Flight feather development: its early specialization during embryogenesis.

BACKGROUND: Flight feathers, a type of feather that is unique to extant/extinct birds and some non-avian dinosaurs, are the most evolutionally advanced type of feather. In general, feather types are formed in the second or later generation of feathers at the first and following molting, and the first molting begins at around two weeks post hatching in chicken. However, it has been stated in some previous reports that the first molting from the natal down feathers to the flight feathers is much earlier than that for other feather types, suggesting that flight feather formation starts as an embryonic event. The aim of this study was to determine the inception of flight feather morphogenesis and to identify embryological processes specific to flight feathers in contrast to those of down feathers. RESULTS: We found that the second generation of feather that shows a flight feather-type arrangement has already started developing by chick embryonic day 18, deep in the skin of the flight feather-forming region. This was confirmed by shh gene expression that shows barb pattern, and the expression pattern revealed that the second generation of feather development in the flight feather-forming region seems to start by embryonic day 14. The first stage at which we detected a specific morphology of the feather bud in the flight feather-forming region was embryonic day 11, when internal invagination of the feather bud starts, while the external morphology of the feather bud is radial down-type. CONCLUSION: The morphogenesis for the flight feather, the most advanced type of feather, has been drastically modified from the beginning of feather morphogenesis, suggesting that early modification of the embryonic morphogenetic process may have played a crucial role in the morphological evolution of this key innovation. Co-optation of molecular cues for axial morphogenesis in limb skeletal development may be able to modify morphogenesis of the feather bud, giving rise to flight feather-specific morphogenesis of traits.

Two Types of Etiological Mutation in the Limb-Specific Enhancer of Shh.

An enhancer named MFCS1 regulates Sonic hedgehog (Shh) expression in the posterior mesenchyme of limb buds. Several mutations in MFCS1 induce ectopic Shh expression in the anterior limb bud, and these result in preaxial polydactyly (PPD). However, the molecular basis of ectopic Shh expression remains elusive, although some mutations are known to disrupt the negative regulation of Shh expression in the anterior limb bud. Here, we analyzed the molecular mechanism of ectopic Shh expression in PPD including in a mouse mutation-hemimelic extra toes (Hx)-and in other MFCS1 mutations in different species. First, we generated transgenic mouse lines with a LacZ reporter cassette flanked with tandem repeats of 40 bp MFCS1 fragments harboring a mutation. The transgenic mouse line with the Hx-type fragment showed reporter expression exclusively in the anterior, but not in the posterior margins of limb buds. In contrast, no specific LacZ expression was observed in lines carrying the MFCS1 fragment with other mutations. Yeast one-hybrid assays revealed that the msh-like homeodomain protein, MSX1, bound specifically to the Hx sequence of MFCS1. Thus, PPD caused by mutations in MFCS1 has two major types of molecular etiology: loss of a cis-motif for negative regulation of Shh, and acquisition of a new cis-motif binding to a preexisting transcription factor, as represented by the Hx mutation.

Functional roles of Aves class-specific cis-regulatory elements on macroevolution of bird-specific features.

Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (>99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.

Evidence for an amphibian sixth digit.

INTRODUCTION: Despite the great diversity in digit morphology reflecting the adaptation of tetrapods to their lifestyle, the number of digits in extant tetrapod species is conservatively stabilized at five or less, which is known as the pentadactyl constraint. RESULTS: We found that an anuran amphibian species, Xenopus tropicalis (western clawed frog), has a clawed protrusion anteroventral to digit I on the foot. To identify the nature of the anterior-most clawed protrusion, we examined its morphology, tissue composition, development, and gene expression. We demonstrated that the protrusion in the X. tropicalis hindlimb is the sixth digit, as is evident from anatomical features, development, and molecular marker expression. CONCLUSION: Identification of the sixth digit in the X. tropicalis hindlimb strongly suggests that the prehallux in other Xenopus species with similar morphology and at the same position as the sixth digit is also a vestigial digit. We propose here that the prehallux seen in various species of amphibians generally represents a rudimentary sixth digit.

AP-2ß is a transcriptional regulator for determination of digit length in tetrapods.

The species-specific morphology of digits in the tetrapod limb, including the length and number of metacarpal, metatarsal, and phalangeal bones, suggests that a common developmental mechanism for digit formation is modified in a species-specific manner. Here, we examined the function of the AP-2ß transcription factor in regulating digit length in the chicken autopod. Mutations in the gene encoding AP-2ß are associated with Char syndrome, a human autosomal dominant disorder. Char syndrome patients exhibit autopod skeletal defects, including loss of phalanges and shortened fingers, suggestive of a function for AP-2ß in normal digit development. The ectopic expression of two different dominant-negative forms of chick AP-2ß, equivalent to mutant forms associated with human Char syndrome, in the developing chick hindlimb bud resulted in defective digit formation, including reductions in the number and length of phalanges and metatarsals. A detailed analysis of the AP-2ß expression pattern in the limb bud indicated a correlation between the pattern/duration of AP-2ß expression in the limb mesenchyme and digit length in three amniote species, the chicken, mouse and gecko. In addition, we found that AP-2ß expression was downstream of Fgf signals from the apical ectodermal ridge, which is crucial in digit morphogenesis, and that excessive AP-2ß function resulted in dysregulated digit length. Taken together, these results suggest that AP-2ß functions as a novel transcriptional regulator for digit morphogenesis.

Evolutionary and developmental aspects of avian-specific traits in limb skeletal pattern.

The two sets of paired appendages, called limbs, are locomotory organs in tetrapods that are used for various functions (e.g., walking, running, crawling, digging, climbing, diving, swimming, and flying). Unlike such organs as the eye, which contain specialized tissues such as the lens and photoreceptor, the limb does not have any specialized cells or tissues, but consists of common tissues, such as bone, cartilage, muscle, blood vessels, and dermis. However, limb morphology is highly specialized and varies to provide species-specific modes of locomotion. As do the vertebrae and skull, the limb skeleton varies in morphology among species. The diversity of limb skeletal morphology provides examples of material for studies on morphogenesis. Avian forelimbs have evolved into wings for flight. The skeletal pattern in the avian limb has many traits that are unique among extant species of vertebrates; some of such traits are avian-specific, others are shared with more basal members of Theropoda, to which Aves belongs. Since such avian traits generally form during ontogenic development, determining when and how they appear in the developing embryonic limbs or limb buds provides important insights into the mechanisms underlying the generation of vertebrate morphological diversity. Here, we present an overview of several features of the skeletal pattern in the avian limb and discuss the developmental mechanisms responsible for their unique and lineage-specific traits.

Heterochronically early decline of Hox expression prior to cartilage formation in the avian hindlimb zeugopod.

The fibula, a zeugopod bone in the hindlimb, exhibits various morphologies in tetrapod species. The fibula in some species has a similar length with the other zeugopod element, the tibia, while other species have obvious differences in the sizes of the two elements. In the avian hindlimb, for example, the fibula is extremely short, thin, and truncated. Basic morphology of the fibula is established during development, and cartilage primordium of the bone emerges in a certain region defined by a distinct combination of expression of Hox genes (Hox code). In order to elucidate how the different morphologies are produced from a region that is defined as the fixed Hox code, we examined spatial and temporal patterns of Hoxd11/Hoxd12 expression in the developing limb bud, which defines the region from which the fibula emerges, in comparison with the sites of precartilaginous mesenchymal condensations representing regions for cartilage formation among chick, mouse, and gecko embryos. We found that in the chick hindlimb, expression of Hoxd11/Hoxd12 decreased and disappeared from the presumptive zeugopod region before cartilage formation. This heterochronically early decline of expression of Hox genes is strongly correlated with the peculiar trait of the fibula in the avian hindlimb, since in the other species examined, expression of those genes continued after the onset of cartilage formation. This is morphological phenotype-related because the early disappearance was not seen in the chick forelimb. Our results suggest that temporal change of the Hox code governs diversification in morphology of homologous structures among related species.

Embryological evidence identifies wing digits in birds as digits 1, 2, and 3.

The identities of the digits of the avian forelimb are disputed. Whereas paleontological findings support the position that the digits correspond to digits one, two, and three, embryological evidence points to digit two, three, and four identities. By using transplantation and cell-labeling experiments, we found that the posteriormost digit in the wing does not correspond to digit four in the hindlimb; its progenitor segregates early from the zone of polarizing activity, placing it in the domain of digit three specification. We suggest that an avian-specific shift uncouples the digit anlagen from the molecular mechanisms that pattern them, resulting in the imposition of digit one, two, and three identities on the second, third, and fourth anlagens.

Morphogenetic change of the limb bud in the hand plate formation.

The vertebrate hand plate is flattened and paddle shaped; that is, it is wide along the anteroposterior (AP) axis (thumb to little finger) and thin along the dorsoventral axis (back of hand to palm). To learn how the hand plate develops its three-dimensional architecture, we observed morphological changes in the distal limb bud of the chick embryo at stages 23-27 and the gecko embryo 11-13 days after oviposition. Cell population of the posterior distal limb bud expanded more than that of the anterior one in the chick embryo. Taken together with the observation that these two cell populations did not show significant differences in their expansion along the proximodistal axis, we propose that the cell population in the posterior limb bud contributes more to the morphogenetic increase along the AP axis, which widens the limb bud for the formation of the hand plate. Our observation that more mitoses were oriented anteroposteriorly than dorsoventrally in the chick embryo at around stage 25 suggests that the oriented cell division contributes to the morphogenetic increase along the AP axis.