Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws

SUMMARY Paleontological evidence indicates that the evolutionary diversification of mammals early in the Cenozoic era was characterized by an adaptive radiation of distal limb structures. Likewise, neontological data show that morphological variation in distal limb integumentary appendages (e.g., nails, hooves, and claws) can be observed not only among distantly related mammalian taxa but also among closely related species within the same clade. Comparative analysis of nail, claw, and hoof morphogenesis reveals relatively subtle differences in mesenchymal and epithelial patterning underlying these adult differences in distal limb appendage morphology. Furthermore, studies of regulatory gene expression during vertebrate claw development demonstrate that many of the signaling molecules involved in patterning ectodermal derivatives such as teeth, hair, and feathers are also involved in organizing mammalian distal limb appendages. For example, Bmp4 signaling plays an important role during the recruitment of mesenchymal cells into the condensations forming the terminal phalanges, whereas Msx2 affects the length of nails and claws by suppressing proliferation of germinal epidermal cells. Evolutionary changes in the form of distal integumentary appendages may therefore result from changes in gene expression during formation of mesenchymal condensations ( Bmp4 , posterior Hox genes), induction of the claw fold and germinal matrix ( shh ), and/or proliferation of epidermal cells in the claw matrix ( Msx1 , Msx2 ). The prevalence of convergences and parallelisms in nail and claw structure among mammals underscores the existence of multiple morphogenetic pathways for evolutionary change in distal limb appendages.     

X chromosomes of American marsupials contain minimal amounts of euchromatin

The karyotypes of four South American didelphid marsupials, representing diploid numbers of 2n = 14 and 18, have been analyzed by a variety of banding techniques. The 2n = 14 karyotypes display a high degree of homoeology, but there also exist distinct similarities between the 2n = 14 and 2n = 18 karyotypes. The interspecific differences found are due to centric fissions, pericentric inversions, and variations in the amount and composition of the constitutive heterochromatin. Contrary to the evolutionary conservation of the banding patterns in all autosomal arms, there are multiple differences in the number and chromosomal location of the nucleolus organizer regions. In species with X-linked nucleolus organizers, the 18S + 28S ribosomal RNA genes escape inactivation in female cells. Measurements on the X chromosomes of Marmosa fuscata and Micoureus demerarae unexpectedly reveal the lowest quantities of euchromatin so far determined in the X chromosomes of mammals: 1.5% and 1.8%, respectively, of their haploid female genomes. This is significantly less than the amount of euchromatin in the basic X chromosomes of other marsupials (3%) or eutherians (5%).     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Morphological Variation of the Forelimb and Claw in Neotropical Sigmodontine Rodents (Rodentia: Cricetidae)

The limbs of mammals exhibit a variety of morphologies that reflect the diversity of their habitats and their functional needs, including subtle structural differences in their distal limb integumentary appendages (hooks, claws, adhesive pads). Little is known about structure and function of claws of sigmodontine rodents. Here, we analyze claw shape and forelimb skeleton morphology of 25 species of sigmodontine rodents with different locomotory types (ambulatory, fossorial, natatorial, quadrupedal saltatorial, and scansorial), taking into account their phylogenetic affinities. Qualitative differences in claw shape were examined using digital photographs, and quantitative measurements were made for length, height, and curvature of the claws of all digits, and dimensions of other forelimb skeletal elements. Our results show that both phylogeny and ecological categories explain substantial components of the morphological variation in sigmodontine rodents. Qualitative analysis reveals that non-specialized forms (ambulatory, quadrupedal saltatorial, and scansorial) tend to have high and strongly curved claws, whereas highly specialized forms (fossorial and natatorial) tend to have elongate and smoothly curved claws. However, the quantitative analysis differentiated the fossorial and scansorial by variables related to claw, and natatorial by variables related to bones of the forelimb. No variables that could differentiate ambulatory or quadrupedal saltatorial forms were found, demonstrating that these forms show a generalized morphological pattern. This study indicates that both historical and ecological factors contribute to the evolution of claw length in these groups.     

An investigation of ecological correlates with hand and foot morphology in callitrichid primates

Studies of primate taxonomy and phylogeny often depend on comparisons of limb dimensions, yet there is little information on how morphology correlates and contributes to foraging strategies and ecology. Callitrichid primates are ideal for comparative studies as they exhibit a range of body size, limb proportions and diet. Many callitrichid species exhibit a high degree of exudativory, and to efficiently exploit these resources, they are assumed to have evolved morphologies that reflect a level of dependence on these resources. We tested assumptions by considering measurements of limb proportion and frictional features of the volar surfaces in preserved specimens of 25 species with relation to published life history and ecological data. The degree of exudativory and utilization of vertical substrates during foraging were found to correlate both with size and with size‐corrected foot and hand dimensions. Smaller species, which engage in greater degrees of exudativory, had proportionally longer hands and feet and more curved claw‐like tegulae (nails) on their digits to facilitate climbing on vertical substrates. The density of patterned ridges (dermatoglyphs) on the volar surfaces of the hands and feet is higher in more exudativorous genera, suggesting a role in climbing on vertical tree trunks during foraging. Dermatoglyph comparisons suggest that ridges on the soles and palms may facilitate food procurement by enhancing frictional grip during exudate feeding. Volar pad features corroborate taxonomic relationships described from dental morphology. Am J Phys Anthropol 152:447–458, 2013. © 2013 Wiley Periodicals, Inc.     

Body size and scaling of the hands and feet of prosimian primates

The hands and feet of primates fulfill a variety of biological roles linked with food acquisition and positional behavior. Current explanations of shape differences in cheiridial morphology among prosimians are closely tied to body size differences. Although numerous studies have examined the relationships between body mass and limb morphology in prosimians, no scaling analysis has specifically considered hand and foot dimensions and intrinsic proportions. In this study, we present such an analysis for a sample of 270 skeletal specimens distributed over eight prosimian families. The degree of association between size and shape was assessed using nonparametric correlational techniques, while the relationship between each ray element length and body mass (from published data and a body mass surrogate) was tested for allometric scaling. Since tarsiers and strepsirrhines encompass many taxa of varying degrees of phylogenetic relatedness, effective degrees of freedom were calculated, and comparisons between families were performed to partially address the problem of statistical nonindependence and "phylogenetic inertia." Correlational analyses indicate negative allometry between relative phalangeal length (as reflected by phalangeal indices) and body mass, except for the pollex and hallux. Thus, as size increases, there is a significant decrease in the relative length of the digits when considering all prosimian taxa sampled. Regression analyses show that while the digital portion of the rays scales isometrically with body mass, the palmar/plantar portion of the rays often scales with positive allometry. Some but not all of these broadly interspecific allometric patterns remain statistically significant when effective degrees of freedom are taken into account. As is often the case in interspecific scaling, comparisons within families show different scaling trends in the cheiridia than those seen across families (i.e., lorisids, indriids, and lemurids exhibit rather different allometries). The interspecific pattern of positive allometry that appears to best characterize the metapodials of prosimians, especially those of the foot, parallels differences found in the morphology of the volar skin. Indeed, relatively longer metapodials appear to covary with flatter and more coalesced volar pads, which in turn slightly improve frictional force for animals that are at a comparative disadvantage while climbing because of their larger mass. Despite the essentially isometric relationship found between digit length and body mass across prosimians, examination of the residual variation reveals that tarsiers and Daubentonia possess, relative to their body sizes, remarkably long fingers. Such marked departures between body size and finger length observed in these particular primates are closely linked with specialized modes of prey acquisition and manipulation involving the hands.     

Reduced phenotypic covariation in marsupial limbs and the implications for mammalian evolution

As serially homologous structures, mammalian fore‐ and hindlimbs ancestrally share a common developmental and genetic architecture. As a result, mammalian fore‐ and hindlimbs are predicted to be highly integrated in the absence of selective pressures to form divergent limb morphologies. Marsupials experience such a divergent selective pressure to form a robust forelimb to power a post‐natal crawl to the teat. In this study, phenotypic covariation in marsupials was assessed to determine if specialization for the crawl did indeed reduce integration between their fore‐ and hindlimbs. To explore the evolution of mammalian limb integration, phenotypic covariation in representative eutherians and monotremes was also examined. Phenotypic covariation in limbs was quantified morphometrically, and analysed with correlational and phylogenetic methods. Results indicate that marsupials generally have relatively high levels of within‐limb phenotypic covariation, and low levels between limbs, in contrast to the pattern reconstructed for the mammalian ancestor. Our findings support the hypothesis that pressure to specialize in one limb (either the fore‐ or the hindlimb) can reduce phenotypic covariation between limbs, and that reduced limb phenotypic covariation is derived in marsupials. Further research is needed to test the effect that these differences in limb phenotypic covariation had on the evolution of the major mammalian groups. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 22–36.     

The dark side of coloration: Ecogeographical evidence supports Gloger's rule in American marsupials

Geographical distribution of color phenotypes and associations with ecological predictors remains poorly understood. An important geographic pattern concerning this topic is Gloger's rule, which predicts the increase of pigmentation in endothermic animals from cold and dry to warm and wet environments. Didelphid marsupials exhibit a variety of color patterns, ranging from light and dark uniform to more complex colorations. However, surprisingly little is known about the adaptive significance of dark coloration in this singular group of mammals. Using a phylogenetic comparative approach, we investigated whether coloration in different body regions of didelphids (i.e., dorsum and face) is associated with variables representing heat and humidity of the environment, as predicted by Gloger's rule. We demonstrated that Gloger's rule explains the interspecific color variation in American marsupials, especially when considering the facial region. Thus, dark coloration was more frequent among didelphid species occupying warm and wet environments than cold and dry environments. We also discuss the selective forces that can potentially explain coat color variation in didelphid marsupials, including camouflage, pathogen resistance, and pleiotropy hypotheses.     

A microscopic study of the marmoset claw and nail

W. E. Le Gros Clark concluded in his study of the problem of the primate claw that the essential difference between claw and nail is the presence of a terminal matrix associated with a deep layer in the claw, whereas neither terminal matrix nor deep layer exists in the nail. He demonstrated that the marmoset claw (which tips every digit except the hallux) has a thin deep layer and a recognizable terminal matrix. The present paper reports and discusses evidence that the marmoset nail also has a deep layer and terminal matrix. Although the importance of these structures in the claw is not disputed, it appears that these can no longer be considered absolute differences between claw and nail. On the basis of this evidence, it cannot be claimed that the presence of the deep layer and terminal matrix determines the distinctive shape of the claw as opposed to the nail.     

Avian skin development and the evolutionary origin of feathers

The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (beta) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (beta) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of "four-winged" dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), "feathered feet." We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the beta keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High-line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers.     

The use of hand morphology in the taxonomy of galagos

Measurements of volar (hand) pad area were made for 244 specimens, representing 12 species and 4 genera of galagos (sub-family Galaginae). When corrected for body weight, statistically significant differences were identified, at both the genus and species levels in the areas of the volar pads. Most informative, in terms of taxonomic differences, were measurements of two of the five pads; interdigjtal pad number 4 and thenar pad number 5. Closely related species were distinguishable on the basis of these measurements. The thick-tailed galago (Otolemur crassicaudatus) was separate from Garnett's galago (O. garnettii) and the specific status of the silver greater galago (O. argentatus) was supported. Likewise, the two needle-clawed galagos (Euoticus elegantulus andE. pallidus), recently separated on mitochodrial DNA grounds, were found to differ significantly in their volar pad morphology. These studies show that comparative studies of volar hand pad morphology provide a novel approach to the re-assessment of galago taxonomy, and may be applicable also in taxonomic studies of other prosimian groups.     

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.     

Iridescent colour production in hairs of blind golden moles (Chrysochloridae)

Relative to other metazoans, the mammalian integument is thought to be limited in colour. In particular, while iridescence is widespread among birds and arthropods, it has only rarely been reported in mammals. Here, we examine the colour, morphology and optical mechanisms in hairs from four species of golden mole (Mammalia: Chrysochloridae) that are characterized by sheens ranging from purple to green. Microspectrophotometry reveals that this colour is weak and variable. Iridescent hairs are flattened and have highly reduced cuticular scales, providing a broad and smooth surface for light reflection. These scales form multiple layers of light and dark materials of consistent thickness, strikingly similar to those in the elytra of iridescent beetles. Optical modelling suggests that the multi-layers produce colour through thin-film interference, and that the sensitivity of this mechanism to slight changes in layer thickness and number explains colour variability. While coloured integumentary structures are typically thought to evolve as sexual ornaments, the blindness of golden moles suggests that the colour may be an epiphenomenon resulting from evolution via other selective factors, including the ability to move and keep clean in dirt and sand.     

Intraspecific Variation in the Vocalizations and Hand Pad Morphology of Southern Lesser Bush Babies (Galago moholi): A Comparison with G. senegalensis

Previous studies have shown the taxonomic value of vocal repertoires and hand (volar) pad characteristics in the classification of cryptic nocturnal primates such as bush babies. However, no study included quantitative comparisons within the geographical range of any one species. We investigated levels of intraspecific variation in calls and hand pad characteristics of the southern lesser bush baby (Galago moholi), using the northern lesser bush baby (Galago senegalensis) for interspecific comparisons. Examination of calls recorded from different regions along a transect of 1500 km across southern Africa revealed low levels of intraspecific variation in Galago moholi, whereas comparisons with homologous call-types in G. senegalensis revealed them to be significantly different. Volar pad measurements across the ranges of both species also showed low levels of intraspecific variation and relatively high interspecific variation. These findings demonstrate that vocal and volar pad characteristics can be used as consistent measures of difference between species that look almost identical. These methods provide a practical means of distinguishing between cryptic species, whether in the field, in captivity, or, in the case of volar pads, of preserved specimens.     

Morphological relationships between vertebrate claw unguals and sheaths and the functional morphology of these structures

The link between claw morphology and function has been historically difficult to quantify, analyze, and interpret. A confounding factor is the ambiguous morphological relationship between the ungual and the sheath and whether one structure or the other is more useful for inferring function from morphology. In this study, the functional morphology of vertebrate claws is analyzed using sheath and ungual measurements taken from modern claw specimens spanning birds and mammals. Claw measurements were chosen for their potential biomechanical significance and a revised, expanded categorization of claw function is used. When corresponding claw measurements from the ungual and sheath are compared independently, some features are highly correlated whereas others are not. A principal component analysis of the claw measurements reveals that some of the morphological disparity is related to functional differences; however, different functional categories are not clearly separated based solely on morphology. A linear discriminant analysis incorporating a supervised dimensionality reduction method (J-function) successfully classifies 94.52% of the claw specimens to their documented functional categories. When the posterior probabilities of each classification are examined, and the next highest probabilities are considered, the analysis can successfully classify 98.63% of the claw specimens. Sheath measurements perform better than ungual measurements but combining measurements from both structures perform better than considering either structure individually. Both structures contribute valuable morphological information when it comes to inferring claw function from morphology.     

Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis

Epithelial-mesenchymal feedback signaling is the key to diverse organogenetic processes such as limb bud development and branching morphogenesis in kidney and lung rudiments. This study establishes that the BMP antagonist gremlin (Grem1) is essential to initiate these epithelial-mesenchymal signaling interactions during limb and metanephric kidney organogenesis. A Grem1 null mutation in the mouse generated by gene targeting causes neonatal lethality because of the lack of kidneys and lung septation defects. In early limb buds, mesenchymal Grem1 is required to establish a functional apical ectodermal ridge and the epithelial-mesenchymal feedback signaling that propagates the sonic hedgehog morphogen. Furthermore, Grem1-mediated BMP antagonism is essential to induce metanephric kidney development as initiation of ureter growth, branching and establishment of RET/GDNF feedback signaling are disrupted in Grem1-deficient embryos. As a consequence, the metanephric mesenchyme is eliminated by apoptosis, in the same way as the core mesenchymal cells of the limb bud.     

Evolution of the genomic rate of recombination in mammals

Rates of recombination vary considerably between species. Despite the significance of this observation for evolutionary biology and genetics, the evolutionary mechanisms that contribute to these interspecific differences are unclear. On fine physical scales, recombination rates appear to evolve rapidly between closely related species, but the mode and tempo of recombination rate evolution on the broader scale is poorly understood. Here, we use phylogenetic comparative methods to begin to characterize the evolutionary processes underlying average genomic recombination rates in mammals. We document a strong phylogenetic effect in recombination rates, indicating that more closely related species tend to have more similar average rates of recombination. We demonstrate that this phylogenetic signal is not an artifact of errors in recombination rate estimation and show that it is robust to uncertainty in the mammalian phylogeny. Neutral evolutionary models present good fits to the data and we find no evidence for heterogeneity in the rate of evolution in recombination across the mammalian tree. These results suggest that observed interspecific variation in average genomic rates of recombination is largely attributable to the steady accumulation of neutral mutations over evolutionary time. Although single recombination hotspots may live and die on short evolutionary time scales, the strong phylogenetic signal in genomic recombination rates indicates that the pace of evolution on this scale may be considerably slower.     

The angular process of Monodelphis domestica (Didelphidae, Marsupialia) and its relation to the middle ear: an ontogenetic and evolutionary morphologic study

In most marsupials, the angular process is inflected medially. By using an ontogenetic series of Monodelphis domestica, the development of this characteristic structure has been described. In contrast with the eutherian mammals, in marsupials there is retained a close connection between the dentale and the tympanicum and goniale; it is well known that these 2 elements of the middle ear are derived from the angulare and prearticulare of the reptilian lower jaw. At the neonatal stage, the dentale and tympanicum are both relatively vertically orientated; during the following 2 weeks, they take an increasingly oblique position, which is primarily caused by the rapid growth of the braincase. Only after the eruption of the first teeth, the ascending ramus of the dentale takes a more and more vertical position, whereas the angular process remains with its tip near the medioventral floor of the tympanic bulla. The bulla shows at this place a rectangular fenestra which is covered by a membrane of loose connective tissue; the tip of the angular process, which is always free of muscular insertions, maintains contacts with this fenestra throughout life. During juvenile and adult life stages, the process becomes somewhat removed from the fenestra for obvious reasons, but at a gape of about 40 to 50 degrees it inevitably must touch the "inferior tympanic membrane" and possibly also the tympanic ring. It is speculated that the relationship between the angular process and the tympanic bulla represents a specific form-function complex for sound transmission, which may be a modified retention from archaic mammalian conditions. Further details of the ontogenetic development of the tympanic region have been described which may be of some relevance for the evolutionary morphology of mammals: The tympanic process of the petrosal, which fixes the posterior end of the tympanic ring, is formed by 'Zuwachsknochen' (additional bone) but not by cartilage. The styloid process remains cartilaginous throughout life: its free tip ends in the lateral wall of the tympanic cavity and it is closely connected with the collum mallei and the posterior end of the tympanicum; it guides the chorda tympani and may therefore be homologous with the cartilage of Spence. The cartilage of Paauw is interpreted in terms of functional morphology. A model of evolutionary transformation of the dentale-tympanicum complex in mesozoic mammals in outlined on the basis of the ontogenetic findings in Monodelphis and other didelphid and dasyurid marsupials.     

Some distal limb structures develop in mice lacking Sonic hedgehog signaling

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.     

Wls-mediated Wnts differentially regulate distal limb patterning and tissue morphogenesis

Wnt proteins are diffusible morphogens that play multiple roles during vertebrate limb development. However, the complexity of Wnt signaling cascades and their overlapping expression prevent us from dissecting their function in limb patterning and tissue morphogenesis. Depletion of the Wntless (Wls) gene, which is required for the secretion of various Wnts, makes it possible to genetically dissect the overall effect of Wnts in limb development. In this study, the Wls gene was conditionally depleted in limb mesenchyme and ectoderm. The loss of mesenchymal Wls prevented the differentiation of distal mesenchyme and arrested limb outgrowth, most likely by affecting Wnt5a function. Meanwhile, the deletion of ectodermal Wls resulted in agenesis of distal limb tissue and premature regression of the distal mesenchyme. These observations suggested that Wnts from the two germ layers differentially regulate the pool of undifferentiated distal limb mesenchyme cells. Cellular behavior analysis revealed that ectodermal Wnts sustain mesenchymal cell proliferation and survival in a manner distinct from Fgf. Ectodermal Wnts were also shown for the first time to be essential for distal tendon/ligament induction, myoblast migration and dermis formation in the limb. These findings provide a comprehensive view of the role of Wnts in limb patterning and tissue morphogenesis.