Molecular mechanisms of cell fate specification

细胞命运规范的分子机制

• 批准号: 7318848
• 负责人: LYNNE M ANGERER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7318848
• 关键词: Molecular; mechanisms; cell; fate; specification

Our laboratory investigates mechanisms of cell fate specification and patterning along the animal?vegetal (A?V) axis of the sea urchin (Strongylocentrotus purpuratus) embryo. Our major focus is to understand the gene regulatory networks and signaling pathways that specify ectodermal domains, which that derive from an undifferentiated pre-ectoderm and form the aboral, oral, neural and ciliagenic ectodermal territories. DEVELOPMENT OF RESOURCES AND TOOLS FOR MINING AND ANNOTATING THE SEA URCHIN GENOME. (33%; Zheng Wei, Staff Scientist). A major achievement was the creation of a gene list, using the gene prediction program, Genscan, which contributed to a composite gene list for the annotation of nearly 10,000 genes in the sea urchin genome. We used the gene predictions to design a whole-genome microarray for temporal profiling of mRNA expression at 5 embryonic stages, using 5 probes per gene on high density arrays (Nimblegen, Inc). The temporal profiles of a large number of well-studied genes have been confirmed and an expression database for about 28,000 different predicted genes is available to the research community for further studies. Several groups have expressed interest in using these microarrays as tools to characterize effects of experimental manipulations at the level of gene expression, and to identify additional genes in regulatory networks. MECHANISMS OF SPECIFICATION OF CELL FATES IN THE ANIMAL POLE DOMAIN (30%; Lynne Angerer, Senior Scientist; Shunsuke Yaguchi, Visiting Fellow). We are interested in understanding the gene regulatory networks underlying the initial specification of cell fates in the animal pole domain (APD) of the sea urchin embryo. This region is special because APD regulatory genes are refractory to repression by vegetal signals. The APD contains the precursors to the 6 serotonergic neurons of the embryonic nervous system and our specific goal is to identify genes constituting the core neurogenic gene regulatory network. The major approach this year has been to exploit the newly completed genome sequence to identify candidate genes encoding transcription factors that are expressed in neurogenic ectoderm. To date, 58 such genes have been identified in the sea urchin genome, 34 of which are expressed during embryogenesis in the APD, some exclusively in this region and some at unexpectedly early stages, well before gastrulation. Interestingly, four genes have been identified as encoding transcription factors that operate at the beginning of neural cell specification in a diverse set of organisms, including the coelenterates, an extremely ancient group in evolution, suggesting that these genes constitute part of the core neurogenic regulatory network of metazoa. The regulatory relationships among candidate neurogenic factors will be tested by blocking translation of each mRNA with morpholino antisense oligonucleotides (MASO) and assaying the consequent effects on expression of other genes using whole mount in situ hybridization and quantitative PCR. TGF-BETA SIGNALING IN ENDODERM DEVELOPMENT (33%; Aditya Sethi, Visiting Fellow). We have found that a TGF-beta signaling pathway is required for the development of early endoderm in the sea urchin embryo. Blocking this pathway with a small molecule inhibitor of the receptor, Alk4, results in significant delays and reduced expression of a specific set of early endoderm marker genes and in gastrulation. We have identified the ligand as activin B, the only TGF-beta, besides nodal, that functions through Alk4 and is expressed in the early embryo. Abrogation of activinB synthesis by either of two morpholino antisense oligonucleotides produces the same phenotype as does Alk4 inhibition, at both molecular and morphological levels. ActivinB signals are required in a subset of blastomeres fated to become endoderm, strongly suggesting that it is the long-sought early signaling pathway from underlying micromeres, which is thought to be one of the first signals in endoderm development downstream of beta-catenin nuclearization. SOXB1 AND NUCLEAR BETA-CATENIN CROSS-REGULATORY MECHANISMS (4%; Zheng Wei, Staff Scientist). SoxB1 is a key regulator of endomesoderm specification during late cleavage/early blastula stages. SoxB1 functions at the top of the endomesoderm gene regulatory network as an inhibitor of signaling through the canoniical Wnt signaling pathwy, by antagonizing nuclear beta-catenin. Conversely, beta-catenin, acting as a transcription cofactor, clears SoxB1 both by repression of transcription and, unexpectedly, through spatially regulated turnover of SoxB1 protein. We are testing whether mutual antagonism involves direct physical interaction between SoxB1 and beta?catenin. Yeast two-hybrid assays show that both full-length and the C-terminal half of SoxB1, required for beta-catenin-dependent SoxB1 turnover, can interact with sea urchin beta-catenin. Co-immunoprecipitation assays will test whether these proteins interact in the sea urchin embryo and may identify additional interacting proteins involved in critical early cross-regulation of SoxB1 and beta-catenin.

我们的实验室研究了海胆(Strongyloctrotus PurPuratus)胚胎的细胞命运指定和沿动物-植物(A？V)轴的模式形成的机制。我们的主要重点是了解特定外胚层区域的基因调控网络和信号通路，外胚层区域来源于未分化的前外胚层，形成流产的、口腔的、神经的和纤毛的外胚层区域。 开发用于挖掘和注释海胆基因组的资源和工具。(33%；郑伟，工作人员科学家)。一项主要成就是使用基因预测程序Genscan创建了一个基因列表，该程序为海胆基因组中近10,000个基因的注释提供了一个复合基因列表。我们使用基因预测设计了一个全基因组微阵列，用于在高密度阵列上使用每个基因5个探针的时间图谱分析5个胚胎阶段的mRNA表达(NimbleGen，Inc.)。大量研究充分的基因的时间分布已经得到确认，大约28,000个不同预测基因的表达数据库可供研究界进一步研究。有几个小组表示有兴趣使用这些微阵列作为工具，在基因表达水平上表征实验操作的效果，并在调控网络中识别更多的基因。 动物极地领域中细胞命运的指定机制(30%；Lynne Angerer，高级科学家；Shunsuke Yaguchi，访问研究员)。我们感兴趣的是理解海胆胚胎动物极域(APD域)细胞命运最初指定的基因调控网络。这个区域是特殊的，因为apd调节基因对植物信号的抑制是难以实现的。Apd含有胚胎神经系统6个5-羟色胺能神经元的前体，我们的具体目标是鉴定构成核心神经源性基因调控网络的基因。今年的主要方法是利用新完成的基因组序列来识别编码转录因子的候选基因，这些转录因子在神经性外胚层中表达。到目前为止，已在海胆基因组中鉴定出58个这样的基因，其中34个基因在apd的胚胎发育过程中表达，一些只在这个区域表达，另一些基因在出人意料的早期阶段，很久以前就在原肠形成之前表达。有趣的是，有四个基因被确定为编码转录因子，这些转录因子在包括腔肠动物在内的各种生物中在神经细胞规格开始时工作，腔肠动物是进化中的一个极其古老的群体，这表明这些基因构成了后生动物的核心神经发生调控网络的一部分。候选神经源性因子之间的调控关系将通过用吗啉反义寡核苷酸(Maso)阻断每个mRNA的翻译，并使用整体原位杂交和定量PCR来分析其对其他基因表达的影响。 内胚层发育中的转化生长因子-β信号(33%；Aditya Sethi，访问研究员)。我们发现，在海胆胚胎的早期内胚层的发育过程中需要一条转化生长因子-β信号通路。用受体的小分子抑制剂Alk4阻断这一途径会导致一组特定的早期内胚层标记基因和原肠形成的显著延迟和表达减少。我们已经确定该配体为激活素B，除了结节外，唯一的转化生长因子-β通过Alk4发挥作用，并在早期胚胎中表达。在分子和形态水平上，用两种吗啉基反义寡核苷酸中的任何一种来阻断ActivinB的合成都会产生与Alk4抑制相同的表型。ActivinB信号在一组注定要成为内胚层的卵裂球中是必需的，这强烈表明它是从潜在的微粒中寻找已久的早期信号通路，被认为是β-连环素核化下游内胚层发育的第一批信号之一。 SOXB1和核β-连环蛋白的交叉调节机制(4%；郑伟，工作人员科学家)。SoxB1是卵裂晚期/早期囊胚期内胚层规范的关键调节因子。SoxB1位于内胚层基因调控网络的顶端，通过拮抗核β-连环蛋白，通过经典的Wnt信号通路抑制信号传递。相反，β-连环蛋白作为转录辅助因子，通过抑制转录和意外地通过空间调节的SoxB1蛋白周转来清除SoxB1。我们正在测试相互拮抗是否涉及SoxB1和β-连环蛋白之间的直接物理相互作用。酵母双杂交分析表明，SoxB1的全长和C末端的一半都可以与海胆β-连环蛋白相互作用，这是依赖于β连环蛋白的SoxB1转化所必需的。免疫共沉淀分析将测试这些蛋白质是否在海胆胚胎中相互作用，并可能识别参与SoxB1和β-catenin关键早期交叉调节的其他相互作用蛋白。