Loss of carpal elements in crocodilian limb evolution: morphogenetic model corroborated by palaeobiological data

The comparison of bone homology between the manus of an Early Cretaceous fossil crocodile and that of the extant species Alligator mississippiensis supports explicidy, for the first time, the hypothesis of carpal loss in crocodilian limb evolution. This hypothesis, based on a developmental model of the organization of the tetrapod limb, is in accordance with the fossil evidence, and may supersede traditional Haeckelian views based on recapitulatory paradigms. The homologous relationships of carpal elements reveal the existence of two carpal patterns- one plesiomorphic and one apomorphic-in the crocodilian lineage. Phylogenetic change is explained causally by alterations of the osteogenesis of the distal carpals 2 and 3, which remain unossified in extant crocodile adults. This implies that crocodilian limb evolution is constrained by a process of paedomorphosis. This modification of the architecture of the crocodilian hand is a terminal event of its evolutionary history, affecting only eusuchian crocodiles. The results of this study contest the traditional view that the skeletal pattern of the crocodilian limb has been conserved unchanged since the Triassic.     

Appendicular skeleton in Bachia bicolor (Squamata: Gymnophthalmidae): osteology,limb reduction and postnatal skeletal ontogeny

The osteology of the appendicular skeleton and its postnatal development are described in Bachia bicolor, a serpentiform lizard with reduced limbs. The pectoral girdle is well developed and the forelimb consists of a humerus, ulna, radius, five carpal elements (ulnare, radiale, distal carpals 4–3, centrale), four metacarpals (II, III, IV, V) and phalanges (phalangeal formula X‐2‐2‐2‐2). In the hindlimb, the femur is small and slender, and articulates distally with a series of ossified amorphous and extremely reduced elements that correspond to a fibula, tibia and proximal and distal tarsals 4 and 3. The pelvic girdle consists of ischium, pubis and ilium, but its two halves are widely separated; the ilium is the least reduced element. We describe the ossification and development during postnatal skeletal ontogeny, especially of epiphyseal secondary centres, ossifications of carpal elements, apophyseal ossifications and sesamoids. Compared to other squamates, B. bicolor shows an overall reduction in limb size, an absence of skeletal elements, a fusion of carpal elements, an early differentiation of apophyseal centres, and a low number of sesamoids and apophyseal centres. These observations suggest that the reductions are produced by heterochronic changes during postnatal development and probably during embryonic development; therefore the appendicular skeleton exhibits a pattern of paedomorphic features.     

Patterns of chondrogenesis and calcification in the developing limb of the lizard,Calotes versicolor

The first skeletal condensation appears deep at the base of the limb bud near the somites, when the apical ectodermal ridge (AER) is maximally developed. Thereafter the skeletal elements generally appear in a proximodistal sequence but most of the mesopodial cartilages appear well after the metapodial ones and one of them, tarsalia-1, even after the phalangeal ones. The skeletal elements that fuse or “disappear” during the development are the cartilaginous condensation of fibulare, and the precartilaginous condensation of the distal centrale in the tarsus, and possibly the mesenchymatous condensation of the intermedium in the carpus. The calcification of all the long cartilages is perichondral and osseous while that of all the mesopodial and other cartilages, like epiphyses and sesamoids, is endochondral and nonosseous except the partly osseous astragalus and fibulare. The limbs of the mature adult have many sesamoids and metaplastic calcifications. The AER starts regressing after the appearance of the first skeletal condensation but is retained on the digital tips, though in a moderately regressed condition, almost till the time of the appearance of all the phalangeal condensations. These studies on the mesopodium differ with most studies on reptilian and avian mesopodia in favoring the view that very few skeletal condensations fuse or disappear during the development. They thus raise important issues concerning the ontogeny and phylogeny of the pentadactyl limb. While the AER has a substantial role in the limb morphogenesis, it most probably is not responsible for the information to mesoderm regarding the number, size, shape and relative position of the skeletal elements in the limb.     

Analysis of highly phosphorylated inositols in avian and crocodilian erythrocytes

Both morphological and paleontological characteristics support the hypothesis of a monophyletic origin of crocodilian and avian groups. However, while the erythrocytes of all birds studied to date are reported to contain high levels of inositol pentakisphosphate (InsP(5)), which acts as an allosteric effector of hemoglobin, this molecule has not been reported in crocodilian erythrocytes. In this study we compare the highly phosphorylated inositols in crocodilian and avian erythrocytes using a particularly sensitive analytical procedure. Our aim was to obtain new data which might provide further evidence for the monophyletic origin, or otherwise, of crocodiles and birds. We studied three avian and three crocodilian species. The erythrocytes of the three bird species contained low levels of inositol-3,4,5,6-tetrakisphosphate and inositol-1,3,4,6-tetrakisphosphate, thought to be precursors of Ins(1,3,4,5,6)P(5). The crocodilian erythrocytes studied contained Ins(1,3,4,5,6)P(5) and InsP(6) in higher concentrations than those found in mammal erythrocytes and in other more active cells such as macrophages. Our data provide further evidence of the similarity between crocodilian and avian groups and agree with the hypothesis that both groups evolved from a common ancestor. The process by which the function of inositol phosphates changed from that of intracellular signaling to hemoglobin allosteric effector is discussed.     

Postnatal allometry of the skeleton in Tupaia glis (Scandentia: Tupaiidae) and Galea musteloides (Rodentia: Caviidae)--a test of the three-segment limb hypothesis

During the evolution of therian mammals, the two-segmented, sprawled tetrapod limbs were transformed into three-segmented limbs in parasagittal zig-zag configuration (three-segment limb hypothesis). As a consequence, the functional correspondence of limb segments has changed (now: scapula to thigh, upper arm to shank, fore arm plus hand to foot). Therefore, the scapula was taken into account in the current study of the postnatal growth of the postcranial skeleton in two small mammalian species (Tupaia glis, Galea musteloides). Comparisons were made between the functionally equivalent elements and not in the traditional way between serially homologous segments. This study presents a test of the three-segment limb hypothesis which predicts a greater ontogenetic congruence in the functionally equivalent elements in fore and hind limbs than in the serially homologous elements. A growth sequence, with decreasing regression coefficients from proximal to distal, was observed in both species under study. This proximo-distal growth sequence is assumed to be ancestral in the ontogeny of eutherian mammals. Different reproductive modes have evolved within eutherian mammals. To test the influence of different life histories on ontogenetic scaling during postnatal growth, one species with altricial juveniles (Tupaia glis) assumed to be the ancestral mode of development for eutherians and one species with derived, precocial young (Galea musteloides) were selected. The growth series covered postnatal development from the first successive steps with a lifted belly to the adult locomotory pattern; thus, functionally equivalent developmental stages were compared. The higher number of allometrically positive or isometrically growing segments in the altricial mammalian species was interpreted as a remnant of the fast growth period in the nest without great locomotor demands, and the clearly negative allometry in nearly all segments in the precocial young was interpreted as a response to the demand on early locomotor activity. Different life histories seem to have a strong influence on postnatal ontogenetic scaling; the effects of the developmental differences are still observable when comparing adults of the two species.     

Ossification patterns in the tetrapod limb – conservation and divergence from morphogenetic events

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore‐ and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step‐by‐step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore‐ and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.     

Comparative ossification sequence and skeletal development of the postcranium of palaeognathous birds (Aves: Palaeognathae)

Palaeognaths constitute one of the most basal lineages of extant birds, and are also one of the most morphologically diverse avian orders. Their skeletal development is relatively unknown, in spite of their important phylogenetic position. Here, we compare the development of the postcranial skeleton in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana) and elegant crested‐tinamou (Eudromia elegans), focusing on ossification. All of these taxa are characterized by element loss in the appendicular skeleton, but there are several developmental mechanisms through which this loss occurs, including failure to chondrify, failure to ossify and fusion of cartilages prior to ossification. Further evidence is presented here to support a reduction in size of skeletal elements resulting in a delay in the timing of ossification. This study provides an important first look at the timing and sequence of postcranial ossification in palaeognathous birds, and discusses the influence of changes in the pattern of skeletal development on morphological evolution.     

Exploring the Relationship between Skeletal Mass and Total Body Mass in Birds

Total body mass (TBM) is known to be related to a number of different osteological features in vertebrates, including limb element measurements and total skeletal mass. The relationship between skeletal mass and TBM in birds has been suggested as a way of estimating the latter in cases where only the skeleton is known (e.g., fossils). This relationship has thus also been applied to other extinct vertebrates, including the non-avian pterosaurs, while other studies have used additional skeletal correlates found in modern birds to estimate TBM. However, most previous studies have used TBM compiled from the literature rather than from direct measurements, producing values from population averages rather than from individuals. Here, we report a new dataset of 487 extant birds encompassing 79 species that have skeletal mass and TBM recorded at the time of collection or preparation. We combine both historical and new data for analyses with phylogenetic control and find a similar and well-correlated relationship between skeletal mass and TBM. Thus, we confirm that TBM and skeletal mass are accurate proxies for estimating one another. We also look at other factors that may have an effect on avian body mass, including sex, ontogenetic stage, and flight mode. While data are well-correlated in all cases, phylogeny is a major control on TBM in birds strongly suggesting that this relationship is not appropriate for estimating the total mass of taxa outside of crown birds, Neornithes (e.g., non-avian dinosaurs, pterosaurs). Data also reveal large variability in both bird skeletal and TBM within single species; caution should thus be applied when using published mass to test direct correlations with skeletal mass and bone lengths.     

Morphological integration versus ecological plasticity in the avian pelvic limb skeleton

Understanding patterns and distributions of morphological traits is essential for discerning underpinning processes of morphological variation. We report on the variation in the avian pelvic limb skeleton. Length and width variables were measured in the skeletons of 236 avian species in order to examine the importance of body mass, ecological factors, phylogeny and integration in the formation of specific hindlimb morphology. Scaling relationships with body mass were analyzed across Aves and in individual avian subclades. Principal component analysis and multiple regressions were performed to examine the relationship between morphology, ecology, and phylogeny. Finally, the occurrence of within‐limb morphological integration was tested by partial correlation analysis of the residuals from element lengths vs. body mass and correlation analysis of avian hindlimb proportions. Body mass is the greatest contributor to variation, and it strongly influences variation in avian skeletal lengths. Lengthening of the leg typically comes from disproportionate increases in tibiotarsal and tarsometatarsal length. Partial correlation analysis showed that only these two elements are distinctly integrated consistently across all bird taxa, whereas relation of femur and third toe to other limb elements displays no clear pattern. Hence, morphological integration of all elements is not a prerequisite for limb design, and variation between taxa is mainly to be found in femoral and digital length. Furthermore, variation in tibiotarsal relative length is much lower than in other elements likely due to geometric constrains. Clear ecological adaptations are obscured by multifunctionality of the avian hindlimb, and phylogeny significantly constrains the morphology. Finally, when looking at relative lengths segmented limbs meet the requirements of many‐to‐one‐mapping of phenotype to functional property, in line with a common concept of evolvability of function and morphology. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Dlx5 regulates chondrocyte differentiation at multiple stages

Endochondral ossification, in which cartilaginous templates are progressively replaced by marrow and bone, represents the dominant mode of development of the axial and appendicular skeleton of vertebrates. Chondrocyte differentiation within the cartilaginous core of these skeletal elements is tightly regulated, both spatially and temporally. Here, we describe the expression of Dlx5 in the cartilaginous core of limb skeletal elements in chicken and mouse embryos. We find that Dlx5 is one of the earliest genes expressed in condensing limb mesenchyme that will give rise to the limb skeleton. Later, when proliferating and differentiating chondrocytes are found in spatially distinct regions of the cartilaginous model, Dlx5 is expressed in the zone of hypertrophy and in proliferating chondrocytes that are poised to differentiate. Consistent with this pattern of expression, we show that forced expression of Dlx5 potentiates early and late chondrocyte differentiation and inhibits proliferation in cultured cells. Examination of the limbs of mutant Dlx5 mouse embryos revealed that they displayed a delay in chondrocyte maturation compared with wild type littermates. Taken together, our data reveal a positive role for Dlx5 during multiple stages of chondrocyte differentiation and, along with previous studies of Dlx5 and osteogenesis, identify Dlx5 as a general regulator of differentiation in the mouse skeleton.     

Agenesis of the scapula in Emx2 homozygous mutants

The shoulder and pelvic girdles represent the proximal bones of the appendicular skeleton that connect the anterior and posterior limbs to the body trunk. Although the limb is a well-known model in developmental biology, the genetic mechanisms controlling the development of the more proximal elements of the appendicular skeleton are still unknown. The knock-out of Pax1 has shown that this gene is involved in patterning the acromion, while the expression pattern candidates Hoxc6 as a gene involved in scapula development. Surprisingly, we have found that scapula and ilium do not develop in Emx2 knock-out mice. In the homozygous mutants, developmental abnormalities of the brain cortex, the most anterior structure of the primary axis of the body, are associated with important defects of the girdles, the more proximal elements of the secondary axis. These abnormalities suggest that the molecular mechanisms patterning the more proximal elements of the limb axis are different from those patterning the rest of appendicular skeleton. While Hox genes specify the different segments of the more distal part of the appendicular skeleton forming the limb, Emx2 is concerned with the more proximal elements constituting the girdles.     

Coevolution of caudal skeleton and tail feathers in birds

Birds are capable of a wide range of aerial locomotor behaviors in part because of the derived structure and function of the avian tail. The tail apparatus consists of a several mobile (free) caudal vertebrae, a terminal skeletal element (the pygostyle), and an articulated fan of tail feathers that may be spread or folded, as well as muscular and fibroadipose structures that facilitate tail movements. Morphological variation in both the tail fan and the caudal skeleton that supports it are well documented. The structure of the tail feathers and the pygostyle each evolve in response to functional demands of differing locomotor behaviors. Here, I test whether the integument and skeleton coevolve in this important locomotor module. I quantified feather and skeletal morphology in a diverse sample of waterbirds and shorebirds using a combination of linear and geometric morphometrics. Covariation between tail fan shape and skeletal morphology was then tested using phylogenetic comparative methods. Pygostyle shape is found to be a good predictor of tail fan shape (e.g., forked, graduated), supporting the hypothesis that the tail fan and the tail skeleton have coevolved. This statistical relationship is used to reconstruct feather morphology in an exemplar fossil waterbird, Limnofregata azygosternon. Based on pygostyle morphology, this taxon is likely to have exhibited a forked tail fan similar to that of its extant sister clade Fregata, despite differing in inferred ecology and other aspects of skeletal anatomy. These methods may be useful in reconstructing rectricial morphology in other extinct birds and thus assist in characterizing the evolution of flight control surfaces in birds. J. Morphol. 275:1431–1440, 2014. © 2014 Wiley Periodicals, Inc.     

Flapping before Flight: High Resolution,Three-Dimensional Skeletal Kinematics of Wings and Legs during Avian Development

Some of the greatest transformations in vertebrate history involve developmental and evolutionary origins of avian flight. Flight is the most power-demanding mode of locomotion, and volant adult birds have many anatomical features that presumably help meet these demands. However, juvenile birds, like the first winged dinosaurs, lack many hallmarks of advanced flight capacity. Instead of large wings they have small “protowings”, and instead of robust, interlocking forelimb skeletons their limbs are more gracile and their joints less constrained. Such traits are often thought to preclude extinct theropods from powered flight, yet young birds with similarly rudimentary anatomies flap-run up slopes and even briefly fly, thereby challenging longstanding ideas on skeletal and feather function in the theropod-avian lineage. Though skeletons and feathers are the common link between extinct and extant theropods and figure prominently in discussions on flight performance (extant birds) and flight origins (extinct theropods), skeletal inter-workings are hidden from view and their functional relationship with aerodynamically active wings is not known. For the first time, we use X-ray Reconstruction of Moving Morphology to visualize skeletal movement in developing birds, and explore how development of the avian flight apparatus corresponds with ontogenetic trajectories in skeletal kinematics, aerodynamic performance, and the locomotor transition from pre-flight flapping behaviors to full flight capacity. Our findings reveal that developing chukars (Alectoris chukar) with rudimentary flight apparatuses acquire an “avian” flight stroke early in ontogeny, initially by using their wings and legs cooperatively and, as they acquire flight capacity, counteracting ontogenetic increases in aerodynamic output with greater skeletal channelization. In conjunction with previous work, juvenile birds thereby demonstrate that the initial function of developing wings is to enhance leg performance, and that aerodynamically active, flapping wings might better be viewed as adaptations or exaptations for enhancing leg performance.     

The evolutionary transformation of phyllopodous to stenopodous limbs in the Branchiopoda (Crustacea)--is there a common mechanism for early limb development in arthropods?

Arthropods and in particular crustaceans show a great diversity concerning their limb morphology. This makes the homologization of limbs and their parts and our understanding of evolutionary transformations of these limb types problematical. To address these problems we undertook a comparative study of the limb development of two representatives of branchiopod crustaceans, one with phyllopodous the other with stenopodous trunk limbs. The trunk limb ontogeny of a 'larger branchiopod', Cyclestheria hislopi ('Conchostraca') and the raptorial cladoceran Leptodora kindtii (Haplopoda) has been examined by various methods such as SEM, Hoechst fluorescent stain and expression of the Distal-less gene. The early ontogeny of the trunk limbs in C. hislopi and L. kindtii is similar. In both species the limbs are formed as ventrally placed, elongate, subdivided limb buds. However, in C. hislopi, the portions of the early limb bud end up constituting the endites and the endopod of the phyllopodous filtratory limb in the adult, whereas in L. kindtii, similar limb bud portions end up constituting the actual segments in the segmented, stenopodous, and raptorial trunk limbs of the adults. Hence, the portions of the limbs corresponding to the endites of the phyllopodous trunk limbs in C. hislopi (and other 'larger branchiopods') are homologous to the segments of the stenopodous trunk limbs in L. kindtii. It is most parsimonious to assume that the segmented trunk limbs in L. kindtii have developed from phyllopodous limbs with endites and not vice versa. This study has demonstrated at least one way in which segmented limbs have been derived from phyllopodous, multi-lobate limbs during evolution. Similar pathways can be assumed for the evolution of stenopodous, segmented and uniramous limbs in other crustaceans. Irrespective of the differences in the adult limb morphology, the early patterning of arthropod limbs seems to follow a similar principle.     

Sequential changes in weight of the skeleton and in length of long limb bones of Macaca mulatta.

In a collection of 274 monkeys (Macaca mulatta) the relative weight of the dry, fat-free skeleton, expressed as a proportion of total body weight, increases significantly throughout the gestational period to approximately 6% with only random variation after birth. The weight of the fetal skeleton increases exponentially with age. In the postnatal period the skeletal weight increases asymptotically to adulthood, which is considered to be 6.5 years of age. Equations for estimating skeletal weight are presented. Of four subdivisions of the skeleton, the skul contributes the greatest proportion of total skeletal weight in the fetal stage with the proportion decreasing to adulthood. The contributions of the other subdivisions, postcranial axial, superior limb, and inferior limb, and inferior limb, are nearly equal in the fetal stage, with that of only the inferior limb increasing to adulthood, when it makes up the greatest proportion of total skeletal weight. Until the last third of the gestational period, the humerus is longer than the femur and the radius longer than the tibia. Thereafter, the inferior limbs grow at a faster rate than the superior limbs, resulting in an intermembral index of approximately 95% by birth and less than 90% by adulthood.     

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.     

SEM studies on the early larval development of Triops cancriformis (Bosc)(Crustacea: Branchiopoda, Notostraca)

We investigated early larval development in the notostracan Triops cancriformis (Bosc, 1801–1802) raised from dried cysts under laboratory conditions. We document the five earliest stages using scanning electron microscopy. The stage I larva is a typical nauplius, lecithotropic and without trunk limbs. The stage II larva is feeding and has trunk limb precursors and a larger carapace. Stage III larvae have larger trunk limbs and a more adult shape. Stage IV larvae have well developed trunk limbs, and stage V larvae show atrophy of the antennae. We describe the ontogeny of selected features such as trunk limbs and carapace, discuss ontogeny and homologization of head appendages, follow the development of the feeding mechanism, and discuss trunk limb ontogeny.     

The patterns and modes of the evolution of disparity in Mesozoic birds

The origin of birds from non-avian theropod dinosaurs is one of the greatest transitions in evolution. Shortly after diverging from other theropods in the Late Jurassic, Mesozoic birds diversified into two major clades—the Enantiornithes and Ornithuromorpha—acquiring many features previously considered unique to the crown group along the way. Here, we present a comparative phylogenetic study of the patterns and modes of Mesozoic bird skeletal morphology and limb proportions. Our results show that the major Mesozoic avian groups are distinctive in discrete character space, but constrained in a morphospace defined by limb proportions. The Enantiornithines, despite being the most speciose group of Mesozoic birds, are much less morphologically disparate than their sister clade, the Ornithuromorpha—the clade that gave rise to living birds, showing disparity and diversity were decoupled in avian history. This relatively low disparity suggests that diversification of enantiornithines was characterized in exhausting fine morphologies, whereas ornithuromorphs continuously explored a broader array of morphologies and ecological opportunities. We suggest this clade-specific evolutionary versatility contributed to their sole survival of the end-Cretaceous mass extinction.     

Loss of FGF receptor 1 signaling reduces skeletal muscle mass and disrupts myofiber organization in the developing limb

The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. We asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the adjacent somites of Hamburger and Hamilton (HH) stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of dnFGFR1. Within 48 h following injection of dnFGFR1, we could detect no obvious changes in skeletal muscle precursor cell migration into the hind limb as compared to control limbs infected with an empty RCAN virus. However, by 3 days following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50% compared to controls there were no detectable effects on myofiber diameter. The loss in myofiber density was, however, accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It is noteworthy that long-bone development was unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3 signaling was not disrupted. Our data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast number and plays a role in myofiber organization.