CONTROL OF METAMORPHOSIS IN MOLLUSC LARVAE

长期目标是提高信息和通信技术的实用性和“连通性”， 一个容易培养的鲍（软体动物）幼虫作为模型系统， 生物医学研究 这些幼虫的发育是在 通过外源形态发生素的化学感受识别;受体， 已经发现，参与这种控制的信号传导器和途径 与哺乳动物系统中的对应物密切同源。 我们建议 通过描述链路，增加模型系统的连通性 化学感受信号识别/转导机制之间的联系 在分子水平上表征， 调节特定靶基因表达的机制，细胞 分化和增殖。 这项建议的具体目标是：（1）确定“主要 它们可能编码的“反应”基因和转录调节因子， 鲍幼鲍变态和仔虾早期生长对环境影响 对外源性形态发生素的化学感受识别;（2）识别 这些基因和转录激活因子的分子机制 与基因和组织特异性顺式作用DNA（和mRNA）相互作用 调控序列，以控制必需的“二级”的表达， 反应”的靶基因，导致变态;（3）确定，通过 G蛋白和磷蛋白cDNA的分析， 受体调节的G蛋白和蛋白质 磷酸化抑制了幼虫产生的诱导信号 识别外源性化学形态发生素;（4）建立 化学感受信号依赖基因机制的相关性 本研究发现的表达与哺乳动物中的相应机制有关。 形态发生剂诱导的初级应答基因转录物及其cDNA将被 通过（a）使用高度保守的PCR扩增和杂交纯化 对应于哺乳动物转录因子（c-myc， c-fos、c-jun和同源异型蛋白基因家族），以及通过（B）直接分离 通过消减杂交。 这些将被克隆和测序， 其蛋白质产物的作用机制和位置， 原位杂交和用抗- 正义RNA。 这些形态发生素诱导的转录因子的作用机制 将进一步通过对监管机构的足迹分析来确定其特征。 它们在靶基因组DNA序列上相互作用的位点 （二次反应）基因。 拟议研究的结果应该是重要的， 详细了解分子机制， 外源形态发生素和调节化学物质的化学感受识别 信号控制基因表达、分化和细胞 在多细胞生物中的增殖。 这种联系是根本的 在正常发育过程中的生物医学重要性，激素 调节，伤口愈合，抗体合成，长期记忆， 癌 因此，拟议的研究应增加 鲍幼虫作为一个具有高度连通性的模型系统，以研究 这些地区

The long-term objective is to increase the usefulness and "connectivity" of easily cultured Haliotis (molluscan) larvae as a model system for biomedical research. Development in these larvae is synchronously induced by chemosensory recognition of an exogenous morphogen; the receptors, signal transducers and pathways involved in this control have been found to be closely homologous to counterparts in mammalian systems. We propose to increase the connectivity of this model system by characterizing the link between the chemosensory signal recognition/transduction mechanisms already characterized at the molecular level, and the developmental and genetic mechanisms that regulate specific target gene expression, cellular differentiation and proliferation. The specific aims of this proposal are: (1) to identify the "primary response" genes and transcription regulators they may encode, that control larval metamorphosis and early post-larval growth in Haliotis in response to chemosensory recognition of exogenous morphogens; (2) to identify the molecular mechanisms by which these genes and transcription activators interact with gene- and tissue-specific cis-acting DNA (and mRNA) regulatory sequences to control the expression of essential "secondary response" target genes, leading to metamorphosis; (3) to identify, through the analysis of G protein and phosphoprotein cDNAs, the centrally important mechanisms by which receptor-regulated G proteins and protein phosphorylations transduce the inductive signals generated by larval recognition of exogenous chemical morphogens; and (4) to establish the relatedness of the mechanisms of chemosensory signal-dependent gene expression found in this research to corresponding mechanisms in mammals. Morphogen-induced primary response gene transcripts and their cDNAs will be purified by (a) PCR-amplification and hybridization using highly conserved oligonucleotides, corresponding to mammalian transcription factors (c-myc, c-fos, c-jun, and homeoprotein gene families), and by (b) direct isolation by subtractive hybridization. These will be cloned and sequenced, and the mechanisms and locations of action of their protein products determined by in situ hybridization, and blockade of target gene induction with anti- sense RNA. The mechanisms of these morphogen-induced transcription factors will be further characterized by footprinting analyses of the regulatory sites with which they interact on the genomic DNA sequences of their target (secondary response) genes. Results of the proposed studies should be significant in providing the most detailed understanding yet available of the molecular mechanisms by which chemosensory recognition of exogenous morphogens and regulatory chemical signals controls gene expression, differentiation and cellular proliferation in multicellular organisms. This link is of fundamental biomedical importance in the processes of normal development, hormonal regulation, wound healing, antibody synthesis, long-term memory, and cancer. The proposed research thus should increase the usefulness of the Haliotis larvae as a model system with high connectivity to research in these areas.