Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic ... more Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic development, both skeletal elements start out with similar lengths. We examined molecular markers of cartilage differentiation in chicken embryos. We found that the distal end of the fibula expresses Indian Hedgehog (IHH), undergoing terminal cartilage differentiation, and almost no Parathyroid-related-protein (PTHrP), which is required to develop a proliferative growth plate (epiphysis). Reduction of the distal fibula may be influenced earlier by its close contact with the nearby fibulare, which strongly expresses PTHrP. The epiphysis-like fibulare however then separates from the fibula, which fails to maintain a distal growth plate, and fibular reduction ensues. Experimental downregulation of IHH signaling at a post-morphogenetic stage led to a tibia and fibula of equal length: The fibula is longer than in controls and fused to the fibulare, while the tibia is shorter and bent. We propose that the presence of a distal fibular epiphysis may constrain greater growth in the tibia. Accordingly, many Mesozoic birds show a fibula that has lost its distal epiphysis, but remains almost as long as the tibia, suggesting that loss of the fibulare preceded and allowed subsequent evolution of great fibulo-tibial disparity
The anklebone (astragalus) of dinosaurs presents a characteristic upward projection, the ‘ascendi... more The anklebone (astragalus) of dinosaurs presents a characteristic upward projection, the ‘ascending process’ (ASC). The ASC is present in modern birds, but develops a separate ossification centre, and projects from the calcaneum in most species. These differences have been argued to make it non-comparable to dinosaurs. We studied ASC development in six different orders of birds using traditional techniques and spin–disc microscopy for whole-mount immunofluorescence. Unexpectedly, we found the ASC derives from the embryonic intermedium, an ancient element of the tetrapod ankle. In some birds it comes in contact with the astragalus, and, in others, with the calcaneum. The fact that the intermedium fails to fuse early with the tibiale and develops an ossification centre is unlike any other amniotes, yet resembles basal, amphibian-grade tetrapods. The ASC originated in early dinosaurs along changes to upright posture and locomotion, revealing an intriguing combination of functional innovation and reversion in its evolution.
Within developmental biology, the digits of the wing of birds are considered on
embryological gro... more Within developmental biology, the digits of the wing of birds are considered on embryological grounds to be digits 2, 3 and 4. In contrast, within paleontology, wing digits are named 1, 2, 3 as a result of phylogenetic analysis of fossil taxa indicating that birds descended from theropod dinosaurs that had lost digits 4 and 5. It has been argued that the development of the wing does not support the conclusion that birds are theropods, and that birds must have descended from ancestors that had lost digits 1 and 5. Here we use highly conserved gene expression patterns in the developing limbs of mouse and chicken, including the chicken talpid2 mutant and polydactylous Silkie breed (Silkie mutant), to aid the assessment of digital identity in the wing. Digit 1 in developing limbs does not express Hoxd12, but expresses Hoxd13. All other digits express both Hoxd12 and Hoxd13. We found this signature expression pattern identifies the anteriormost digit of the wing as digit 1, in accordance with the hypothesis these digits are 1, 2 and 3, as in theropod dinosaurs. Our evidence contradicts the long-standing argument that the development of the wing does not support the hypothesis that birds are living dinosaurs
From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ... more From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal–anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal–anterior ossification does not support the dinosaur–bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal–posterior ossification develops from a cartilage referred to as “element x,” but its position corresponds to distal carpal 3. The proximal–posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal–posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.
Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the
... more Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation
Specialized morphologies of bird feet have
evolved several times independently as different group... more Specialized morphologies of bird feet have evolved several times independently as different groups have become zygodactyl, semi-zygodactyl, heterodactyl, pamprodactyl or syndactyl. Birds have also convergently evolved similar modes of development, in a spectrum that goes from precocial to altricial. Using the new context provided by recent molecular phylogenies, we compared the evolution of foot morphology and modes of development among extant avian families. Variations in the arrangement of toes with respect to the anisodactyl ancestral condition have occurred only in altricial groups. Those groups represent four independent events of super-altriciality and many independent transformations of toe arrangements (at least four zygodactyl, three semi-zygodactyl, one heterodactyl, one pamprodactyl group, and several syndactyl). We propose that delayed skeletal maturation due to altriciality facilitates the epigenetic influence of embryonic muscular activity over developing toes, allowing for repeated evolution of innovations in their morphology.
Fossil evidence documenting the evolutionary transition from theropod dinosaurs to birds indicate... more Fossil evidence documenting the evolutionary transition from theropod dinosaurs to birds indicates unambiguously that the digits of the wing of birds are digits 1, 2, and 3. However, some embryological evidence suggests that these digits are 2, 3, and 4. This apparent lack of correspondence has been described as the greatest challenge to the widely accepted theropod–bird link (Zhou 2004. Naturwissenschaften 91:455–471). Here we review the pertinent literature regarding the debate on the origin of birds and wing digital identity and the evidence in favor of a 1, 2, 3 identity of the wing digits. Recent molecular evidence shows that the expression of Hoxd12 and Hoxd13 in the developing wing supports the theropod–bird link. In the chicken foot and in the mouse hand and foot, digit 1 is the only digit to combine the expression of Hoxd13 with the absence of expression of Hoxd12. The same is observed in the anterior digit of the wing, suggesting it is a digit 1, as expected for a theropod. Nevertheless, Galis et al. (2005. J Exp Zool (Mol Dev Evol) in press), argue that Hoxd12 and Hoxd13 expression patterns in mutant limbs do not allow distinguishing the most anterior digit in the bird wing from digit 2. They also argue that constraints to the evolution of limb development support the 2, 3, 4 identity of the wing digits. However, the case put forward by Galis et al. is biased and flawed with regard to interpretation of mutant limbs, developmental mechanisms, stages observed, and the description of the evolutionary variation of limb development. Importantly, Galis et al. do not present evidence from wild-type limbs that counters the conclusions of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B(1):85–89), and fail to provide molecular evidence to specifically support the hypothesis that the wing digits are 2, 3, and 4. The expression of Hoxd12 and Hoxd13 in the developing wing is consistent with the hypothesis that birds are living dinosaurs; this view can lead to a greater understanding of the actual limits to the evolutionary variation of limb development
Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but t... more Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but they develop at embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11 expression in crocodilians, the closest living relatives of birds. Methodology/Principal Findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1. Conclusions/Significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.
Among theropod species the arm tends to become proportionately smaller as size increases. (Humeru... more Among theropod species the arm tends to become proportionately smaller as size increases. (Humerus=0.3876femur"8°7, R(r2)=0.89). This trend may reflect common descent from an ancestor in which the arm grew alometrically slower than the body. Conservation of this developmental trend could have been allowed in large theropods since arms not functional in predation are compensated by a large body size. This hypothesis predicts the fossil rec-ord will show developmental sequences of theropods in which earlier stages show arms proportionately longer than those of later stages.
Background: The homology of the digits in the bird wing is a high-profile controversy in developm... more Background: The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. Results: We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Conclusion: Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.
The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing... more The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing backwards) evolved independently in different extant bird taxa. To understand the origin of this trait in modern birds, we investigated the development of the zygodactyl foot of the budgerigar (Psittaciformes). We compared its muscular development with that of the anisodactyl quail (Galliformes) and show that while the musculus abductor digiti IV (ABDIV) becomes strongly developed at HH36 in both species, the musculus extensor brevis digiti IV (EBDIV) degenerates and almost disappears only in the budgerigar. The asymmetric action of those muscles early in the devel- opment of the budgerigar foot causes retroversion of digit IV (dIV). Paralysed budgerigar embryos do not revert dIV and are anisodactyl. Both molecular phylogenetic analysis and palaeontological information suggest that the ancestor of passerines could have been zygodactyl. We followed the development of the zebra finch (Passeriformes) foot muscles and found that in this species, both the primordia of the ABDIV and of the EBDIV fail to develop. These data suggest that loss of asymmetric forces of muscular activity exerted on dIV, caused by the absence of the ABDIV, could have resulted in secondary anisodactyly in Passeriformes.
A highly conserved spatio-temporal pattern
of cartilage formation reveals that the digits of the ... more A highly conserved spatio-temporal pattern of cartilage formation reveals that the digits of the bird wing develop from positions that become digits 2, 3, and 4 in other amniotes. However, the morphology of the digits of early birds like Archaeopteryx corresponds to that of digits 1, 2, and 3 of other archosaurs. A hypothesis is that a homeotic ‘‘frameshift’’ occurred, such that in the bird wing, digits 1, 2, and 3 develop from the embryological positions of digits 2, 3, and 4. Experimental homeotic transformations of single digits are well-documented, but frame-shifts of more than one digit are not. We investigated the pattern of cartilage formation in the development of Cyclopamine-treated wings. When Cyclopamine was applied between stages 18 and 21, morphologies that normally develop from positions 2 and 3 developed from positions 3 and 4. The serial shift of digit identity toward posterior confirms a mechanistic possibility that was previously inferred from the evolutionary history of birds.
Abstract: Limusaurus is a remarkable herbivorous ceratosaur unique among theropods in having digi... more Abstract: Limusaurus is a remarkable herbivorous ceratosaur unique among theropods in having digits II, III and IV, with only a small metacarpal vestige of digit I. This raises interesting questions regarding the controversial identity of avian wing digits. The early tetanuran ancestors of birds had tridactyl hands with digital morphologies corresponding to digits I, II & III of other dinosaurs. In bird embryos, however, the pattern of cartilage formation indicates that their digits develop from positions that become digits II, III, & IV in other ...
Digit identity in the avian wing is a classical
example of conflicting anatomical and embryologic... more Digit identity in the avian wing is a classical example of conflicting anatomical and embryological evidence regarding digit homology. Anatomical in conjunction with phylogenetic evidence supports the hypothesis that the three remaining digits in the bird wing are digits 1, 2, and 3. At the same time, various lines of embryological evidence support the notion that these digits develop in positions that normally produce digits 2, 3, and 4. In recent years, gene expression as well as experimental evidence was published that supports the hypothesis that this discrepancy arose from a digit identity shift in the evolution of the bird wing. A similar but less well-known controversy has been ongoing since the late 19th century regarding the identity of the digits of the three-toed Italian skink, Chalcides chalcides. Comparative anatomy identifies these digits as 1, 2, and 3, while embryological evidence suggests their derivation from embryological positions 2, 3, and 4. Here we re-examine this evidence and add gene expression data to determine the identity of the three digits of C. chalcides. The data confirm that the adult and the embryological evidence for digit identity are in conflict, and the expression of Hoxd11 suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4. We conclude that in C. chalcides, and likely in its close relatives, a digit identity frame shift has occurred, similar to the one in avian evolution. This result suggests that changes in of digit identity might be a more frequent consequence of digit reduction than previously assumed.
Asymmetric regulation of Hox gene expression pre-dates the appearance of tetrapod digits, and was... more Asymmetric regulation of Hox gene expression pre-dates the appearance of tetrapod digits, and was co-opted in the development of ‘thumbness’. This asymmetric expression correlates with independent morphological evolutionary variation of digit 1.
The neural crest is a unique cell population among embryonic cell types, displaying properties of... more The neural crest is a unique cell population among embryonic cell types, displaying properties of both ectodermal and mesodermal lineages. Most of the recent studies examining the neural crest have been performed in avian embryos. Only in the first half of this century were amphibians extensively used. We first summarize this important older source of information, reviewing studies made since the turn of the century. Due to the increasingly detailed in cellular and molecular knowledge of the early development of Xenopus laevis, the remainder of the review focuses on this species. We describe the route of migration and fate of the neural crest and propose a new model of neural crest induction in which prospective cells are induced independently of the neural plate by a double gradient of a morphogen that patterns the entire ectoderm. This model is also discussed in a more general context in connection with the dorsoventral patterning of the neural tube. Finally, we discuss some ideas...
Theropod dinosaurswere the dominant predators in most Mesozoic era terrestrial ecosystems1. Early... more Theropod dinosaurswere the dominant predators in most Mesozoic era terrestrial ecosystems1. Early theropod evolution is currently interpreted as the diversification of various carnivorous and cursorial taxa, whereas the acquisition of herbivorism, together with the secondary loss of cursorial adaptations, occurredmuch later among advanced coelurosaurian theropods1,2. A new, bizarre herbivorous basal tetanuran from the Upper Jurassic of Chile challenges this conception. The new dinosaur was discovered at Ayse´n, a fossil locality in the Upper Jurassic Toqui Formation of southern Chile (General Carrera Lake)3,4. The site yielded abundant and exquisitely preserved three-dimensional skeletons of small archosaurs. Several articulated individuals ofChilesaurus at different ontogenetic stages have been collected, as well as less abundant basal crocodyliforms, and fragmentary remains of sauropod dinosaurs (diplodocids and titanosaurians).
Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic ... more Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic development, both skeletal elements start out with similar lengths. We examined molecular markers of cartilage differentiation in chicken embryos. We found that the distal end of the fibula expresses Indian Hedgehog (IHH), undergoing terminal cartilage differentiation, and almost no Parathyroid-related-protein (PTHrP), which is required to develop a proliferative growth plate (epiphysis). Reduction of the distal fibula may be influenced earlier by its close contact with the nearby fibulare, which strongly expresses PTHrP. The epiphysis-like fibulare however then separates from the fibula, which fails to maintain a distal growth plate, and fibular reduction ensues. Experimental downregulation of IHH signaling at a post-morphogenetic stage led to a tibia and fibula of equal length: The fibula is longer than in controls and fused to the fibulare, while the tibia is shorter and bent. We propose that the presence of a distal fibular epiphysis may constrain greater growth in the tibia. Accordingly, many Mesozoic birds show a fibula that has lost its distal epiphysis, but remains almost as long as the tibia, suggesting that loss of the fibulare preceded and allowed subsequent evolution of great fibulo-tibial disparity
The anklebone (astragalus) of dinosaurs presents a characteristic upward projection, the ‘ascendi... more The anklebone (astragalus) of dinosaurs presents a characteristic upward projection, the ‘ascending process’ (ASC). The ASC is present in modern birds, but develops a separate ossification centre, and projects from the calcaneum in most species. These differences have been argued to make it non-comparable to dinosaurs. We studied ASC development in six different orders of birds using traditional techniques and spin–disc microscopy for whole-mount immunofluorescence. Unexpectedly, we found the ASC derives from the embryonic intermedium, an ancient element of the tetrapod ankle. In some birds it comes in contact with the astragalus, and, in others, with the calcaneum. The fact that the intermedium fails to fuse early with the tibiale and develops an ossification centre is unlike any other amniotes, yet resembles basal, amphibian-grade tetrapods. The ASC originated in early dinosaurs along changes to upright posture and locomotion, revealing an intriguing combination of functional innovation and reversion in its evolution.
Within developmental biology, the digits of the wing of birds are considered on
embryological gro... more Within developmental biology, the digits of the wing of birds are considered on embryological grounds to be digits 2, 3 and 4. In contrast, within paleontology, wing digits are named 1, 2, 3 as a result of phylogenetic analysis of fossil taxa indicating that birds descended from theropod dinosaurs that had lost digits 4 and 5. It has been argued that the development of the wing does not support the conclusion that birds are theropods, and that birds must have descended from ancestors that had lost digits 1 and 5. Here we use highly conserved gene expression patterns in the developing limbs of mouse and chicken, including the chicken talpid2 mutant and polydactylous Silkie breed (Silkie mutant), to aid the assessment of digital identity in the wing. Digit 1 in developing limbs does not express Hoxd12, but expresses Hoxd13. All other digits express both Hoxd12 and Hoxd13. We found this signature expression pattern identifies the anteriormost digit of the wing as digit 1, in accordance with the hypothesis these digits are 1, 2 and 3, as in theropod dinosaurs. Our evidence contradicts the long-standing argument that the development of the wing does not support the hypothesis that birds are living dinosaurs
From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ... more From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal–anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal–anterior ossification does not support the dinosaur–bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal–posterior ossification develops from a cartilage referred to as “element x,” but its position corresponds to distal carpal 3. The proximal–posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal–posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.
Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the
... more Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation
Specialized morphologies of bird feet have
evolved several times independently as different group... more Specialized morphologies of bird feet have evolved several times independently as different groups have become zygodactyl, semi-zygodactyl, heterodactyl, pamprodactyl or syndactyl. Birds have also convergently evolved similar modes of development, in a spectrum that goes from precocial to altricial. Using the new context provided by recent molecular phylogenies, we compared the evolution of foot morphology and modes of development among extant avian families. Variations in the arrangement of toes with respect to the anisodactyl ancestral condition have occurred only in altricial groups. Those groups represent four independent events of super-altriciality and many independent transformations of toe arrangements (at least four zygodactyl, three semi-zygodactyl, one heterodactyl, one pamprodactyl group, and several syndactyl). We propose that delayed skeletal maturation due to altriciality facilitates the epigenetic influence of embryonic muscular activity over developing toes, allowing for repeated evolution of innovations in their morphology.
Fossil evidence documenting the evolutionary transition from theropod dinosaurs to birds indicate... more Fossil evidence documenting the evolutionary transition from theropod dinosaurs to birds indicates unambiguously that the digits of the wing of birds are digits 1, 2, and 3. However, some embryological evidence suggests that these digits are 2, 3, and 4. This apparent lack of correspondence has been described as the greatest challenge to the widely accepted theropod–bird link (Zhou 2004. Naturwissenschaften 91:455–471). Here we review the pertinent literature regarding the debate on the origin of birds and wing digital identity and the evidence in favor of a 1, 2, 3 identity of the wing digits. Recent molecular evidence shows that the expression of Hoxd12 and Hoxd13 in the developing wing supports the theropod–bird link. In the chicken foot and in the mouse hand and foot, digit 1 is the only digit to combine the expression of Hoxd13 with the absence of expression of Hoxd12. The same is observed in the anterior digit of the wing, suggesting it is a digit 1, as expected for a theropod. Nevertheless, Galis et al. (2005. J Exp Zool (Mol Dev Evol) in press), argue that Hoxd12 and Hoxd13 expression patterns in mutant limbs do not allow distinguishing the most anterior digit in the bird wing from digit 2. They also argue that constraints to the evolution of limb development support the 2, 3, 4 identity of the wing digits. However, the case put forward by Galis et al. is biased and flawed with regard to interpretation of mutant limbs, developmental mechanisms, stages observed, and the description of the evolutionary variation of limb development. Importantly, Galis et al. do not present evidence from wild-type limbs that counters the conclusions of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B(1):85–89), and fail to provide molecular evidence to specifically support the hypothesis that the wing digits are 2, 3, and 4. The expression of Hoxd12 and Hoxd13 in the developing wing is consistent with the hypothesis that birds are living dinosaurs; this view can lead to a greater understanding of the actual limits to the evolutionary variation of limb development
Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but t... more Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but they develop at embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11 expression in crocodilians, the closest living relatives of birds. Methodology/Principal Findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1. Conclusions/Significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.
Among theropod species the arm tends to become proportionately smaller as size increases. (Humeru... more Among theropod species the arm tends to become proportionately smaller as size increases. (Humerus=0.3876femur"8°7, R(r2)=0.89). This trend may reflect common descent from an ancestor in which the arm grew alometrically slower than the body. Conservation of this developmental trend could have been allowed in large theropods since arms not functional in predation are compensated by a large body size. This hypothesis predicts the fossil rec-ord will show developmental sequences of theropods in which earlier stages show arms proportionately longer than those of later stages.
Background: The homology of the digits in the bird wing is a high-profile controversy in developm... more Background: The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. Results: We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Conclusion: Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.
The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing... more The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing backwards) evolved independently in different extant bird taxa. To understand the origin of this trait in modern birds, we investigated the development of the zygodactyl foot of the budgerigar (Psittaciformes). We compared its muscular development with that of the anisodactyl quail (Galliformes) and show that while the musculus abductor digiti IV (ABDIV) becomes strongly developed at HH36 in both species, the musculus extensor brevis digiti IV (EBDIV) degenerates and almost disappears only in the budgerigar. The asymmetric action of those muscles early in the devel- opment of the budgerigar foot causes retroversion of digit IV (dIV). Paralysed budgerigar embryos do not revert dIV and are anisodactyl. Both molecular phylogenetic analysis and palaeontological information suggest that the ancestor of passerines could have been zygodactyl. We followed the development of the zebra finch (Passeriformes) foot muscles and found that in this species, both the primordia of the ABDIV and of the EBDIV fail to develop. These data suggest that loss of asymmetric forces of muscular activity exerted on dIV, caused by the absence of the ABDIV, could have resulted in secondary anisodactyly in Passeriformes.
A highly conserved spatio-temporal pattern
of cartilage formation reveals that the digits of the ... more A highly conserved spatio-temporal pattern of cartilage formation reveals that the digits of the bird wing develop from positions that become digits 2, 3, and 4 in other amniotes. However, the morphology of the digits of early birds like Archaeopteryx corresponds to that of digits 1, 2, and 3 of other archosaurs. A hypothesis is that a homeotic ‘‘frameshift’’ occurred, such that in the bird wing, digits 1, 2, and 3 develop from the embryological positions of digits 2, 3, and 4. Experimental homeotic transformations of single digits are well-documented, but frame-shifts of more than one digit are not. We investigated the pattern of cartilage formation in the development of Cyclopamine-treated wings. When Cyclopamine was applied between stages 18 and 21, morphologies that normally develop from positions 2 and 3 developed from positions 3 and 4. The serial shift of digit identity toward posterior confirms a mechanistic possibility that was previously inferred from the evolutionary history of birds.
Abstract: Limusaurus is a remarkable herbivorous ceratosaur unique among theropods in having digi... more Abstract: Limusaurus is a remarkable herbivorous ceratosaur unique among theropods in having digits II, III and IV, with only a small metacarpal vestige of digit I. This raises interesting questions regarding the controversial identity of avian wing digits. The early tetanuran ancestors of birds had tridactyl hands with digital morphologies corresponding to digits I, II & III of other dinosaurs. In bird embryos, however, the pattern of cartilage formation indicates that their digits develop from positions that become digits II, III, & IV in other ...
Digit identity in the avian wing is a classical
example of conflicting anatomical and embryologic... more Digit identity in the avian wing is a classical example of conflicting anatomical and embryological evidence regarding digit homology. Anatomical in conjunction with phylogenetic evidence supports the hypothesis that the three remaining digits in the bird wing are digits 1, 2, and 3. At the same time, various lines of embryological evidence support the notion that these digits develop in positions that normally produce digits 2, 3, and 4. In recent years, gene expression as well as experimental evidence was published that supports the hypothesis that this discrepancy arose from a digit identity shift in the evolution of the bird wing. A similar but less well-known controversy has been ongoing since the late 19th century regarding the identity of the digits of the three-toed Italian skink, Chalcides chalcides. Comparative anatomy identifies these digits as 1, 2, and 3, while embryological evidence suggests their derivation from embryological positions 2, 3, and 4. Here we re-examine this evidence and add gene expression data to determine the identity of the three digits of C. chalcides. The data confirm that the adult and the embryological evidence for digit identity are in conflict, and the expression of Hoxd11 suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4. We conclude that in C. chalcides, and likely in its close relatives, a digit identity frame shift has occurred, similar to the one in avian evolution. This result suggests that changes in of digit identity might be a more frequent consequence of digit reduction than previously assumed.
Asymmetric regulation of Hox gene expression pre-dates the appearance of tetrapod digits, and was... more Asymmetric regulation of Hox gene expression pre-dates the appearance of tetrapod digits, and was co-opted in the development of ‘thumbness’. This asymmetric expression correlates with independent morphological evolutionary variation of digit 1.
The neural crest is a unique cell population among embryonic cell types, displaying properties of... more The neural crest is a unique cell population among embryonic cell types, displaying properties of both ectodermal and mesodermal lineages. Most of the recent studies examining the neural crest have been performed in avian embryos. Only in the first half of this century were amphibians extensively used. We first summarize this important older source of information, reviewing studies made since the turn of the century. Due to the increasingly detailed in cellular and molecular knowledge of the early development of Xenopus laevis, the remainder of the review focuses on this species. We describe the route of migration and fate of the neural crest and propose a new model of neural crest induction in which prospective cells are induced independently of the neural plate by a double gradient of a morphogen that patterns the entire ectoderm. This model is also discussed in a more general context in connection with the dorsoventral patterning of the neural tube. Finally, we discuss some ideas...
Theropod dinosaurswere the dominant predators in most Mesozoic era terrestrial ecosystems1. Early... more Theropod dinosaurswere the dominant predators in most Mesozoic era terrestrial ecosystems1. Early theropod evolution is currently interpreted as the diversification of various carnivorous and cursorial taxa, whereas the acquisition of herbivorism, together with the secondary loss of cursorial adaptations, occurredmuch later among advanced coelurosaurian theropods1,2. A new, bizarre herbivorous basal tetanuran from the Upper Jurassic of Chile challenges this conception. The new dinosaur was discovered at Ayse´n, a fossil locality in the Upper Jurassic Toqui Formation of southern Chile (General Carrera Lake)3,4. The site yielded abundant and exquisitely preserved three-dimensional skeletons of small archosaurs. Several articulated individuals ofChilesaurus at different ontogenetic stages have been collected, as well as less abundant basal crocodyliforms, and fragmentary remains of sauropod dinosaurs (diplodocids and titanosaurians).
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Papers by Alexander O Vargas
embryological grounds to be digits 2, 3 and 4. In contrast, within paleontology, wing digits are named
1, 2, 3 as a result of phylogenetic analysis of fossil taxa indicating that birds descended from theropod
dinosaurs that had lost digits 4 and 5. It has been argued that the development of the wing does not
support the conclusion that birds are theropods, and that birds must have descended from ancestors
that had lost digits 1 and 5. Here we use highly conserved gene expression patterns in the developing
limbs of mouse and chicken, including the chicken talpid2 mutant and polydactylous Silkie breed
(Silkie mutant), to aid the assessment of digital identity in the wing. Digit 1 in developing limbs does
not express Hoxd12, but expresses Hoxd13. All other digits express both Hoxd12 and Hoxd13. We
found this signature expression pattern identifies the anteriormost digit of the wing as digit 1, in
accordance with the hypothesis these digits are 1, 2 and 3, as in theropod dinosaurs. Our evidence
contradicts the long-standing argument that the development of the wing does not support the
hypothesis that birds are living dinosaurs
non-opposable hallux of early theropod dinosaurs. An important morphological difference with
early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic
musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and
retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and
have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular
markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly
delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased
plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of
embryonic muscular activity in the evolutionary origin of a crucial adaptation
evolved several times independently as different groups have
become zygodactyl, semi-zygodactyl, heterodactyl, pamprodactyl
or syndactyl. Birds have also convergently
evolved similar modes of development, in a spectrum that
goes from precocial to altricial. Using the new context provided
by recent molecular phylogenies, we compared the
evolution of foot morphology and modes of development
among extant avian families. Variations in the arrangement
of toes with respect to the anisodactyl ancestral condition
have occurred only in altricial groups. Those groups represent
four independent events of super-altriciality and many
independent transformations of toe arrangements (at least
four zygodactyl, three semi-zygodactyl, one heterodactyl,
one pamprodactyl group, and several syndactyl). We propose
that delayed skeletal maturation due to altriciality
facilitates the epigenetic influence of embryonic muscular
activity over developing toes, allowing for repeated evolution
of innovations in their morphology.
some embryological evidence suggests that these digits are 2, 3, and 4. This apparent lack of correspondence has been described as the greatest challenge to the widely accepted theropod–bird
link (Zhou 2004. Naturwissenschaften 91:455–471). Here we review the pertinent literature regarding the debate on the origin of birds and wing digital identity and the evidence in favor of a 1,
2, 3 identity of the wing digits. Recent molecular evidence shows that the expression of Hoxd12 and Hoxd13 in the developing wing supports the theropod–bird link. In the chicken foot and in the mouse hand and foot, digit 1 is the only digit to combine the expression of Hoxd13 with the absence of expression of Hoxd12. The same is observed in the anterior digit of the wing, suggesting it is a digit 1, as expected for a theropod. Nevertheless, Galis et al. (2005. J Exp Zool (Mol Dev Evol) in press), argue that Hoxd12 and Hoxd13 expression patterns in mutant limbs do not allow distinguishing the most anterior digit in the bird wing from digit 2. They also argue that constraints to the evolution of limb development support the 2, 3, 4 identity of the wing digits. However, the case put forward by Galis et al. is biased and flawed with regard to interpretation of mutant limbs, developmental
mechanisms, stages observed, and the description of the evolutionary variation of limb development. Importantly, Galis et al. do not present evidence from wild-type limbs that counters the conclusions of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B(1):85–89), and fail to provide molecular evidence to specifically support the hypothesis that the wing digits are 2, 3, and 4. The expression of Hoxd12 and Hoxd13 in the developing wing is consistent with the hypothesis that birds are living dinosaurs; this view can lead to a greater understanding of the actual limits to the evolutionary variation of limb development
embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic
frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from
embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is
also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect
closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11
expression in crocodilians, the closest living relatives of birds.
Methodology/Principal Findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator
HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl
forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1.
Conclusions/Significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit
1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator
and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the
hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.
evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and
ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of
digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1,
2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by
some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling
digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2
could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and
HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition
of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis
shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings.
Results: We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the
rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh
signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift
is induced.
Conclusion: Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity
of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift
hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally
possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.
of cartilage formation reveals that the digits of the bird wing
develop from positions that become digits 2, 3, and 4 in other
amniotes. However, the morphology of the digits of early birds
like Archaeopteryx corresponds to that of digits 1, 2, and 3
of other archosaurs. A hypothesis is that a homeotic ‘‘frameshift’’
occurred, such that in the bird wing, digits 1, 2, and
3 develop from the embryological positions of digits 2, 3,
and 4. Experimental homeotic transformations of single digits
are well-documented, but frame-shifts of more than one digit
are not. We investigated the pattern of cartilage formation
in the development of Cyclopamine-treated wings. When
Cyclopamine was applied between stages 18 and 21,
morphologies that normally develop from positions 2 and
3 developed from positions 3 and 4. The serial shift of
digit identity toward posterior confirms a mechanistic
possibility that was previously inferred from the evolutionary
history of birds.
example of conflicting anatomical and embryological evidence
regarding digit homology. Anatomical in conjunction with
phylogenetic evidence supports the hypothesis that the three
remaining digits in the bird wing are digits 1, 2, and 3. At the
same time, various lines of embryological evidence support the
notion that these digits develop in positions that normally
produce digits 2, 3, and 4. In recent years, gene expression as
well as experimental evidence was published that supports the
hypothesis that this discrepancy arose from a digit identity shift
in the evolution of the bird wing. A similar but less well-known
controversy has been ongoing since the late 19th century
regarding the identity of the digits of the three-toed Italian skink,
Chalcides chalcides. Comparative anatomy identifies these
digits as 1, 2, and 3, while embryological evidence suggests
their derivation from embryological positions 2, 3, and 4. Here
we re-examine this evidence and add gene expression data to
determine the identity of the three digits of C. chalcides. The
data confirm that the adult and the embryological evidence for
digit identity are in conflict, and the expression of Hoxd11
suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4.
We conclude that in C. chalcides, and likely in its close
relatives, a digit identity frame shift has occurred, similar to the
one in avian evolution. This result suggests that changes in of
digit identity might be a more frequent consequence of digit
reduction than previously assumed.
basal tetanuran from the Upper Jurassic of Chile challenges this
conception. The new dinosaur was discovered at Ayse´n, a fossil locality in the Upper Jurassic Toqui Formation of southern Chile (General Carrera Lake)3,4. The site yielded abundant and exquisitely preserved three-dimensional skeletons of small archosaurs. Several articulated individuals ofChilesaurus at different ontogenetic stages have been collected, as well as less abundant basal crocodyliforms, and fragmentary remains of sauropod dinosaurs (diplodocids and titanosaurians).
embryological grounds to be digits 2, 3 and 4. In contrast, within paleontology, wing digits are named
1, 2, 3 as a result of phylogenetic analysis of fossil taxa indicating that birds descended from theropod
dinosaurs that had lost digits 4 and 5. It has been argued that the development of the wing does not
support the conclusion that birds are theropods, and that birds must have descended from ancestors
that had lost digits 1 and 5. Here we use highly conserved gene expression patterns in the developing
limbs of mouse and chicken, including the chicken talpid2 mutant and polydactylous Silkie breed
(Silkie mutant), to aid the assessment of digital identity in the wing. Digit 1 in developing limbs does
not express Hoxd12, but expresses Hoxd13. All other digits express both Hoxd12 and Hoxd13. We
found this signature expression pattern identifies the anteriormost digit of the wing as digit 1, in
accordance with the hypothesis these digits are 1, 2 and 3, as in theropod dinosaurs. Our evidence
contradicts the long-standing argument that the development of the wing does not support the
hypothesis that birds are living dinosaurs
non-opposable hallux of early theropod dinosaurs. An important morphological difference with
early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic
musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and
retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and
have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular
markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly
delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased
plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of
embryonic muscular activity in the evolutionary origin of a crucial adaptation
evolved several times independently as different groups have
become zygodactyl, semi-zygodactyl, heterodactyl, pamprodactyl
or syndactyl. Birds have also convergently
evolved similar modes of development, in a spectrum that
goes from precocial to altricial. Using the new context provided
by recent molecular phylogenies, we compared the
evolution of foot morphology and modes of development
among extant avian families. Variations in the arrangement
of toes with respect to the anisodactyl ancestral condition
have occurred only in altricial groups. Those groups represent
four independent events of super-altriciality and many
independent transformations of toe arrangements (at least
four zygodactyl, three semi-zygodactyl, one heterodactyl,
one pamprodactyl group, and several syndactyl). We propose
that delayed skeletal maturation due to altriciality
facilitates the epigenetic influence of embryonic muscular
activity over developing toes, allowing for repeated evolution
of innovations in their morphology.
some embryological evidence suggests that these digits are 2, 3, and 4. This apparent lack of correspondence has been described as the greatest challenge to the widely accepted theropod–bird
link (Zhou 2004. Naturwissenschaften 91:455–471). Here we review the pertinent literature regarding the debate on the origin of birds and wing digital identity and the evidence in favor of a 1,
2, 3 identity of the wing digits. Recent molecular evidence shows that the expression of Hoxd12 and Hoxd13 in the developing wing supports the theropod–bird link. In the chicken foot and in the mouse hand and foot, digit 1 is the only digit to combine the expression of Hoxd13 with the absence of expression of Hoxd12. The same is observed in the anterior digit of the wing, suggesting it is a digit 1, as expected for a theropod. Nevertheless, Galis et al. (2005. J Exp Zool (Mol Dev Evol) in press), argue that Hoxd12 and Hoxd13 expression patterns in mutant limbs do not allow distinguishing the most anterior digit in the bird wing from digit 2. They also argue that constraints to the evolution of limb development support the 2, 3, 4 identity of the wing digits. However, the case put forward by Galis et al. is biased and flawed with regard to interpretation of mutant limbs, developmental
mechanisms, stages observed, and the description of the evolutionary variation of limb development. Importantly, Galis et al. do not present evidence from wild-type limbs that counters the conclusions of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B(1):85–89), and fail to provide molecular evidence to specifically support the hypothesis that the wing digits are 2, 3, and 4. The expression of Hoxd12 and Hoxd13 in the developing wing is consistent with the hypothesis that birds are living dinosaurs; this view can lead to a greater understanding of the actual limits to the evolutionary variation of limb development
embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic
frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from
embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is
also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect
closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11
expression in crocodilians, the closest living relatives of birds.
Methodology/Principal Findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator
HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl
forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1.
Conclusions/Significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit
1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator
and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the
hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.
evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and
ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of
digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1,
2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by
some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling
digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2
could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and
HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition
of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis
shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings.
Results: We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the
rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh
signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift
is induced.
Conclusion: Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity
of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift
hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally
possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.
of cartilage formation reveals that the digits of the bird wing
develop from positions that become digits 2, 3, and 4 in other
amniotes. However, the morphology of the digits of early birds
like Archaeopteryx corresponds to that of digits 1, 2, and 3
of other archosaurs. A hypothesis is that a homeotic ‘‘frameshift’’
occurred, such that in the bird wing, digits 1, 2, and
3 develop from the embryological positions of digits 2, 3,
and 4. Experimental homeotic transformations of single digits
are well-documented, but frame-shifts of more than one digit
are not. We investigated the pattern of cartilage formation
in the development of Cyclopamine-treated wings. When
Cyclopamine was applied between stages 18 and 21,
morphologies that normally develop from positions 2 and
3 developed from positions 3 and 4. The serial shift of
digit identity toward posterior confirms a mechanistic
possibility that was previously inferred from the evolutionary
history of birds.
example of conflicting anatomical and embryological evidence
regarding digit homology. Anatomical in conjunction with
phylogenetic evidence supports the hypothesis that the three
remaining digits in the bird wing are digits 1, 2, and 3. At the
same time, various lines of embryological evidence support the
notion that these digits develop in positions that normally
produce digits 2, 3, and 4. In recent years, gene expression as
well as experimental evidence was published that supports the
hypothesis that this discrepancy arose from a digit identity shift
in the evolution of the bird wing. A similar but less well-known
controversy has been ongoing since the late 19th century
regarding the identity of the digits of the three-toed Italian skink,
Chalcides chalcides. Comparative anatomy identifies these
digits as 1, 2, and 3, while embryological evidence suggests
their derivation from embryological positions 2, 3, and 4. Here
we re-examine this evidence and add gene expression data to
determine the identity of the three digits of C. chalcides. The
data confirm that the adult and the embryological evidence for
digit identity are in conflict, and the expression of Hoxd11
suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4.
We conclude that in C. chalcides, and likely in its close
relatives, a digit identity frame shift has occurred, similar to the
one in avian evolution. This result suggests that changes in of
digit identity might be a more frequent consequence of digit
reduction than previously assumed.
basal tetanuran from the Upper Jurassic of Chile challenges this
conception. The new dinosaur was discovered at Ayse´n, a fossil locality in the Upper Jurassic Toqui Formation of southern Chile (General Carrera Lake)3,4. The site yielded abundant and exquisitely preserved three-dimensional skeletons of small archosaurs. Several articulated individuals ofChilesaurus at different ontogenetic stages have been collected, as well as less abundant basal crocodyliforms, and fragmentary remains of sauropod dinosaurs (diplodocids and titanosaurians).