The importance of gonadotropins and androgens for spermatogenesis is generally accepted in vertebrates, but the role played by specific hormones has not been clarified. Under cultivation conditions, male Japanese eels (Anguila japonica) have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia. In the present study, a recently developed organ-culture system for eel testes was used to determine in vitro effects ofvarious steroid hormones on spermatogenesis. After 9 days of culture in serum-free, chemically defined medium. containing 11-ketotestosterone (10 ng/ml), a major androgen in male eels, type A and early-type B spermatogonia began mitosis, producing late-type B spermatogonia. After 18 days, zygotene spermatocytes with synaptonemal complexes appeared, indicating that melosis had already started by this time. In testis fragments cultured for 21 days, round spermatids and spermatozoa were observed with spermatogenic cells at all stages of development. Addition of 11-ketotestosterone to the culture medium also caused a marked cytological activation of Sertoli cells. No other steroid hormones tested had such stimulatory effects. These results, together with our earlier observations, suggest the following sequence for the hormonal induction of spermatogenesis Wi eel testes; gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which, in turn, activates the Sertoli cells leading to the completion of spermatogenesis. This is, thus, an example of an animal system in which all stages of spermatogenesis have been induced by hormonal manipulation in vitro.The formation of sperm, spermatogenesis, is an extended process that begins with the proliferation of spermatogonia and proceeds through the extensive morphological changes that convert the haploid spermatid into a mature, functional spermatozoon. Although it is generally accepted that the principal stimuli for vertebrate spermatogenesis are pituitary gonadotropins and androgens, the specific role played by individual hormones has not been clarified (1-4). A number of factors complicating in vivo investigations of the mechanisms involved in the spermatogenesis can be eliminated in in vitro organ (5, 6) and cell (7-10) culture systems in which the direct effects of various factors, including hormonal influences, upon the spermatogenic cells and testes can be investigated.Under conditions of cultivation, male Japanese and European eel have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia (11)(12)(13)(14)(15). It has been reported that in both species a single injection of exogenous human chorionic gonadotropin induces all stages of spermatogenesis in vivo (12,14,15). This injection also caused an increase in plasma levels of 11-ketotestosterone (12, 15). Thus, the eel testis provides an excellent system for studying the mechanism by which spermatogenesis is regulated. In the present study, we have used a recently developed organ culture system for eel te...

The ascidian chordate Ciona intestinalis is an established model organism frequently exploited to examine cellular development and a rapidly emerging model organism with a strong potential for developmental systems biology studies. However, there is no standardized developmental table for this organism. In this study, we made the standard web-based image resource called FABA: Four-dimensional Ascidian Body Atlas including ascidian's three-dimensional (3D) and cross-sectional images through the developmental time course. These images were reconstructed from more than 3,000 high-resolution real images collected by confocal laser scanning microscopy (CLSM) at newly defined 26 distinct developmental stages (stages 1-26) from fertilized egg to hatching larva, which were grouped into six periods named the zygote, cleavage, gastrula, neurula, tailbud, and larva periods. Our data set will be helpful in standardizing developmental stages for morphology comparison as well as for providing the guideline for several functional studies of a body plan in chordate.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential CiBra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Received March 22, 1999; revised version accepted May 3, 1999. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain (Herrmann et al. 1990;Kispert et al. 1995;Conlon et al. 1996). In vertebrates, Brachyury is initially expressed throughout the presumptive mesoderm, and during later stages the expression pattern is gradually restricted to the developing notochord and tailbud. Brachyury notochord differentiation is essential in all vertebrates that have been studied, including mice, frogs, and zebrafish (for review, see Herrmann and Kispert 1994;Smith 1997;Papaioannou and Silver 1998).Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, Halocynthia roretzi (Yasuo and Satoh 1993) and Ciona intestinalis (Corbo et al. 1997a). The spatial and temporal patterns of the gene expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm (Nakatani and Nishida 1994). Overexpression of the Halocynthia Brachyury gene (As-T) via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage (Yasuo and Satoh 1998). In addition, misexpression of As-T also causes transformation of endoderm and neuronal lineages into notochord (Yasuo and Satoh 1998). These results indicate that the ascidian Brachyury gene is a critical determinant of the notochord. Here we report that the misexpression of the Brachyury gene (Ci-Bra) of C. intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra overexpression. We isolated and characterized 38 different notochord-specific genes that may include potential targets of the ascidian Brachyury. Results and DiscussionThe fork head/HNF-3␤ gene of C. intestinalis (Ci-fkh) is expressed in the endoderm, endodermal strand, notochord, and ventral ependymal cells of the neural tube (Corbo et al. 1997b). A 2.6-kb genomic DNA fragment from the 5Ј-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into one-cell embryos (Fig. 1A). The Ci-Bra gene was misexpr...

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.