Kunifumi Tagawa

Novel pattern of Brachyury gene expression in hemichordate embryos

Together with echinoderms and chordates, hemichordates constitute the third major group of the deuterostomes, which share a number of common developmental features. The Brachyury gene is responsible for the formation of notochord, the most defining feature of chordates. Therefore, isolation and characterization of the hemichordate homolog of Brachyury is key to understand the origin and evolution of chordates. Here we show that the hemichordate Brachyury gene (PfBra) is expressed in two regions of the gastrula and young tornaria larva, the archenteron invagination region and the stomodeum invagination region.

Hemichordate genomes and deuterostome origins

Acorn worms, also known as enteropneust (literally, ‘gut-breathing’) hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal ‘gill’ slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain.

T-box genes encode a novel family of sequence-specific activators that appear to play crucial roles in various processes of animal development. Although most of the T-box genes are involved in the mesoderm formation of chordate embryos, mammalian T-Brain is expressed in the developing central nervous system, and defines molecularly distinct domains within the cerebral cortex. Here we report the first invertebrate T-Brain homologue from the hemichordate acorn worm, Ptychodera flava, which we designate Pf-Tbrain. Developmental expression of Pf-Tbrain was examined by whole mount in situ hybridization to various stages of P. flava embryos. A weak, broad in situ hybridization signal of the Pf-Tbrain transcript is first detected during gastrulation in cells around the archenteron, but this signal disappears as gastrulation proceeds. At mid-gastrula an intense signal appears in several apical ectoderm cells of the gastrula. This signal becomes restricted to the apical region, where the eyespots or the light-sensory organ of the tornaria larva form. Expression of Pf-Tbrain in the apical sensory organ of the tornaria and vertebrate T-Brain in the forebrain suggests an evolutionary relationship between the non-chordate deuterostome larval apical sensory organ and the chordate forebrain.

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.

Developmental expression of the hemichordate otx ortholog.

The phylogenetic location of hemichordates is unique because they seem to fill an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva.

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

SUMMARY 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.

Group B Sox Genes That Contribute to Specification of the Vertebrate Brain are Expressed in the Apical Organ and Ciliary Bands of Hemichordate Larvae

Abstract 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.

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

Abstract 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.

The Spawning and Early Development of the Hawaiian Acorn Worm (Hemichordate), Ptychodera flava

Abstract The spawning and early embryogenesis of the hemichordate, Ptychodera flava, in Hawaii are described in detail and illustrated with photographs of living material. Natural spawning in the evenings of early December was induced by a shift of seawater temperature from about 22°C to about 26°C. The fertilized egg divides equally and slowly at first, reaching 8 cells at about 5 hr after insemination at room temperature (20-24°C). Divisions then appear to become slightly unequal and by 9 hr the embryo has divided into about 100 cells. The blastocoel forms during cleavage as an irregular space that, when viewed from the side, tends to appear oblate and ultimately appear crescent-shaped as the vegetal plate thickens into the blastocoel. The archenteron forms at about 18 hr as a cleft beginning at the vegetal pole and extending into the vegetal plate. As development proceeds, the embryo expands and by 24 hr forms a typical deuterostome gastrula with an outer sphere of ectoderm and a inner tube of endoderm connected at the blastopore. An out-pocketing of the gut appears at the tip of the archenteron over the next 4 hr to form the protocoel which will become the proboscis coelom. Approaching 40 hr the gut becomes asymmetric and over the next few hr contacts the ectoderm to form a mouth. Hatching occurs during this time at about 45 hr of development. Morphogenesis continues to produce an early tornaria larva by about 60 hr.

Conserved Expression Pattern of BMP-2/4 in Hemichordate Acorn Worm and Echinoderm Sea Cucumber Embryos

Abstract 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 nonchor-date 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.