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XATH-1, a basic/helix-loop-helix transcription factor and a homolog of Drosophila atonal and mammalian MATH-1, is expressed specifically in the dorsal hindbrain during Xenopus neural development. In order to investigate the role of XATH-1 in the neuronal differentiation process, we have examined the effects of XATH-1 overexpression during Xenopus development. XATH-1 induces the expression of neuronal differentiation markers, such as N-tubulin, within the neural plate as well as within nonneural ectodermal progenitor populations, resulting in the appearance of process-bearing neurons within the epidermis. The related basic/helix-loop-helix genes neurogenin-related-1 and neuroD are not induced in response to XATH-1 overexpression within the embryo, suggesting that XATH-1 may activate an alternate pathway of neuronal differentiation. In further contrast to neurogenin-related-1 and neuroD, high-level expression of general neural markers expressed earlier in development, such as N-CAM, is not induced by XATH-1 overexpression. Competent ectodermal progenitors therefore respond to ectopic XATH-1 expression by initiating a distinct program of neuronal differentiation.

An experiment conducted to study skeletal muscle development in the mouse mutant peroneal muscular atrophy (pma) muscular indicate that the formation of secondary myotubes is not nerve-dependent. A higher ratio of secondary to primary myotubes was recorded in the pma as compared to normal strains. Furthermore, no difference was observed between aneural and normal muscles in terms of myotube ratio. These results, along with similar studies conducted on chicks, indicate that innervation doe not directly affect secondary myotube formation.

The mouse mutants peroneal muscular atrophy and muscular dysgenesis were used to study the effects of muscle innervation, electrical activity and contraction on the development of myotubes during embryonic development. During embryonic day 15, the ratio of secondary to primary myotubes was three times greater than normal. Furthermore, administration of tetrodoxin to induce paralysis significantly reduced the number of secondary myotubes and resulting in a smaller ratio. These results indicate that secondary myotube formation depends on the electrical activity produced by primary myotubes.

An experiment was conducted to test whether the formation of secondary myotubes in aneural fetal muscles is dependent on the presence of primary myotubes or on innervation. Attempts to suppress secondary myotube formation using the neurotoxic agent Beta-bungarotoxin (Beta-BTX) indicated that the influence of Beta-BTX is dependent on the timing of injection. Results provided evidence for the hypothesis that secondary myotube formation is nerve-dependent.

We have investigated the role of the bone morphogenetic protein (BMP) pathway during neural tissue formation in the ascidian embryo. The orthologue of the BMP antagonist, chordin, was isolated from the ascidian Halocynthia roretzi. While both the expression pattern and the phenotype observed by overexpressing chordin or BMPb (the dpp-subclass BMP) do not suggest a role for these factors in neural induction, BMP/CHORDIN antagonism was found to affect neural patterning. Overexpression of BMPb induced ectopic sensory pigment cells in the brain lineages that do not normally form pigment cells and suppressed pressure organ formation within the brain. Reciprocally, overexpressing chordin suppressed pigment cell formation and induced ectopic pressure organ. We show that pigment cell formation occurs in three steps. (1) During cleavage stages ectodermal cells are neuralized by a vegetal signal that can be substituted by bFGF. (2) At the early gastrula stage, BMPb secreted from the lateral nerve cord blastomeres induces those neuralized blastomeres in close proximity to adopt a pigment cell fate. (3) At the tailbud stage, among these pigment cell precursors, BMPb induces the differentiation of specifically the anterior type of pigment cell, the otolith; while posteriorly, CHORDIN suppresses BMP activity and allows ocellus differentiation. Key Words: neural induction; neural patterning; sensory pigment cell; BMP; CHORDIN; bFGF; ascidian.

In C. elegans mail tail, the peripheral sensory structures comprise of nine pairs of rays embedded in a cuticular fan. Each ray has its own specific morphological and functional identity as defined by a group of regulatory genes. Among them, mab-21 regulates ray 6 formation . Since its isolation, mab-21's cellular role is by large unknown. To establish the role of mab-21 in normal development, we have tried to dissect the elements in the mab-21 locus. A minimal length of genomic fragment that can efficiently rescue the mab-21 mutant male tail was defined. The results showed that mab-21 locus contains many transcriptional regulatory elements including hypodermal, neuronal and activity enhancer. We present data showing that not all these enhancer elements are required for the ray development. Knowing the localization of the gene product is crucial for addressing the mab-21 function. A full length MAB-21::GFP fusion protein was employed as a reporter. We found that the fusion protein can efficiently rescue the mab-21 mutant sensory ray phenotype. The green fluorescence signal could be detected in the hypodermal tissue, neuronal cells as well as a group of ray cells. The expression are reflective to the phenotype we observed in mab-21 mutant; relatively short body length, backward uncoordinated movement and ray fusion. Taken together, we can define the elements in the locus which are required for wild type mab-21 expression and sensory ray differentiation in the male tail. (The research is supported by the Research Grant Council, Hong Kong.)

Fibroblast growth factor (FGF) signaling has been shown to play a key role in the mouse trophoblast development. To dissect the molecular mechanism underlies this process, we have studied the in vivo functions of two potential FGF receptor 2 (FGFR2) downstream targets, Eomesodermin (mEomes), a novel T-box gene, and Cdx2, a mouse homologue of Drosophila caudal. Members of both gene families have been postulated to be regulated by FGF signaling in several organisms. mEomes expression is first detected in the trophectoderm of the blastocyst. In the post-implantation embryos, mEomes is expressed in the extraembryonic ectoderm, the primitive streak, the posterior third of the epiblast, and the nascent mesoderm. Our data show that embryos homozygous for mEomes underwent implantation and induced uterine decidualization, yet failed to develop to egg cylinders due to the lack of trophoblast development. In vitro culture of mutant blastocysts further demonstrate that mEomes is essential for trophoblast growth and is required for trophoblast stem cell (TS) formation. The peri-implantation defect of mEomes-/- embryos is very reminiscent to mutants of FGF4 and FGFR2, suggesting that mEomes may t)e a direct downstream factor mediating FGF4 signaling for trophoblast formation. Cdx2 is also expressed in the trophectoderm of the blastocyst and later' in some trophectoderm derived tissues. Cdx2 mutant embryos die peri-implantation, presumably due to deflects in trophoblast development. To gain more insight into whether these genes function in FGF regulated trophoblast development, we are analyzing trophoblast development and regulation of trophoblast-specific gene expression in single and compound mutant embryos.

Members of protein kinase C family are involved in regulating a broad range of cellular events including growth and differentiation. It has been shown in a variety of cell types that individual isozymes of protein kinase C have cell type-specific expression, and for a given isozyme the expression is locally restricted to particular subcellular sites, indicating functional specificity of these isozymes. Several studies have indicated the involvement of the protein kinase C family in the regulation of mammalian egg activation, start of embryonic transcription, embryonic compaction and blastocoel formation. However, it is not known which protein kinase C isozymes are present during this period of mammalian development. We have used immunocytochemistry and confocal laser scanning microscopy to determine the subcellular localization of eleven members of protein kinase C family ([Alpha], [Beta] I, [Beta] II, [Gamma], [Delta], [Epsilon], [Eta], [Theta], [Iota], [Lambda], and [Zeta]) in 2-, 4-, 8-cell, compacted, and blastocyst stages of the murine preimplantation embryo.

In Drosophila, Gliotactin is necessary for the development of the glial sheath which protects the peripheral nerves from exposure to hemolymph. Gliotactin belongs to a family of transmembrane serine-esterase like proteins which are important for the formation of cellular junctions. Evolutionary analysis of other members of this family of proteins showed that neuroligins were most closely related to Gliotactin. Using a reverse genetic approach we have isolated the vertebrate homolog of Gliotactin which corresponds to Neuroligin 3. Using RT-PCR and immunostaining we have shown that Neuroligins 2 and 3 are expressed in dorsal root ganglia, and in cultured Schwann cells. Neuroligin 3 is expressed in the olfactory ensheathing glia of the olfactory epithelium. In addition, Neuroligin 1 and/or Neuroligin 3 are expressed in astrocytes in developing mouse spinal cord and retina. The glial expression of the neuroligins, close evolutionary relationship, and interactions with related proteins suggests that these proteins may function in glial wrapping of peripheral axons in vertebrates. This work is supported by RHNI, HHMI and BCHRF.

The rat choriocarcinoma cell line (Rcho-1) has long been used as a model for trophoblast giant cell differentiation because it can be manipulated to proliferate as stem cells or differentiate into giant cells. Differentiation is characterized by cessation of mitosis, expression of marker proteins, and by prominent changes in cell size, shape and adhesive behavior. We have characterized the changes in organization of the actin cytoskeleton and focal contacts during Rcho-1 cell differentiation, and correlated these results to analyses of Rcho-1 cell motility. Additionally, we have investigated the roles played by the small GTPases, whose activities are altered during Rcho-1 differentiation. The stem cells have no organized actin stress fibers, but have small, peripheral focal complexes. They also have a high rate of protrusive activity and increased motility: they migrate randomly while forming many protrusions, including filopodia and lamellipodia. This correlates with their low Rho, but high Rac and Cdc42 activities. The differentiated cells, on the other hand, have very prominent stress fibers and large internal and peripheral focal contacts. They also exhibit decreased motility compared to the stem cells, and very little or no membrane protrusive activity. This corresponds with their high Rho but low Rac and Cdc42 activities. In addition, tyrosine phosphorylation of FAK is high in the stem cells, but decreases with differentiation; also FRNK expression is only detected in differentiated cells. Changes in Rcho-1 cells by adenovirus-mediated expression of wild-type and mutant forms of Rho GTPases will be further discussed. NIH grant HD34807.

The congruence of neural and vascular distribution patterns in children and adults(2) has important implications for reconstructive surgery. Anecdotal evidence suggests that this congruence arises during embryogenesis(1). The aim of this study is to produce an accurate spatio-temporal, graphical record of peripheral nerve and blood vessel development, and establish whether these structures maintain a close spatial relationship to one another. Quail embryos from E2.5-7 were examined with the aid of double labeling fluorescence and confocal microscopy. Blood vessels are present in the quail forelimb and change their initial pattern before the in-growth of nerves from the neural tube and dorsal root ganglia. A highly stereotyped sequence of changes in the peripheral pattern of both nerves and blood vessels occurs with both structures maintaining a close spatial relationship. This can be visualised down to the level of single leading axons advancing and branching along major blood vessels. This close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. 1. H. Q. Miao, et al., Journal of Cell Biology 146, 233-241 (1999). 2. G. Taylor, M. Gianoutsos, S. Morris, Plastic and Reconstructive Surgery 94, 1-36 (1994).