd10 transcripts are detected in all digits but the future thumb, a quantitative effect sometimes referred to as "reverse collinearity" (Nelson et al., 1996Nelson C.E. Morgan B.A. Burke A.C. Laufer E. DiMambro E. Murtaugh L.C. Gonzales E. Tessarollo L. Parada L.F. Tabin C. Analysis of Hox gene expression in the chick limb bud.Development. 1996; 122: 1449-1466PubMed Google Scholar). This uneven anterior to posterior (AP) distribution reflects both the graded transcriptional efficiencies of Hoxd genes and the activity of sonic hedgehog (Shh), which is expressed at the posterior margin of the limb bud (Riddle et al., 1993Riddle R.D. Johnson R.L. Laufer E. Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA.Cell. 1993; 75: 1401-1416Abstract Full Text PDF PubMed Scopus (1886) Google Scholar, Drossopoulou et al., 2000Drossopoulou G. Lewis K.E. Sanz-Ezquerro J.J. Nikbakht N. McMahon A.P. Hofmann C. Tickle C. A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling.Development. 2000; 127: 1337-1348PubMed Google Scholar, Harfe et al., 2004Harfe B.D. Scherz P.J. Nissim S. Tian H. McMahon A.P. Tabin C.J. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.Cell. 2004; 118: 517-528Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). These specific distributions of posterior Hoxa and Hoxd transcripts are globally conserved in tetrapod limbs and similar patterns were described for Axolotl and Xenopus (Torok et al., 1998Torok M.A. Gardiner D.M. Shubin N.H. Bryant S.V. Expression of HoxD genes in developing and regenerating axolotl limbs.Dev. Biol. 1998; 200: 225-233Crossref PubMed Scopus (87) Google Scholar, Christen et al., 2003Christen B. Beck C.W. Lombardo A. Slack J.M. Regeneration-specific expression pattern of three posterior Hox genes.Dev. Dyn. 2003; 226: 349-355Crossref PubMed Scopus (55) Google Scholar, Satoh et al., 2006Satoh A. Endo T. Abe M. Yakushiji N. Ohgo S. Tamura K. Ide H. Characterization of Xenopus digits and regenerated limbs of the froglet.Dev. Dyn. 2006; 235: 3316-3326Crossref PubMed Scopus (30) Google Scholar, Ohgo et al., 2010Ohgo S. Itoh A. Suzuki M. Satoh A. Yokoyama H. Tamura K. Analysis of hoxa11 and hoxa13 expression during patternless limb regeneration in Xenopus.Dev. Biol. 2010; 338: 148-157Crossref PubMed Scopus (34) Google Scholar). Comparisons between modern limbs, fossils, and recent sarcopterygian fins reveal likely homologies between proximal and intermediate limb bones and parts of fin skeletons (Coates, 1994Coates M.I. The origin of vertebrate limbs.Dev. Suppl. 1994; : 169-180PubMed Google Scholar, Cohn et al., 2002Cohn M.J. Lovejoy C.O. Wolpert L. Coates M.I. Branching, segmentation and the metapterygial axis: pattern versus process in the vertebrate limb.Bioessays. 2002; 24: 460-465Crossref PubMed Scopus (51) Google Scholar). However, when the most distal parts of the appendages are considered, in particular the digits, structural relationships become problematic and hence homologies between the autopod and ancestral fin elements have been controversial (Coates, 1994Coates M.I. The origin of vertebrate limbs.Dev. Suppl. 1994; : 169-180PubMed Google Scholar, Coates, 1995Coates M.I. Limb evolution. Fish fins or tetrapod limbs—a simple twist of fate?.Curr. Biol. 1995; 5: 844-848Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Coates et al., 2002Coates M.I. Jeffery J.E. Rut M. Fins to limbs: what the fossils say.Evol. Dev. 2002; 4: 390-401Crossref PubMed Scopus (116) Google Scholar, Wagner and Chiu, 2001Wagner G.P. Chiu C.H. The tetrapod limb: a hypothesis on its origin.J. Exp. Zool. 2001; 291: 226-240Crossref PubMed Scopus (77) Google Scholar). Coates and colleagues consider digits as generally related to fin radials (e.g., Coates, 1994Coates M.I. The origin of vertebrate limbs.Dev. Suppl. 1994; : 169-180PubMed Google Scholar, Friedman et al., 2007Friedman M. Coates M.I. Anderson P. First discovery of a primitive coelacanth fin fills a major gap in the evolution of lobed fins and limbs.Evol. Dev. 2007; 9: 329-337Crossref PubMed Scopus (54) Google Scholar), based on shared ontological and anatomical characters (Friedman et al., 2007Friedman M. Coates M.I. Anderson P. First discovery of a primitive coelacanth fin fills a major gap in the evolution of lobed fins and limbs.Evol. Dev. 2007; 9: 329-337Crossref PubMed Scopus (54) Google Scholar). Anatomical criteria, however, do not exclude convergence, and the ontological argument (both structures develop as buds and use similar genetic programs; see Friedman et al., 2007Friedman M. Coates M.I. Anderson P. First discovery of a primitive coelacanth fin fills a major gap in the evolution of lobed fins and limbs.Evol. Dev. 2007; 9: 329-337Crossref PubMed Scopus (54) Google Scholar) does not consider the proximal versus distal parts separately, which makes it only moderately informative regarding the origin of digits. Alternatively, structural and molecular differences have led others to postulate a more recent origin for the autopodial field including the digits, as opposed to that of more proximal limb elements. This hypothesis implies different evolutionary trajectories for distal versus more proximal parts of the tetrapod limb and suggests that the autopod is a neomorphic structure. In this view, the tetrapod limb is made out of two independent pieces bearing distinct ontogenetic and phylogenetic histories (Holmgreen, 1952Holmgreen N. An embryological analysis of the mammalian carpus and its bearing on the question of the origin of the tetrapod limb.Acta Zool. 1952; 33: 1-115Crossref Scopus (31) Google Scholar, Sordino et al., 1995Sordino P. van der Hoeven F. Duboule D. Hox gene expression in teleost fins and the origin of vertebrate digits.Nature. 1995; 375: 678-681Crossref PubMed Scopus (269) Google Scholar, Wagner and Chiu, 2001Wagner G.P. Chiu C.H. The tetrapod limb: a hypothesis on its origin.J. Exp. Zool. 2001; 291: 226-240Crossref PubMed Scopus (77) Google Scholar; reviewed in Wagner and Larsson, 2007Wagner G.P. Larsson H.C.E. Fins and Limbs in the Study of Evolutionary Novelties: Fins into Limbs. The University of Chicago Press, Chicago, USA2007Google Scholar). The conceptual distinction between homology and neomorphy must nevertheless be handled carefully considering the facts that novel structures (1) seldom arise entirely de novo and (2) obligatorily implement preexisting genetic pathways and hence bear genetic signatures related to preexisting morphologies. Support for the latter view came from a comparison of Hox gene expression between tetrapods and fishes, which revealed the absence, in fins, of the clear bimodal signature of proximal versus distal domains. In the zebrafish fin buds, a single phase of Hoxd expression was distinguished, extending to the most distal part of the presumptive endoskeleton, suggesting that the fin to limb transition involved the acquisition of a new phase of Hoxd transcription, functionally associated with the emergence of the autopod and digits (Sordino and Duboule, 1996Sordino P. Duboule D. A molecular approach to the evolution of vertebrate paired appendages.Trends Ecol. Evol. 1996; 2: 114-119Abstract Full Text PDF Scopus (60) Google Scholar). This interpretation was recently challenged after the examination of fish species such as Polyodon, shark (Scyliorhinus), and lungfish (Neoceratodus), whose fins more closely resemble those of tetrapod ancestors, for instance via the presence of a metapterygium (Mabee, 2000Mabee P.M. Developmental Data and Phylogenetic Systematics: Evolution of the Vertebrate Limb.Am. Zool. 2000; 40: 789-800Crossref Google Scholar). In these species, two phases of expression were observed during pectoral fin development (Scyliorhinus, Polyodon) and strong distal Hoxd13 signal was reported in fin radials (Neoceratodus), suggesting the existence of the same proximal and distal expression domains as in tetrapod limbs (Davis et al., 2007Davis M.C. Dahn R.D. Shubin N.H. An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish.Nature. 2007; 447: 473-476Crossref PubMed Scopus (107) Google Scholar, Freitas et al., 2007Freitas R. Zhang G. Cohn M.J. Biphasic Hoxd gene expression in shark paired fins reveals an ancient origin of the distal limb domain.PLoS ONE. 2007; 15: e754Crossref Scopus (91) Google Scholar, Johanson et al., 2007Johanson Z. Joss J. Boisvert C.A. Ericsson R. Sutija M. Ahlberg P.E. Fish fingers: digit homologues in sarcopterygian fish fins.J. Exp. Zool. B Mol. Dev. Evol. 2007; 308: 757-768Crossref PubMed Scopus (89) Google Scholar). In support of this view, Polyodon and shark Hoxd13 transcripts extend more anteriorly than those of Hoxd12, as expected from reverse collinearity (Davis et al., 2007Davis M.C. Dahn R.D. Shubin N.H. An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish.Nature. 2007; 447: 473-476Crossref PubMed Scopus (107) Google Scholar, Freitas et al., 2007Freitas R. Zhang G. Cohn M.J. Biphasic Hoxd gene expression in shark paired fins reveals an ancient origin of the distal limb domain.PLoS ONE. 2007; 15: e754Crossref Scopus (91) Google Scholar). Hoxd expression was also reconsidered in zebrafish pectoral fin buds and both the proximal and distal expression phase were reported to exist in this fish too (Ahn and Ho, 2008Ahn D. Ho R.K. Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.Dev. Biol. 2008; 322: 220-233Crossref PubMed Scopus (86) Google Scholar). Altogether, it was proposed that the two-phased Hoxd expression, including that in the autopod, is an ancestral gnathostome character, rather than a neomorphic trait of tetrapods (see Shubin et al., 2009Shubin N. Tabin C. Carroll S. Deep homology and the origins of evolutionary novelty.Nature. 2009; 457: 818-823Crossref PubMed Scopus (495) Google Scholar). By extension, distal fin cells labeled by the second phase of Hoxd expression could possess an "autopodial identity" (Freitas et al., 2007Freitas R. Zhang G. Cohn M.J. Biphasic Hoxd gene expression in shark paired fins reveals an ancient origin of the distal limb domain.PLoS ONE. 2007; 15: e754Crossref Scopus (91) Google Scholar), implying homology between fin radials and digits (Johanson et al., 2007Johanson Z. Joss J. Boisvert C.A. Ericsson R. Sutija M. Ahlberg P.E. Fish fingers: digit homologues in sarcopterygian fish fins.J. Exp. Zool. B Mol. Dev. Evol. 2007; 308: 757-768Crossref PubMed Scopus (89) Google Scholar). These new expression data for Hoxd genes in fishes imply an ancient origin for the distal Hoxd domain and the presence of structures homologous to digits in fishes. However, a careful examination of these data sets calls for some caution in several respects. The description of multiple expression phases (see Ahn and Ho, 2008Ahn D. Ho R.K. Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.Dev. Biol. 2008; 322: 220-233Crossref PubMed Scopus (86) Google Scholar) does not rely upon clear temporal and/or topological observations, and Hoxd11 "proximal" and "distal" expression in fins refers to a unique and continuous domain, unlike in tetrapods, wherein two distinct positive areas of the limb are visible concomitantly, separated by a zone of no (or low) Hox activity (see below). Also, while it is clear that Hoxd expression in Polyodon fins becomes distal (Davis et al., 2007Davis M.C. Dahn R.D. Shubin N.H. An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish.Nature. 2007; 447: 473-476Crossref PubMed Scopus (107) Google Scholar), a similar trend occurs in the developing mouse limb within the proximal domain itself (i.e., during the early phase) before the autopod is formed (e.g., Nelson et al., 1996Nelson C.E. Morgan B.A. Burke A.C. Laufer E. DiMambro E. Murtaugh L.C. Gonzales E. Tessarollo L. Parada L.F. Tabin C. Analysis of Hox gene expression in the chick limb bud.Development. 1996; 122: 1449-1466PubMed Google Scholar, Tarchini and Duboule, 2006Tarchini B. Duboule D. Control of Hoxd genes' collinearity during early limb development.Dev. Cell. 2006; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In other words, should the autopod be absent, expression of tetrapod Hox genes during the early proximal phase would develop into a "distal" domain as observed in Polyodon. In this latter case, a single extended expression domain seems to include all of the future endoskeletal components, and it is thus unclear how the Hox expression patterns in the Polyodon fin would relate to the bimodal pattern of the tetrapod limb. It should also be noted that Hoxa11 and Hoxa13 transcripts are expressed in the same domain in fin buds (Sordino et al., 1995Sordino P. van der Hoeven F. Duboule D. Hox gene expression in teleost fins and the origin of vertebrate digits.Nature. 1995; 375: 678-681Crossref PubMed Scopus (269) Google Scholar, van der Hoeven et al., 1996van der Hoeven F. Sordino P. Fraudeau N. Izpisua-Belmonte J.-C. Duboule D. Teleost HoxD and HoxA genes: Comparison with tetrapods and functional evolution of the HoxD complex.Mech. Dev. 1996; 54: 9-21Crossref PubMed Scopus (82) Google Scholar, Metscher et al., 2005Metscher B.D. Takahashi K. Crow K. Amemiya C. Nonaka D.F. Wagner G.P. Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbs.Evol. Dev. 2005; 7: 186-195Crossref PubMed Scopus (55) Google Scholar, Davis et al., 2007Davis M.C. Dahn R.D. Shubin N.H. An autopodial-like pattern of Hox expression in the fins of a basal actinopterygian fish.Nature. 2007; 447: 473-476Crossref PubMed Scopus (107) Google Scholar), a situation drastically different from tetrapods wherein the two domains segregate early on to label proximal versus distal parts. Therefore, qualifications such as "distal," "proximal," "early," or "late," while helpful in an ontogenetic context, are of little use whenever phylogenetic issues are being considered, in particular when trying to homologize expression patterns between structures as distinct as fins and limbs. This problem can be partially addressed by considering a deeper level of comparison, that of the underlying regulatory circuits, rather than looking at the resulting expression patterns. Extensive genetic analysis of the HoxD locus in vivo has shown that the proximal and distal expression phases are mechanistically disconnected from one another (Figure 1). Therefore, the morphological boundary, in tetrapod limbs, between those elements that can be clearly homologized with an ancestral fin and those that cannot matches a region of transition between two completely distinct regulatory modules controlling different, yet partially overlapping, subsets of Hoxd genes. Different sets of regulatory sequences are indeed located on opposite sides of the gene cluster (Spitz et al., 2003Spitz F. Gonzalez F. Duboule D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster.Cell. 2003; 113: 405-417Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, Spitz et al., 2005Spitz F. Herkenne C. Morris M.A. Duboule D. Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes.Nat. Genet. 2005; 37: 889-893Crossref PubMed Scopus (125) Google Scholar; see Deschamps, 2007Deschamps J. Ancestral and recently recruited global control of the Hox genes in development.Curr. Opin. Genet. Dev. 2007; 17: 422-427Crossref PubMed Scopus (51) Google Scholar), such that the proximal domain (the forearm) is controlled by sequences mapping telomeric to the cluster, whereas the distal domain (the digits) is driven by enhancers lying on the centromeric side (Gonzalez et al., 2007Gonzalez F. Duboule D. Spitz F. Transgenic analysis of Hoxd gene regulation during digit development.Dev. Biol. 2007; 306: 847-859Crossref PubMed Scopus (86) Google Scholar). Because Hoxd13 is "the closest" to the centromeric enhancers, it shows preferential interactions and is thus expressed twice as strongly as Hoxd12. This difference impacts the AP distribution of Hoxd13 in presumptive digits and causes it to be expressed in future digit I (the thumb), where other Hoxd genes are not transcribed. This biased interaction of centromeric enhancers with posterior Hoxd genes thus leads to the observed reverse collinearity in digits (Montavon et al., 2008Montavon T. Le Garrec J.F. Kerszberg M. Duboule D. Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness.Genes Dev. 2008; 22: 346-359Crossref PubMed Scopus (118) Google Scholar). The distal regulation triggered by these global enhancers also affects Lunapark (Lnp) and Evx2, two genes located on the centromeric side of the cluster (Spitz et al., 2003Spitz F. Gonzalez F. Duboule D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster.Cell. 2003; 113: 405-417Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) and expressed in the same presumptive digit domain. In tetrapods, some of these enhancers have been identified, in particular two sequences referred to as CsB (part of the GCR sequence; Spitz et al., 2003Spitz F. Gonzalez F. Duboule D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster.Cell. 2003; 113: 405-417Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) and CsC (part of the Prox sequence; Gonzalez et al., 2007Gonzalez F. Duboule D. Spitz F. Transgenic analysis of Hoxd gene regulation during digit development.Dev. Biol. 2007; 306: 847-859Crossref PubMed Scopus (86) Google Scholar), which can, on their own, elicit expression in distal limbs in a transgenic context. While CsB is highly conserved in all vertebrate genomes, the CsC sequence is found neither in fugu, nor in tetraodon, whereas it is present in birds, Xenopus, and Anolis (Gonzalez et al., 2007Gonzalez F. Duboule D. Spitz F. Transgenic analysis of Hoxd gene regulation during digit development.Dev. Biol. 2007; 306: 847-859Crossref PubMed Scopus (86) Google Scholar; data not shown). What do we know about Hoxd gene regulation during pectoral fin development, which could help ascertain homologies? Unfortunately, a quantitative assessment of Hox transcripts in budding fins is lacking, which makes the comparison with reverse collinearity, as mechanistically defined in tetrapods, illegitimate. In addition, the pufferfish CsB sequence, when introduced into a mouse transgenic context, is unable to elicit expression in distal limbs, whereas it triggers expression in neuronal populations corresponding to another expected regulatory potential of this sequence (Spitz et al., 2003Spitz F. Gonzalez F. Duboule D. A global control region defines a chromosomal regulatory landscape containing the HoxD cluster.Cell. 2003; 113: 405-417Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Finally, the zebrafish Lnp gene, even though located at the same relative distance from Hoxd13, does not show expression in developing fins (Ahn and Ho, 2008Ahn D. Ho R.K. Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.Dev. Biol. 2008; 322: 220-233Crossref PubMed Scopus (86) Google Scholar) equivalent to that seen in the mouse limb bud. Given the absence of regulatory and quantitative analyses of posterior Hoxd gene regulation, the lack of apparent reverse collinearity in zebrafish (Ahn and Ho, 2008Ahn D. Ho R.K. Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.Dev. Biol. 2008; 322: 220-233Crossref PubMed Scopus (86) Google Scholar), and the conflicting data regarding genes known to be coregulated in the distal expression phase in tetrapods (Lnp and Evx), the conclusion that this distinctive tetrapod digit regulation may have a counterpart in fishes (see Shubin et al., 2009Shubin N. Tabin C. Carroll S. Deep homology and the origins of evolutionary novelty.Nature. 2009; 457: 818-823Crossref PubMed Scopus (495) Google Scholar) should perhaps await more evidence. A defining feature of tetrapods (Gaffney, 1979Gaffney E.S. Tetrapod monophyly: a phylogenetic analysis.Bull. Carnegie Mus. Nat. Hist. 1979; 13: 92-105Google Scholar) is the presence of an interface between the digits and the radius/ulna, a collection of nodular-shaped mesopodial elements (the carpals/tarsals) that form the wrist and ankles and allow the hands and feet to properly articulate with the rest of the limbs (see Coates et al., 2002Coates M.I. Jeffery J.E. Rut M. Fins to limbs: what the fossils say.Evol. Dev. 2002; 4: 390-401Crossref PubMed Scopus (116) Google Scholar, Wagner and Chiu, 2001Wagner G.P. Chiu C.H. The tetrapod limb: a hypothesis on its origin.J. Exp. Zool. 2001; 291: 226-240Crossref PubMed Scopus (77) Google Scholar, Johanson et al., 2007Johanson Z. Joss J. Boisvert C.A. Ericsson R. Sutija M. Ahlberg P.E. Fish fingers: digit homologues in sarcopterygian fish fins.J. Exp. Zool. B Mol. Dev. Evol. 2007; 308: 757-768Crossref PubMed Scopus (89) Google Scholar). In terms of adaptive value, the emergence of fully functional limbs thus required the coevolution of a mesopodium (carpus/tarsus) so as to optimize the use of hands and feet in land-based locomotion (Carroll, 1997Carroll R.L. Patterns and Processes of Vertebrate Evolution. Cambridge University Press, Cambridge1997Google Scholar). In this context, the mesopodium was an essential novelty to be added to a sarcopterygian fin, either as a new structure or via the transformation of preexisting elements. We would like to propose that the evolution of a mesopodium was made possible by the existence of two independent Hox regulatory modules as opposed to a single ancestral one. Hoxd expression levels influence both the pattern of mesenchymal condensations and their subsequent growth capacities during limb development, in a dose-dependent manner. They have also been associated with the induction of growth plates (Boulet and Capecchi, 2004Boulet A.M. Capecchi M.R. Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod.Development. 2004; 131: 299-309Crossref PubMed Scopus (96) Google Scholar). The segregation of the two Hoxd domains in tetrapods creates a no-Hoxd land, situated between two series of elongated bones (the ulna-radius and the metacarpals), precisely at the future mesopodial position. Consequently, this region includes those cells producing the lowest amount of posterior Hox transcripts during limb development (Nelson et al., 1996Nelson C.E. Morgan B.A. Burke A.C. Laufer E. DiMambro E. Murtaugh L.C. Gonzales E. Tessarollo L. Parada L.F. Tabin C. Analysis of Hox gene expression in the chick limb bud.Development. 1996; 122: 1449-1466PubMed Google Scholar, Reno et al., 2008Reno P.L. McCollum M.A. Cohn M.J. Meindl R.S. Hamrick M. Lovejoy C.O. Patterns of correlation and covariation of anthropoid distal forel
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