FATE SPECIFICATION ALONG THE SEA URCHIN EMBRYO A-V AXIS

VEB（极早期囊胚）基因是第一个严格的合子基因 在海胆胚胎中被激活;它们大约在 80%的胚胎，但不是在植物极周围的区域。 关于VEB 因此，信息分配提供了一个最早的分子 母亲指定的动植物轴的迹象。 几行 有证据表明，VEB基因活性的不对称性是由以下因素调节的： 一个或多个母体因子，其活性类似地分布在 卵和早期胚胎。 此外，一种VEB基因SpAN编码一种astacin- 家族蛋白酶，具有激活SpBMP 4的功能， TGFb超家族的成员。 因此，SpAN基因是一个强有力的候选者， 对于在非常早期的开发中的转换点， 母体基因调控信息与空间调控的 一种生长因子，可能在特化中具有重要的早期功能 细胞命运的沿着动物-植物（AV）轴，这取决于细胞- 细胞相互作用 为了检验这一总体假设，我们正在（目的一）研究 SpAN和第二个VEB基因（SpHE，其编码SpAN的空间调控）， 孵化酶），以努力了解分子机制， 创建AV轴和（目的II）检查的潜在作用 SpAN/SpBMP 4作为形态发生素。 顺式和反式作用元件 调节SpAN和SpHE转录的因子正在通过免疫组化分析。 海胆胚胎中启动子转基因构建体的体内测定和 蛋白质-DNA相互作用的体外测定。 SpAN/SpBMP 4在 将通过分离编码 SpBMP 4受体（SpBMP 4R）;通过表征发育 SpBMP 4和SpBMP 4R肽的表达;以及通过过表达， SpAN，SpBMP 4和SpAN基因的表达错误和显性负干扰， SpBMP 4R功能。 (Aim III）如果合适， VEB基因被鉴定，其功能在空间上受到限制，我们 将进行类似的实验，以测试发展的影响， 干扰其功能。

The VEB (very early blastula) genes are the first strictly zygotic genes activated in the sea urchin embryo; they are transcribed in approximately 80% of the embryo, but not in a region around the vegetal pole. The VEB message distribution thus provides one of the earliest molecular indications of the maternally specified animal-vegetal axis. Several lines of evidence suggest that the asymmetry of VEB gene activity is regulated by one or more maternal factors whose activity is similarly disposed in the egg and early embryo. Furthermore, one VEB gene, SpAN, encodes an astacin- family protease with demonstrated function in activating SpBMP4, a growth factor of the TGFb superfamily. Thus, the SpAN gene is a strong candidate for a switch point in very early development which converts prelocalized maternal gene regulatory information to spatially regulated production of a growth factor likely to have an important early function in specification of cell fates along the animal-vegetal (AV) axis, which depends on cell- cell interactions. To test this overall hypothesis we are (Aim I) studying spatial regulation of SpAN and a second VEB gene (SpHE, which encodes the hatching enzyme) in an effort to understand the molecular mechanism that creates the AV axis and (Aim II) examining the potential role of SpAN/SpBMP4 as a morphogen. The cis-acting elements and trans-acting factors which regulate SpAN and SpHE transcription are being analyze by in vivo assays of promoter transgene constructs in sea urchin embryos and by in vitro assays of protein-DNA interaction. The role of SpAN/SpBMP4 in fate specification will be studied by isolating cDNA clones encoding the receptor for SpBMP4 (SpBMP4R); by characterizing the developmental expression of SpBMP4 and SpBMP4R peptides; and by overexpression, misexpression and dominant negative interference with SpAN, SpBMP4 and SpBMP4R function. (Aim III) if suitable candidate maternal regulators of the VEB genes are identified, whose functions are spatially restricted, we shall carry out similar experiments to test the developmental effects of perturbing their function.