Conserved and divergent expression patterns of markers of axial development in reptilian embryos: Chinese soft-shell turtle and Madagascar ground gecko.

The processes of development leading up to gastrulation have been markedly altered during the evolution of amniotes, and it is uncertain how the mechanisms of axis formation are conserved and diverged between mouse and chick embryos. To assess the conservation and divergence of these mechanisms, this study examined gene expression patterns during the axis formation process in Chinese soft-shell turtle and Madagascar ground gecko preovipositional embryos. The data suggest that NODAL signaling, similarly to avian embryos but in contrast to eutherian embryos, does not have a role in epiblast and hypoblast development in reptilian embryos. The posterior marginal epiblast (PME) is the initial molecular landmark of axis formation in reptilian embryos prior to primitive plate development. Ontogenetically, PME may be the precursor of the primitive plate, and phylogenetically, Koller's sickle and posterior marginal zone in avian development may have been derived from the PME. Most of the genes expressed in the mouse anterior visceral endoderm (AVE genes), especially signaling antagonist genes, are not expressed in the hypoblast of turtle and gecko embryos, though they are expressed in the avian hypoblast. This study proposes that AVE gene expression in the hypoblast and the visceral endoderm could have been independently established in avian and eutherian lineages, similar to the primitive streak that has been independently acquired in these lineages.

Reevaluating Emx gene phylogeny: homopolymeric amino acid tracts as a potential factor obscuring orthology signals in cyclostome genes.

BACKGROUND: Vertebrate Emx genes, retained as multiple copies, are expressed in a nested pattern in the early embryonic forebrain and required for its regionalization. This pattern seems to have originated in a vertebrate common ancestor; however, a previous analysis, reporting two lamprey Emx genes, claimed independent Emx gene duplications in both cyclostome (extant jawless fish) and gnathostome (jawed vertebrate) lineages after their divergence. This scenario is neither parsimonious nor consistent with the hypothesis that genome expansion occurred before the cyclostome-gnathostome split, which is supported by recent genome-wide analyses. RESULTS: We isolated and sequenced cDNA of two hagfish Emx genes and performed intensive molecular phylogenetic analyses, including the hagfish and/or lamprey Emx genes. The lamprey genes tended to attract each other in inferred phylogenetic trees, an effect that tended to be relaxed on inclusion of the hagfish genes. The results of these analyses suggest that cyclostome EmxB is orthologous to gnathostome Emx2, which was also supported by conserved synteny. Homopolymeric amino acid (HPAA) tracts represent a remarkable feature of the lamprey Emx sequences, and a comparative genome-wide scan revealed that lamprey proteins exhibit a unique pattern of HPAA tract accumulation. CONCLUSIONS: Our analysis, including hagfish Emx genes, suggests that gene duplications gave rise to Emx1, -2 and -3 before the cyclostome-gnathostome split. We propose that independent HPAA tract accumulations in multiple ancient duplicates, as identified in lamprey Emx gene products, may have led to erroneous identification of gene duplication in the lamprey lineage. Overall, our reanalysis favors the scenario that the nested Emx expression pattern in mouse and lamprey shares a common origin.

Role of paraxial mesoderm in limb/flank regionalization of the trunk lateral plate.

To understand the developmental mechanism that determines limb size and the consequent limb-to-trunk proportions in the tetrapod body, we investigated the role of the paraxial mesoderm in the specification of the limb and flank fields in the chick embryo. We found that the paraxial mesoderm subjacent to the limb field can affect the size of the limb bud along the anterior-posterior and proximal-distal axes. We also found that the paraxial mesoderm subjacent to the flank plays roles in suppressing the emergence and growth of the limb bud and in promoting flank-specific apoptosis in the lateral plate mesoderm. Our results suggest that signals from the paraxial mesoderm specify the limb and flank fields in the competent lateral plate mesoderm.

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.

Anterior shift in gene expression precedes anteriormost digit formation in amniote limbs.

In tetrapod limbs, an anteriormost digit has common traits of small, short and less-phalange morphology. In this study, we focused on three genes, Mkp3, Sef and Tsukushi (TSK), which have anterior-specific or anterior-prominent expression patterns in the developing limb bud at the autopod-forming stage. The anterior expression is not fixed in the period of limb development, but the expression domains of Mkp3, Sef and TSK change considerably from the distal domain to the anterior domain. This change in expression domains, anterior shift, of these genes involves maintenance of gene expression in the anterior side and downregulation in the posterior side. Manipulated overdose of fibroblast growth factor (FGF) in the presumptive digit 2 region of chick forelimb bud results in elongation of cartilage elements of digit 2, suggesting that attenuated FGF signaling, which Mkp3, Sef, and TSK negatively regulate, provides digit 2-specific traits of morphology. The anterior expression of Mkp3 and Sef but not TSK is conserved also in limb buds of the mouse and gecko, and the anterior shift of these genes, accumulation of their transcripts in the anterior side and appropriate regulation of strength of FGF signaling may control species-specific morphology of the anteriormost digit.

Normal developmental stages of the Madagascar ground gecko Paroedura pictus with special reference to limb morphogenesis.

A table of developmental stages of the target species is useful for studying the development of any animal. Although tables of developmental stages have been established for several squamates, none has been published for gekkonid lizards. We have established a table of developmental stages for the Madagascar ground gecko Paroedura pictus. The table includes 27 embryonic stages from oviposition to hatching based on chronology and external morphology. The interval from oviposition to hatching is 60 days. Eleven to sixteen somites were observed at oviposition, and 5 to 6 somites were formed each day. Limb bud swellings were recognized by the third day after oviposition. After 2 weeks of incubation, the presumptive autopod was detected by carpal/tarsal cartilage formation. Cartilages in all digits were seen by 3 weeks after oviposition. Skin pigment was visible after 4 weeks incubation, and the skin color pattern was apparent 40 days after oviposition.

Competent stripes for diverse positions of limbs/fins in gnathostome embryos.

Every vertebrate species has its own unique morphology adapted to a particular lifestyle and habitat. Limbs and fins are strikingly diversified in size, shape, and position along the body axis. This diversity in morphology suggests the existence of a variety of embryonic developmental programs. However, comparisons of various embryos suggest common mechanisms underlying limb/fin formation. Here, we report the existence of continuous stripes of competency for appendage formation along the dorsal midline and the lateral trunk of all of the major jawed vertebrate (gnathostome) groups. We also show that the developing fin buds of cartilaginous fish share a mechanism of anterior-posterior axis formation as well as an shh (sonic hedgehog) expression domain in the posterior bud. We hypothesize a continuous distribution of competent stripes that represents the common developmental program at the root of appendage formation in gnathostomes. This schema would have permitted subsequent divergence into various levels of limbs/fins in each animal group.