On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates.

As a group closely related to chordates, hemichordate acorn worms are in a key phylogenic position for addressing hypotheses of chordate origins. The stomochord of acorn worms is an anterior outgrowth of the pharynx endoderm into the proboscis. In 1886 Bateson proposed homology of this organ to the chordate notochord, crowning this animal group "hemichordates." Although this proposal has been debated for over a century, the question still remains unresolved. Here we review recent progress related to this question. First, the developmental mode of the stomochord completely differs from that of the notochord. Second, comparison of expression profiles of genes including Brachyury, a key regulator of notochord formation in chordates, does not support the stomochord/notochord homology. Third, FoxE that is expressed in the stomochord-forming region in acorn worm juveniles is expressed in the club-shaped gland and in the endostyle of amphioxus, in the endostyle of ascidians, and in the thyroid gland of vertebrates. Based on these findings, together with the anterior endodermal location of the stomochord, we propose that the stomochord has evolutionary relatedness to chordate organs deriving from the anterior pharynx rather than to the notochord.

Identification of an intact ParaHox cluster with temporal colinearity but altered spatial colinearity in the hemichordate Ptychodera flava.

BACKGROUND: ParaHox and Hox genes are thought to have evolved from a common ancestral ProtoHox cluster or from tandem duplication prior to the divergence of cnidarians and bilaterians. Similar to Hox clusters, chordate ParaHox genes including Gsx, Xlox, and Cdx, are clustered and their expression exhibits temporal and spatial colinearity. In non-chordate animals, however, studies on the genomic organization of ParaHox genes are limited to only a few animal taxa. Hemichordates, such as the Enteropneust acorn worms, have been used to gain insights into the origins of chordate characters. In this study, we investigated the genomic organization and expression of ParaHox genes in the indirect developing hemichordate acorn worm Ptychodera flava. RESULTS: We found that P. flava contains an intact ParaHox cluster with a similar arrangement to that of chordates. The temporal expression order of the P. flava ParaHox genes is the same as that of the chordate ParaHox genes. During embryogenesis, the spatial expression pattern of PfCdx in the posterior endoderm represents a conserved feature similar to the expression of its orthologs in other animals. On the other hand, PfXlox and PfGsx show a novel expression pattern in the blastopore. Nevertheless, during metamorphosis, PfXlox and PfCdx are expressed in the endoderm in a spatially staggered pattern similar to the situation in chordates. CONCLUSIONS: Our study shows that P. flava ParaHox genes, despite forming an intact cluster, exhibit temporal colinearity but lose spatial colinearity during embryogenesis. During metamorphosis, partial spatial colinearity is retained in the transforming larva. These results strongly suggest that intact ParaHox gene clustering was retained in the deuterostome ancestor and is correlated with temporal colinearity.

Identical genomic organization of two hemichordate hox clusters.

Genomic comparisons of chordates, hemichordates, and echinoderms can inform hypotheses for the evolution of these strikingly different phyla from the last common deuterostome ancestor. Because hox genes play pivotal developmental roles in bilaterian animals, we analyzed the Hox complexes of two hemichordate genomes. We find that Saccoglossus kowalevskii and Ptychodera flava both possess 12-gene clusters, with mir10 between hox4 and hox5, in 550 kb and 452 kb intervals, respectively. Genes hox1-hox9/10 of the clusters are in the same genomic order and transcriptional orientation as their orthologs in chordates, with hox1 at the 3' end of the cluster. At the 5' end, each cluster contains three posterior genes specific to Ambulacraria (the hemichordate-echinoderm clade), two forming an inverted terminal pair. In contrast, the echinoderm Strongylocentrotus purpuratus contains a 588 kb cluster of 11 orthologs of the hemichordate genes, ordered differently, plausibly reflecting rearrangements of an ancestral hemichordate-like ambulacrarian cluster. Hox clusters of vertebrates and the basal chordate amphioxus have similar organization to the hemichordate cluster, but with different posterior genes. These results provide genomic evidence for a well-ordered complex in the deuterostome ancestor for the hox1-hox9/10 region, with the number and kind of posterior genes still to be elucidated.

Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development.

Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.

Diversification of epithelial adherens junctions with independent reductive changes in cadherin form: identification of potential molecular synapomorphies among bilaterians.

The adherens junction (AJ) is the most universal junction found in bilaterian epithelia and may represent one of the earliest types of cell-cell junctions. The adhesion molecules responsible for forming AJs are the classic cadherins (referred to simply as cadherins), whose extracellular domain organization displays marked variety among species examined so far. In this study, we attempted to reconstruct the evolution of cadherin by analyzing new data from several arthropods (two insects, one non-insect hexapod, three crustaceans, and one chelicerate) and previously published sequences for Drosophila melanogaster and other animals. The results of comparative analyses using the BLAST tool and immunohistochemical analyses revealed that the extracellular domain organizations of a decapod, an isopod, a spider, and a starfish cadherin, which are present at AJs in the embryonic epithelia are homologous. Independent reductive changes from the ancestral state were evident in the epithelia of hexapods+branchiopod, vertebrates+urochordates, and a cephalochordate. The form of cadherins in hexapods is more closely related to that of a branchiopod than to that of malacostracan crustaceans, and one of those of vertebrates is more closely related to that of urochordates than to that of a cephalochordate. Although the sampling of taxa is limited at this stage of research, we hypothesize that the reductive events in cadherin structure related to AJ formation in the epithelia may possess information about bilaterian relationships as molecular synapomorphies.

Development and neural organization of the tornaria larva of the Hawaiian hemichordate, Ptychodera flava.

We report scanning and transmission electron microscopic studies of the early development of the Hawaiian acorn worm, Ptychodera flava. In addition, we provide an immunohistochemical identification of the larval nervous system. Development occurs and is constrained within the stout chorion and fertilization envelope that forms upon the release of the cortical granules in the cytoplasm of the egg. The blastula consists of tall columnar blastomeres encircling a small blastocoel. Typical gastrulation occurs and a definitive tornaria is formed compressed within the fertilization envelope. The young tornaria hatches at 44 hr and begins to expand. The major circumoral ciliary band that crosses the dorsal surface and passes preorally and postorally is well developed. In addition, we find a nascent telotroch, as well as a midventral ciliary band that is already clearly developed. The epithelium of tornaria is a mosaic of monociliated and multiciliated cells. Immunohistochemistry with a novel neural marker, monoclonal antibody 1E11, first detects nerve cells at the gastrula stage. In tornaria, 1E11 staining nerve cells occur throughout the length of the ciliary bands, in the apical organ, in a circle around the mouth, in the esophageal epithelium and in circumpylorus regions. Axon(s) and apical processes extend from the nerve cell bodies and run in tracks along the ciliary bands. Axons extending from the preoral and postoral bands extend into the oral field and form a network. The tornaria nervous system with ciliary bands and an apical organ is rather similar to the echinoderm bipinnaria larvae.

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny.

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.

Conserved expression pattern of BMP-2/4 in hemichordate acorn worm and echinoderm sea cucumber embryos.

The auricularia larva of sea cucumbers and tornaria larva of acorn worms share striking developmental and morphological similarities. They are regarded as not only an archetype of the nonchordate deuterostome larva, but also an archetype of the origin of chordates. Here we report the characterization and spatial expression patterns of the BMP-2/4 genes of a hemichordate acorn worm (Pf-bmp2/4) and an echinoderm sea cucumber (Sj-bmp2/4). Both the Pf-bmp2/4 and Sj-bmp2/4 genes exhibited apparently conserved expression in the region of the coelomopore complex. This is in agreement with the homology between their basic larval body plans with respect to coelomogenesis and allows us to discuss the evolutionary counterparts of the coelomopore complex in chordates.

Amphi-Eomes/Tbr1: an amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development.

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode.

Group B sox genes that contribute to specification of the vertebrate brain are expressed in the apical organ and ciliary bands of hemichordate larvae.

We have identified and characterized the sequence and expression of two Group B Sox genes in the acorn worm, Ptychodera flava. One sequence represents a Group B1 Sox gene and is designated Pf-SoxB1; the other is a Group B2 Sox gene and is designated Pf-SoxB2. Both genes encode polypeptides with an HMG domain in the N-terminal half. Whole-mount in situ hybridization to embryonic and larval stages of P. flava shows that the two genes are expressed in rather similar patterns at these stages. Expression is first detected in the cells of the blastula and subsequently localizes to the ectoderm during gastrulation. As the mouth forms, expression becomes concentrated in the stomodeum region. During morphogenesis of the tornaria larva, expression in the stomodeal ectoderm remains prominent around the mouth and under the oral hood. Later the genes are prominently upregulated in the ciliary bands and the apical organ. These results provide additional evidence that genes playing essential roles in the formation of the chordate dorsal central nervous system function in the development of the ciliary bands and apical organ, neural structures of this non-chordate deuterostome larva.

Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi.

During ascidian embryogenesis mesenchymal cells proliferate and have no known function until after metamorphosis, when they give rise to various mesodermal tissues of the adult. Despite their simple lineage, specification mechanisms of the embryonic mesenchyme cells remain to be investigated; this is mainly due to lack of specific molecular markers for this cell type. Here we report that, in Halocynthia roretzi, zygotic expression of a cytoskeletal actin gene (HrCA1) begins at the late gastrula stage and that transcripts were predominantly distributed in embryonic mesenchyme cells, although some expression was observed in special cells of the central nervous system as well as in notochord cells. When HrCA1 expression was examined in cleavage-arrested embryos, it was found only in mesenchyme-lineage blastomeres of arrested 8-cell and later stages, consistent with the predominant expression of HrCA1 being in mesenchyme cells. To examine whether cell-cell contact until the 8-cell stage is required for mesenchyme cell specification, blastomeres were continuously dissociated during the period of 2-8-cell stages and division of the blastomeres was then arrested. Results showed that two of eight dissociated and division-arrested blastomeres from a single fertilized egg (presumably those corresponding to B4.1 cells) expressed HrCA1, suggesting that specification of embryonic mesenchyme cells does not require cell-cell contact until the 8-cell stage.