Yoshito Harada

Mechanism of Self-Sterility in a Hermaphroditic Chordate

Hermaphroditic organisms avoid inbreeding by a system of self-incompatibility (SI). A primitive chordate (ascidian) Ciona intestinalis is an example of such an organism, but the molecular mechanism underlying its SI system is not known. Here, we show that the SI system is governed by two gene loci that act cooperatively. Each locus contains a tightly linked pair of polycystin 1–related receptor (s-Themis) and fibrinogen-like ligand (v-Themis) genes, the latter of which is located in the first intron of s-Themis but transcribed in the opposite direction. These genes may encode male- and female-side self-recognition molecules. The SI system of C. intestinalis has a similar framework to that of flowering plants but utilizing different molecules.

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.

Spatial expression of a forkhead homologue in the sea urchin embryo

Echinoderms are the sister group of the chordates and hemichordates within the deuterostomes. They lack a notochord or any structures obviously homologous with it. To gain insight into developmental mechanisms important in the origin and early evolution of chordates, we investigated sea urchin homologues of chordate genes that are implicated in notochord formation, viz. Brachyury and HNF-3 beta. Here we report the pattern of expression of a sea urchin orthologue of forkhead, Hphnf3 which is present as a single copy per haploid genome. An Hphnf3 transcript of 3.0 kb was first detected at the swimming blastula stage, accumulated maximally at the gastrula and prism-embryo stages, and decreased at the pluteus-larva stage. In situ hybridization signals were found in cells of the vegetal plate of the swimming blastula. During gastrulation, intense staining was evident in the cells surrounding the blastopore, whereas weak staining was detected in the invaginating archenteron. At the prism-embryo stage, the entire archenteron stained intensely; then, at pluteus stage, the larva staining decreased in intensity. The forkhead and Brachyury genes begin to be expressed almost simultaneously in sea urchin embryos, in the vegetal plate at the late blastula stage. After the onset of gastrulation, however, Hphnf3 is expressed in the posterior part of the archenteron, whereas the Brachyury orthologue, HpTa, is expressed in the secondary mesenchyme founder cells, which occupy the anterior tip of archenteron. Hphnf3 may contribute to specification of embryonic cells as archenteron, and the role of HpTa may be directed towards specification of mesodermal founder cells. Except for the basal character of expression in endoderm and endomesoderm, these transcription factors are clearly utilized differently in chordates.

Comprehensive Egg Coat Proteome of the Ascidian Ciona intestinalis Reveals Gamete Recognition Molecules Involved in Self-sterility

Despite central roles of egg coat proteins in gamete recognition, their functions and composition are poorly understood. Here, we report that the proteome of the egg coat in the solitary ascidian Ciona intestinalis, called vitelline coat (VC) fraction, contains more than 800 proteins identified by mass spectrometry-based analyses. Over 100 proteins were enriched in the VC fraction compared with the VC-free egg proteome. The most abundant component in the VC was an apolipoprotein-like protein. The VC contained multiple homologs of mammalian zona pellucida (ZP) proteins, the number of which was unexpectedly large and most of which possessed epidermal growth factor-like repeats. Furthermore, the present study revealed that two fibrinogen-like proteins, v-Themis-A and -B, both of which are expressed in the VC, are the molecules responsible for the two self-sterility loci that were identified by our previous genetic study in this species.

Expression of the Otx gene in the ciliary bands during sea cucumber embryogenesis

Summary: The Otx gene encodes a homeodomain transcription factor that has a highly conserved role in brain formation of both flies and vertebrates. To deduce evolutionary relationship of the chordate central nervous system to the larval or adult nervous system of nonchordate deuterostomes, we characterized the expression of the Otx gene (Sj‐Otx) throughout the entire embryonic and larval development of the sea cucumber Stichopus japonicus. Sj‐Otx transcripts were detected in fertilized eggs and in the posterior part of the archenteron of gastrulae. However, the expression was downregulated as embryos developed into auricularia larvae. Sj‐Otx was expressed again in the ciliary bands of late auricularia larvae, just before metamorphosis to doliolaria larvae. The expression domain corresponded to the domains moving to the mouth during metamorphosis and sinking into the buccal cavity, but not to the five transverse ciliary bands of the doliolaria. The expression gradually disappeared during further development and was not detected in juveniles. These results indicate that the gene responsible for chordate brain formation is expressed in the ciliary bands of auricularia larvae. genesis 27:58–63, 2000.

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.

Characterization of a hemichordate fork head/HNF-3 gene expression

Abstract Based on anatomical and developmental similarities, hemichordates are thought to be most closely related to chordates. However, so far very few developmental genes have been characterized from hemichordates. To gain molecular insight into the developmental mechanisms involved in the origin and evolution of chordates, we investigated the expression of a fork head/HNF-3 (PfHNF3) gene in the acorn worm embryo. Chordate fork head genes are implicated in the formation of endoderm, notochord and floor plate. We found that a PfHNF3 transcript was first detected at the early blastula stage; the signal of in situ hybridization was found in the vegetal plate cells, invaginating endoderm and then in the archenteron. By the late gastrula and into the early tornaria larva stages, an intense signal remained in the anterior region of the archenteron, while the expression in the other regions of archenteron decreased. The intense signal was retained in the pharynx of the tornaria larva. A comparison of the pattern of PfHNF3 with that of HNF-3 genes of sea urchin, ascidian, amphioxus and vertebrate suggests a possible acquisition of new functions of the gene during deuterostome evolution.

Allorecognition mechanisms during ascidian fertilization.

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.

Ascidian Sperm Glycosylphosphatidylinositol-anchored CRISP-like Protein as a Binding Partner for an Allorecognizable Sperm Receptor on the Vitelline Coat

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

The expression of nonchordate deuterostome Brachyury genes in the ascidian Ciona embryo can promote the differentiation of extra notochord cells.

The notochord is a structure present in all chordates and its development requires the transcription of Brachyury. While previous studies have shown that Brachyury is essential for notochord formation in vertebrate embryos, this gene is also expressed during the embryogenesis of nonchordate deuterostomes, hemichordates and echinoderms. Here we report that nonchordate deuterostome Brachyury genes can trigger the differentiation of extra notochord cells when these genes are ectopically expressed in ascidian embryos. The 2.6 kb upstream region of fork head gene (Ci-fkh) of Ciona intestinalis promotes the tissue-specific expression of a reporter gene in endoderm, notochord and nerve cord. By taking advantage of this promoter, we misexpressed the Brachyury gene of ascidian (Ci-Bra), cephalochordate amphioxus (Am(Bb)Bra2), hemichordate acorn worm (PfBra), and echinoderm sea urchin (SpBra), in Ciona embryos. The misexpression of not only the chordate Brachyury, but also the nonchordate deuterostome Brachyury, resulted in the transformation of presumptive endodermal cells into notochord cells. This was confirmed by in situ hybridization experiments using four different notochord-specific probes from Ciona that have different temporal expression patterns. RT-PCR analyses indicated that Ci-Bra was not upregulated by the product of Am(Bb)Bra2, PfBra or SpBra. In situ hybridization showed no ectopic expression of Ci-Bra in the manipulated embryos. These results suggest that the introduction of nonchordate deuterostome Brachyury genes into ascidian embryos can trigger the differentiation of notochord cells in ascidian embryos. Evolutionary alteration in the genetic circuitry, especially downstream of this transcription factor, seems critical for the evolution of notochord and chordate body plan.