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.     

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 Distinctive Nerve Cell Type Common to Diverse Deuterostome Larvae: Comparative Data from Echinoderms,Hemichordates and Amphioxus

Abstract The larval ciliary bands of echinoderm bipinnaria and pluteus larvae and the hemichordate tornaria contain similar multipolar or bipolar nerve cells with unusual apical processes that run across the surface of the band between the bases of its cilia. We report on some distinctive ultrastructural features of these cells. Among these are specialized junctions that occur between the cells' apical processes and adjacent ciliary band cells near the base of each cilium. Such structures are best developed in pluteus larvae. Many nerve cells in the larval spinal cord of amphioxus also have large apical processes that cross the central lumen of the cord. We interpret our observations on these cells in terms of Garstang's hypothesis, which derives the chordate neural tube from a larval ciliary band, and suggest that multipolar cells like those in echinoderm and tornaria bands may be the antecedents of some categories of neurons in the chordate spinal cord.     

Structure of the nervous system in the tornaria larva of<Emphasis Type="Italic"> Balanoglossus proterogonius</Emphasis> (Hemichordata: Enteropneusta) and its phylogenetic implications

The tornaria larva of hemichordates occupies a central position in phylogenetic discussions on the relationships between Echinodermata, Hemichordata, and Chordata. Dipleurula-type larvae (tornaria and echinoderm larvae) are considered to be primary in the life cycle and thus provide a model for the ancestral animal common to all three taxa (the theory of W. Garstang). If the similarities between tornaria and the larvae in Echinodermata result from homology, their nervous systems should be basically similar as well. The present study utilizes anti-serotonin and FMRFamide antisera together with laser scanning microscopy, and transmission electron microscopy, to describe in detail the nervous system of the tornaria of Balanoglossus proterogonius. Serotonin immunoreactive neurons were found in the apical and esophageal ganglia, and in the stomach epithelium. FMRFamide immunoreactive neurons, probably sensory in nature, were detected in the apical ganglion and in the equatorial region of the stomach epithelium. At the ultrastructural level, the apical organ consists of a columnar epithelium of monociliated cells and includes a pair of symmetrical eyespots. The apical ganglion is located at its base and has a well-developed neuropil. Different types of neurons are described in the apical organ, esophagus, and stomach. Comparison with larvae in Echinodermata shows several significant differences in the way the larval nervous system is organized. This calls into question the homology between tornariae and echinoderm larvae. The possibility of convergence between the two larval types is discussed.     

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues

The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals. DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly. Recent evidence also suggests that they are developmentally regulated in chordate embryos. bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata. Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al. 1999). We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development. We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles. In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos. Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog. Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian. Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.     

Roles of Hroth, the ascidian otx gene, in the differentiation of the brain (sensory vesicle) and anterior trunk epidermis in the larval development of Halocynthia roretzi

Otx genes are expressed in the anterior neural tube and endoderm in all of the chordates so far examined. In mouse embryos, important roles of otx genes in the brain development have been well documented. However, roles of otx genes in other chordate species have been less characterized. To advance our understanding about roles of otx genes in chordates, we have studied Hroth, otx of the ascidian, Halocynthia roretzi. Hroth is expressed in the anterior part of the neural tube (the sensory vesicle), the endoderm and anterior epidermis in the development. In this study, we investigated roles of Hroth in the larval development through an antisense morpholino oligonucleotides (MOs) approach. Embryos injected with Hroth-targeting MO (Hroth knockdown embryos) developed into larvae without the adhesive organ, sensory pigment cells and cavity of the sensory vesicle. The tissues, in which defects were observed, are derived from anterior-animal cells of the embryo in early cleavage stages. During cleavage stages, Hroth is also expressed in the endoderm precursors of the vegetal hemisphere. However, Hroth expression in the anterior endoderm precursors do not seem to be essential for the above defects, since MO injection into the anterior-animal but not anterior-vegetal pair cells at the 8-cell stage gave the defects. Analysis of marker gene expression demonstrated that the fate choice of the sensory vesicle precursors and the specification of the sensory vesicle territory occurred normally, but the subsequent differentiation of the sensory vesicle was severely affected in Hroth knockdown embryos. The anterior trunk epidermis including the adhesive organ-forming region was also affected, indicating that anterior epidermal patterning requires Hroth function. Based on these findings, similarities and differences in the roles of otx genes between ascidians and mice are discussed.