Gene expression and digit homology in the chicken embryo wing

The bird wing is of special interest to students of homology and avian evolution. Fossil and developmental data give conflicting indications of digit homology if a pentadactyl "archetype" is assumed. Morphological signs of a vestigial digit I are seen in bird embryos, but no digit-like structure develops in wild-type embryos. To examine the developmental mechanisms of digit loss, we studied the expression of the high-mobility group box containing Sox9 gene, and bone morphogenetic protein receptor 1b (bmpR-1b)-markers for precondensation and prechondrogenic cells, respectively. We find an elongated domain of Sox9 expression, but no bmpR-1b expression, anterior to digit II. We interpret this as a digit I domain that reaches precondensation, but not condensation or precartilage stages. It develops late, when the tissue in which it is lodged is being remodeled. We consider these findings in the light of previous Hoxd-11 misexpression studies. Together, they suggest that there is a digit I vestige in the wing that can be rescued and undergo development if posterior patterning cues are enhanced. We observed Sox9 expression in the elusive "element X" that is sometimes stated to represent a sixth digit. Indeed, incongruity between digit domains and identities in theropods disappears if birds and other archosaurs are considered primitively polydactyl. Our study provides the first gene expression evidence for at least five digital domains in the chick wing. The failure of the first to develop may be plausibly linked to attenuation of posterior signals.     

The evolution of HoxD-11 expression in the bird wing: insights from Alligator mississippiensis

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.     

Limusaurus inextricabilis (Theropoda: Ceratosauria) gives a hand to evolutionary teratology: a complementary view on avian manual digits identities

It is widely accepted that birds are rooted within theropod dinosaurs. However, there is controversy between palaeontological and developmental data regarding manual digit identities of birds and their tetanuran ancestors (I, II and III vs. II, III and IV). To resolve this conflict, the principle of a frame‐shift has been considered. Identities of digits I–III would develop on condensations 2–4. Nevertheless, the discovery of the basal Ceratosauria Limusaurus inextricabilis has been used as a reference to define the digital identity of Tetanurae as II–IV. The new concept of evolutionary teratology states that certain anatomical structures identified in evolutionary lineages are viable developmental anomalies (‘adaptive’ or not), becoming part of the considered groups. The features of Limusaurus' forelimb match teratological characterization. This diagnosis, associated with the variations previously identified in derived Ceratosauria taxa (Carnotaurinae), underline an anatomical and developmental independence (regarding evolutionary conserved mechanisms) compared with Tetanurae and therefore birds. Consequently, Limusaurus should not be used as a reference concerning the identity of avian manuals digits. Evolutionary teratology supports identities I, II and III of the tetanuran manus via a frame‐shift that did not occur in the Ceratosauria lineage. © 2016 The Linnean Society of London     

Hox genes, digit identities and the theropod/bird transition

Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B:86-90) propose that Hox gene expression patterns indicate that the most anterior digit in bird wings is homologous to digit 1 rather than to digit 2 in other amniotes. This interpretation is based on the presence of Hoxd13 expression in combination with the absence of Hoxd12 expression in the second digit condensation from which this digit develops (the first condensation is transiently present). This is a pattern that is similar to that in the developing digit 1 of the chicken foot and the mouse hand and foot. They have tested this new hypothesis by analysing Hoxd12 and Hoxd13 expression patterns in two polydactylous chicken mutants, Silkie and talpid2. They conclude that the data support the notion that the most anterior remaining digit of the bird wing is homologous to digit 1 in other amniotes either in a standard phylogenetic sense, or alternatively in a (limited) developmental sense in agreement with the Frameshift Hypothesis of Wagner and Gautier (1999, i.e., that the developmental pathway is homologous to the one that leads to a digit 1 identity in other amniotes, although it occurs in the second instead of the first digit condensation). We argue that the Hoxd12 and Hoxd13 expression patterns found for these and other limb mutants do not allow distinguishing between the hypothesis of Vargas and Fallon (2005. J Exp Zool (Mol Dev Evol) 304B:86-90) and the alternative one, i.e., the most anterior digit in bird wings is homologous to digit 2 in other amniotes, in a phylogenetic or developmental sense. Therefore, at the moment the data on limb mutants does not present a challenge to the hypothesis, based on other developmental data (Holmgren, 1955. Acta Zool 36:243-328; Hinchliffe, 1984. In: Hecht M, Ostrom JH, Viohl G, Wellnhofer P, editors. The beginnings of birds. Eichst?tt: Freunde des Jura-Museum. p 141-147; Burke and Feduccia, 1997. Science 278:666-668; Kundrát et al., 2002. J Exp Zool (Mol Dev Evol) 294B:151-159; Larsson and Wagner, 2002. J Exp Zool (Mol Dev Evol) 294B:146-151; Feduccia and Nowicki, 2002. Naturwissenschaften 89:391-393), that the digits of bird wings are homologous to digits 2,3,4 in amniotes. We recommend further testing of the hypothesis by comparing Hoxd expression patterns in different taxa.     

Histogenesis in 11-day mouse embryo limb buds explanted in organ culture

Fore- and hindlimb buds from 11-day mouse embryos with 40 to 52 somites (including the four occipital pairs) were explanted in organ culture and submitted to systematic histological analysis. Chondrogenesis occurs normally in culture in all preskeletal rudiments which were already represented by condensed blastemas before explantation. In the proximal territories, the progress of cartilage differentiation occurs according to the normal pattern and can be revealed histologically much earlier than in bulk preparations. In all explanted hindlimbs as well as in forelimbs from embryos with less than 50 somites, a primary coalescence occurs between the IId and IIId digital rays, leading to various fusions from soft tissue syndactyly to oligosyndactyly. This is the result of two combined unfavorable effects of the culture conditions: the lack of simultaneous volumetric growth of the foot- or handplate, which normally would provide the necessary space for the laying down of a pentadactylous pattern, and a loss of cells resulting from abnormal cell death affecting selective mesodermal sites in the zeugopod and in the marginal subridge area, the latter being more severely affected in hindlimb buds. Several observations suggest that the preferential sensitivity of the marginal mesoderm might be related to early changes in the apical ectoderm, which itself becomes excessively necrotic and rapidly looses its pseudostratified configuration. The forelimb buds from embryos with 50 somites and more usually develop a pentadactylous pattern with a better individuation of digital structures. In all explants, the prospective mesoderm of digit I exhibits stronger regulatory tendencies.     

Frame-shifts of digit identity in bird evolution and Cyclopamine-treated wings

SUMMARY 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 "frame-shift" 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.     

Diversity and evolution of male secondary sexual characters in African squeakers and long-fingered frogs

The African frog genera Arthroleptis and Cardioglossa are unique among vertebrates in having males with extremely long third fingers. In some species, this sexual dimorphism is impressive, with male third fingers approaching 40% of body length. However, the diversity of this trait has not been documented thoroughly and several species appear to lack this trait. The present study documents the diversity of male secondary sexual traits in Arthroleptis and Cardioglossa , including elongate third fingers and digital and inguinal spines. Furthermore, it explores hypotheses of trait evolution, including explanations for the absence of male traits. Analyses of covariance suggest that the functional relationship between finger length and snout–vent length (SVL), both within and among species, is different for male finger III than for male fingers I, II, and IV, or for female finger III. Ancestral state reconstruction suggests that all male traits were present in the most recent common ancestor of Arthroleptis and Cardioglossa and that reduction or loss of traits occurred later. Across species, independent contrast analyses find no correlation between SVL and either male relative third digit length or dimorphism in relative third digit length. The number of spines on male fingers II and III are positively correlated but spine number is not correlated with SVL and only weakly correlated with relative third digit length. The diversity of male traits is evolutionarily labile and is not explained by simple hypotheses of character evolution. Arthroleptis and Cardioglossa may thus provide an interesting study system for understanding how changes in sexual selection forces produce male trait diversity.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 553–573.     

Sequential pattern of limb anomalies in Japanese monkeys on Awajishima Island

X-ray examinations of Japanese monkeys (Macaca fuscata) in two groups on Awajishima Island revealed that 11 of 46 monkeys from the Kaminada group and 5 of 37 monkeys from the Shirasaki group had limb anomalies. All the cleft hands, which comprised most of the anomalies in these Awajishima monkeys, involved reduction of one, two, or three fingers. The digital reductions showed a definite pattern: cleft hands with four, three, or two fingers lacking the digital rays III, III & IV, or II, III & IV, respectively. A similar teratological pattern has been recognized in the anomalies of other troops of Japanese monkeys. The presence of such a common teratological pattern among Japanese monkeys may be related to the high incidence of the anomalies and suggests that they may have a common etiological factor.     

The Mississippian Tetrapod Footprint Ichnogenus Palaeosauropus: Extramorphological Variation and Ichnotaxonomy

Palaeosauropus primaevus is a tetrapod footprint ichnotaxon first described from the Upper Mississippian (Visean) Mauch Chunk Formation near Pottsville, Pennsylvania, United States. Our relocation of the type locality and stratigraphic horizon of P. primaevus, a long-available but unstudied collection of tetrapod footprints from these strata, and our new collections allow a much fuller characterization of this ichnotaxon and the range of extramorphological variation encompassed by it. P. primaevus is characterized as the footprints of a quadruped with a pentadactyl pes and a tetradactyl manus, in which the pes frequently oversteps the manus and with which tail drags are common. In the manus, all digits are relatively broad and have rounded tips, digit III is longest, and digit IV is more widely separated from digit III than the other digits are from each other. The pes has five digits that are also wide and blunt-tipped, digit IV is longest, and digit V projects nearly laterally. P. primaevus is the track of a relatively large temnospondyl (～400 mm gleno-acetabular length) and documents the Mississippian presence of such large amphibians long before their body fossil record. Palaeosauropus also occurs in Mississippian strata in Indiana and is distinguished from the geologically younger but similar temnospondyl footprint ichnogenus Limnopus by its relatively narrower manus and pes that lack broad and rounded sole impressions.     

Evolution of digit identity in the three-toed Italian skink Chalcides chalcides: a new case of digit identity frame shift

SUMMARY 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.     

Tbx Genes Specify Posterior Digit Identity through Shh and BMP Signaling

Despite extensive studies on the anterior-posterior (AP) axis formation of limb buds, mechanisms that specify digit identities along the AP axis remain obscure. Using the four-digit chick leg as a model, we report here that Tbx2 and Tbx3 specify the digit identities of digits IV and III, respectively. Misexpression of Tbx2 and Tbx3 induced posterior homeotic transformation of digit III to digit IV and digit II to digit III, respectively. Conversely, misexpression of their mutants VP16 Delta Tbx2 and VP16 Delta Tbx3 induced anterior transformation. In both cases, alterations in the expression of several markers (e.g., BMP2, Shh, and HoxD genes) were observed. In addition, Tbx2 and Tbx3 rescued Noggin-mediated inhibition of interdigital BMP signaling, signaling which is pivotal in establishing digit identities. Hence, we conclude that Tbx3 specifies digit III, and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade.     

The non-avian theropod track Jialingpus from the Cretaceous of the Ordos Basin,China, with a revision of the type material: Implications for ichnotaxonomy and trackmaker morphology

Deposits from the Ordos Basin of mid-western China are rich in body fossils and ichnofossils of Early Cretaceous vertebrates. Thousands of Early Cretaceous sauropod, theropod and bird tracks described since 1958 have been found at several localities in the basin. We report two new sites (Dijiaping and Bawangzhuang) in the Luohe Formation of the Ordos Basin, Shaanxi Province, which contain small theropod footprints that are here referred to the ichnogenus Jialingpus. The assignment is based on pad configurations including (1) the large metatarsophalangeal area positioned in line with the axis of digit III, (2) the subdivision of this part into a small pad behind digit II, which in some specimens is close to the general position of the hallux (digit I), and a large metatarsophalangeal pad behind digit IV, and (3) a distinct inter-pad space between metatarsophalangeal pads and proximal phalangeal pads of digits II and III. We re-describe the type material of the type ichnospecies Jialingpus yuechiensis from the Upper Jurassic Penglaizhen Formation of Sichuan Province, proposing a largely amended diagnosis for this ichnotaxon. The presence of a digit I trace in the holotype, indicating a relatively long hallux, and the large metatarsophalangeal area positioned in line with digit III distinguishes Jialingpus from the ichnogenus Grallator and similar tracks that all lack these features. The only difference between Jialingpus specimens from the Cretaceous of the Ordos Basin and those of the Jurassic Penglaizhen Formation is the larger digit divarication in the Cretaceous taxon. This is the fourth record of Jialingpus in China and the second in Cretaceous strata, with the first being those from the Huangyangquan locality in Xinjiang, China.     

The developmental origin of zygodactyl feet and its possible loss in the evolution of Passeriformes

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 development 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.     

Are there general laws for digit evolution in squamates? The loss and re‐evolution of digits in a clade of fossorial lizards (Brachymeles,Scincinae)

Evolutionary simplification of autopodial structures is a major theme in studies of body‐form evolution. Previous studies on amniotes have supported Morse's law, that is, that the first digit reduced is Digit I, followed by Digit V. Furthermore, the question of reversibility for evolutionary digit loss and its implications for “Dollo's law” remains controversial. Here, we provide an analysis of limb and digit evolution for the skink genus Brachymeles. Employing phylogenetic, morphological, osteological, and myological data, we (a) test the hypothesis that digits have re‐evolved, (b) describe patterns of morphological evolution, and (c) investigate whether patterns of digit loss are generalizable across taxa. We found strong statistical support for digit, but not limb re‐evolution. The feet of pentadactyl species of Brachymeles are very similar to those of outgroup species, while the hands of these lineages are modified (2‐3‐3‐3‐2) and a have a reduced set of intrinsic hand muscles. Digit number variation suggests a more labile Digit V than Digit I, contrary to Morse's law. The observed pattern of digit variation is different from that of other scincid lizards (Lerista, Hemiergis, Carlia). Our results present the first evidence of clade‐specific modes of digit reduction.     

Finding the frame shift: digit loss, developmental variability, and the origin of the avian hand

The origin of the tridactyl hand of crown birds from the pentadactyl hand of those early theropod dinosaurs lying along the avian stem has become a classic, but at times seemingly intractable, historical problem. The point in question is whether the fingers of crown birds represent digits 1-3 as predicted by generalized trends in the fossil record; or digits 2-4, as evidenced by the topology of the embryonic mesenchymal condensations from which the digits develop. The frame shift hypothesis attempted to resolve this paradox by making these signals congruent by means of a homeotic transformation in digital identity, but recently the paleontological support for this hypothesis was questioned. Here, we reassess the frame shift from a paleontological perspective by addressing what predictions a frame shift makes for skeletal morphology, whether the frame shift remains a viable explanation of the known fossil data, and where on the theropod tree the frame shift most likely occurred. Our results indicate that the frame shift remains viable, and based on the inferred pattern of digit loss, the frame shift likely occurred at a deeper position in theropod phylogeny than previously proposed. A new evolutionary model of the frame shift is described in which the early history of the frame-shifted hand is marked by an extended zone of developmental polymorphism. This model provides a new conceptual framework for the role of developmental variability in communicating broad evolutionary patterns on a taxonomically inclusive phylogenetic tree.     

A REEVALUATION OF THE ORIGIN OF PENTADACTYLY

The hypothesis suggested by Coates that pentadactyly arose twice is based on the presumed affinities between Tulerpeton, embolomeres, and amniotes. Flaws were found in the coding of some characters that supported this clade and, once corrected, the matrix fails to support the proposed phylogeny. Furthermore, interpretation of the phylogeny of Coates is difficult because no amniotes and no lissamphibians were included in this analysis. A reappraisal of the affinities of Tulerpeton using a matrix composed of a greater range of taxa suggests that it is a stem-tetrapod, that pentadactyly arose only once, and that the first tetrapod was already pentadactyl.     

Comparative and functional morphology of hominoid fingers.

Comparisons of hominoid metacarpals and phalanges reveal differences, many of which are closely linked to locomotor hand postures. The African apes display features of the metacarpals and phalanges which distinguish them from the other Hominoidea. These features are most evident in digits III and IV. The orangutan hand is demonstrably less well adapted to knuckle-walking and is distinctive in its adaptation to power and hook grasping of vertical and horizontal supports, respectively. Orangutan fingers possess a "double-locking" mechanism (Napier, '60), and a slight ulnad shift in the axis of the hand which results in lengthened phalanges of ray IV. Hylobatid apes are more like orangutans in their finger morphology than any of the other Hominoidea, but exhibit unique features of their own. These include elongate phalanges of fingers II-V. Human metacarpals II-V form two sets composed of II-III, and IV-V. The heads of both metacarpals II and III are characterized by axial torsion. This reflects the enhanced manipulatory role of the third finger in humans. Human distal phalanges are unique in the development of pronounced apical tufts. Multivariate analysis of metacarpal III and proximal III yields variables that array the extant apes along an arboreal-terrestrial axis, from hylobatid apes to male gorillas. The positions of taxa on this discriminant concur with observations on the locomotion of free-ranging apes.     

Evolution of Hoxa-11 Expression in Amphibians: Is the Urodele Autopodium an Innovation?

In this paper we present preliminary results on the expressionof Hoxa-11 in the frog Xenopus laevis and the newt Notophthalmusviridescens. In amniote limbs, Hoxa-11 is expressed exclusivelyin the prospective zeugopodium and is involved in the developmentof zeugopodial character identity. While the Hoxa-11 expressionpattern in Xenopus is similar to those described in mice andchick, the newt Notophthalmus has a phase of autopodial Hoxa-11expression starting with the development of digit III. Expressionis particularly strong in the buds of the postaxial digits.This Hoxa-11 expression pattern is unique among tetrapods andadds to the list of developmental peculiarities of urodele limbdevelopment, which also include an anterior to posterior polarityof digit development. We propose a scenario to explain the evolutionaryorigin of urodele limb development. We assume that recent urodelesare derived from a lineage of amphibians which underwent partialdigit reduction, leaving only two digits intact, most likelydigits III and IV according to Morse's law. This implies thaturodele digits I and II are homologous to amniote digits IIIand IV, and that the postaxial digits of urodeles are an evolutionaryinnovation (re-invention). Supporting evidence for this homologyhypothesis is found in the developmental connections betweenthe basale commune and the postaxial zeugopodial element.