Molecular mechanisms of cell fate specification

我们的实验室研究细胞命运的规范和模式的机制沿着动物？植物（A？V）海胆（Strongylocentrotus purpuratus）胚胎的轴。我们的主要重点是了解基因调控网络和信号通路，指定外胚层域，这是来自未分化的前外胚层，并形成aboral，oral，neural和ciliagenic外胚层领土。 海胆基因组挖掘和注释的资源和工具的开发。(33%;郑伟，研究员）。一项重大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。我们使用基因预测来设计全基因组微阵列，用于在5个胚胎阶段对mRNA表达进行时间分析，在高密度阵列上使用每个基因5个探针（Nimblegen，Inc）。大量经过充分研究的基因的时间分布已经得到证实，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。几个小组表示有兴趣使用这些微阵列作为工具，以表征实验操作的影响，在基因表达水平，并确定其他基因的调控网络。 在动物极地领域细胞命运的特异性机制（30%; Lynne Angerer，高级科学家; Shunsuke Yaguchi，访问学者）。我们有兴趣了解海胆胚胎动物极域（APD）中细胞命运初始规范的基因调控网络。这个区域是特殊的，因为APD调节基因是难治性的植物信号的阻遏。APD包含胚胎神经系统的6个多巴胺能神经元的前体，我们的具体目标是确定构成核心神经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鉴定编码在神经源性外胚层中表达的转录因子的候选基因。迄今为止，已经在海胆基因组中鉴定出58个这样的基因，其中34个在APD的胚胎发生期间表达，一些仅在该区域中表达，另一些在出乎意料的早期阶段表达，早在原肠胚形成之前。有趣的是，四个基因已被确定为编码转录因子，这些转录因子在多种生物体（包括腔肠动物（进化中极其古老的群体））的神经细胞特化开始时发挥作用，这表明这些基因构成了核心神经原性的一部分。后生动物的调节网络。通过用吗啉代反义寡核苷酸（MASO）阻断每个mRNA的翻译，并使用整体原位杂交和定量PCR分析对其他基因表达的影响，来检测候选神经源性因子之间的调控关系。 内胚层发育中的TGF-β信号传导（33%; Aditya Sethi，客座研究员）。我们发现，TGF-β信号通路是海胆胚胎早期内胚层发育所必需的。用受体的小分子抑制剂Alk 4阻断该途径，导致一组特定的早期内胚层标记基因和原肠胚形成的显著延迟和表达减少。我们已经鉴定了激活素B的配体，它是除nodal外唯一通过Alk 4发挥作用并在早期胚胎中表达的TGF-β。通过两种吗啉代反义寡核苷酸中的任一种来消除激活素B的合成，在分子和形态学水平上产生与Alk 4抑制相同的表型。激活素B信号在注定成为内胚层的卵裂球亚组中是必需的，强烈表明它是长期寻求的来自潜在微粒体的早期信号传导途径，这被认为是β-连环蛋白核化下游内胚层发育中的第一个信号之一。 SOXB 1和核β-连环蛋白交叉调节机制（4%; Zheng Wei，研究员）。SoxB 1是卵裂后期/囊胚早期内中胚层特化的关键调节因子。SoxB 1在内中胚层基因调控网络的顶端发挥作用，通过拮抗核β-连环蛋白，作为典型Wnt信号通路的信号传导抑制剂。相反，β-连环蛋白，作为一个转录辅因子，清除SoxB 1的转录抑制，并出乎意料地，通过空间调控的周转SoxB 1蛋白。我们正在测试相互拮抗作用是否涉及SoxB 1和β之间的直接物理相互作用？连环蛋白。酵母双杂交试验表明，全长和C-末端的一半的SoxB 1，需要β-连环蛋白依赖的SoxB 1营业额，可以与海胆β-连环蛋白相互作用。免疫共沉淀试验将测试这些蛋白质是否在海胆胚胎中相互作用，并可能确定参与SoxB 1和β-连环蛋白关键早期交叉调节的其他相互作用蛋白质。

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.