Molecular mechanisms of cell fate specification

我们关注的主要问题是：1)海胆胚胎的动物极域的上游调控成分是什么，它含有承载着长而不动的纤毛的神经和细胞？2)在早期卵裂和囊胚阶段，内胚层规范和适时原肠形成所需的信号传递是什么？3)胚胎能够改变其形态以适应不断变化的食物浓度的分子基础是什么？在每种情况下，我们分析基因组中所有预测基因的表达，以获得对这些过程背后的基因调控网络的完整了解。 对于apd规范，Six3是必要的，也是足够的(45%)(郑伟、山口纯子、矢口顺介、林恩·安杰尔) 在过去的几年里，我们利用生物信息学和分子筛选的方法，鉴定了许多编码调控蛋白的基因，这些基因在海胆胚胎的初级神经源性区域中特异表达。其中最早的两个，FoxQ2和Six3，控制着外胚层图案的早期决定。2008年进行的实验表明，Six3对于所有已知的apd发育特征是必要的，也是充分的，包括在胚胎发育过程中形成的所有神经元，以及之前发现的绝大多数apd特异调控蛋白，包括FoxQ2。我们确定了依赖于Six3的基因的完整调控谱，并发现Six3还可以抑制形成胚胎其余部分的规范Wnt和转化生长因子-β信号。我们的工作揭示了Six3在海胆胚胎apd和脊椎动物前脑中的功能有一些共同的特点。因为我们的研究已经确定了许多额外的调控基因，这些基因依赖于Six3并在动作电位中表达，这些基因可能有助于理解脊椎动物的前脑发育。报道这项工作的手稿(魏等人)正在审查中。 FoxQ2是一个沿着初级和次级发育轴协调细胞命运的检查点(5%)(Shunsuke和Junko Yaguchi) FoxQ2负性调节转化生长因子-β信号，转化生长因子-β信号是除动物外胚层外，沿着外胚层所有区域的第二轴形成图案所必需的。为了使转化生长因子-β信号发生，FoxQ2必须从外侧外胚层中去除，这一事件依赖于典型的Wnt，主轴信号系统。FoxQ2提供了一个检查点，协调沿着主轴和副轴的图案(Yaguchi等人，发育细胞14，97-107,2008)。它还参与了动作电位中不同类型细胞的分化，并可能介导了Six3s的作用。全基因组筛查显示，这是近500个基因表达所必需的。今年的成果为确定依赖于FoxQ2的GRN以及依赖于Six3的GRN在动物极点的神经源性领域的结构和细胞类型特异性提供了强大的动力。 ActivinB/ALK4/5/7和Delta/Notch是诱导内胚层和内胚层规范的早期信号(25%)(Adi Sethi，Radhika Wikramanayake) 先前的实验表明，异位和正常的内胚层诱导都需要ActivinB，并且这种信号因子执行先前未知的、但长期寻找的早期微米信号的所有内胚层诱导功能。他的工作是第一次将ActivinB信号与内胚层基因调控(GRN)网络的特定组件联系起来，这是所有胚胎中最大和最发达的网络，并为其运作带来了新的见解。在这一年里，为回应审查而进行的更多实验证实了以前的工作，正在审查一份经过改进和修订的手稿。先前阻断Notch功能的工作揭示了这一途径的新功能，即在内胚层区域内分离两个初级胚层，内胚层和中胚层。在2008年，我们发现，微丝Delta信号不仅激活了主要的色素细胞GRN调节因子GCM，而且还抑制了早期内胚层指定所需的几个关键基因。Delta信号转导内胚层的时间比以往报道的要早，并且是早期内胚层特定基因表达所必需的。因为宏微米Delta依赖于先前的微微米Delta信号，所以已经开发出在特定的卵裂球中敲除它的新方法。这些措施包括注射吗啡阻断单个卵裂球中Delta的翻译，或使用反义内含子RNA诱导特定卵裂球中Delta转录的丢失。初步实验表明，这两种方法都将是有用的。 多巴胺能神经元调节胚胎对食物密度的反应(25%)(黛安·亚当斯) 一项新的研究旨在了解幼虫通过调整手臂结构来最大限度地摄取食物来感知和反应食物浓度的分子途径。这是一个连接幼虫发育生物学、幼虫生理学和幼虫扩散生态学的高风险项目。Diane发现，口腔后骨骼杆长对食物密度的反应是由多巴胺调节的，多巴胺能神经元正确地定位在口腔后手臂的底部，以调节感觉反应。为了确定食物感知途径的组成部分，她正在使用微阵列分析对食物浓度以及多巴胺拮抗剂和激动剂的整个基因组反应。她还与加州理工大学的安德鲁·卡梅伦博士建立了合作关系，以寻找在幼虫阶段对食物感觉可能起作用的G偶联蛋白受体。

The major questions we focus on are: 1) What are the upstream regulatory components that specify the animal pole domain (APD) of the sea urchin embryo, which contains nerves and cells bearing long, immotile cilia? 2) What are the signals transmitted during early cleavage and blastula stages that are required for endomesoderm specification and timely gastrulation? 3) What is the molecular basis for the embryos ability to change its morphology to adapt to changing food concentrations? In each case, we assay the expression of all predicted genes in the genome in order to gain a complete understanding of the gene regulatory networks that underlie these processes. Six3 is necessary and sufficient for APD specification (45%) (Zheng Wei, Junko Yaguchi, Shunsuke Yaguchi, Lynne Angerer) During the last several years, using bioinformatics approaches and molecular screens, we identified many genes encoding regulatory proteins expressed specifically in the primary neurogenic domain of the sea urchin embryo. Two of the earliest, FoxQ2 and Six3, control early decisions in ectodermal patterning. Experiments conducted during 2008 showed that Six3 is necessary and sufficient for all known features of APD development, including all neurons formed during embryogenesis and for the large majority of APD-specific regulatory proteins previously identified, including FoxQ2. We identified the full regulatory repertoire of genes that depends on Six3, and discovered that Six3 can also suppress canonical Wnt and TGF-beta signals that pattern the rest of the embryo. Our work revealed that Six3 function in the sea urchin embryo APD and the vertebrate forebrain share some features. Because our studies have identified many additional regulatory genes that are Six3-dependent and expressed in the APD, they are likely to facilitate understanding of vertebrate forebrain development. A manuscript reporting this work (Wei et al.) is in review. FoxQ2 is a checkpoint coordinating cell fate specification along the primary and secondary developmental axes (5%)(Shunsuke and Junko Yaguchi) FoxQ2 negatively regulates TGF-beta signaling, which is required for patterning along the secondary axis of all regions of ectoderm except that at the animal pole. In order for TGf-beta signaling to occur, FoxQ2 must be eliminated from the lateral ectoderm, an event that depends on canonical Wnt, the primary axis signaling system. FoxQ2 provides a checkpoint coordinating patterning along the primary and secondary axes (Yaguchi et al., Developmental Cell 14, 97-107, 2008). It also functions in the differentiation of different cell types in the APD and may mediate Six3s role. Whole-genome screens show that is required for the expression of nearly 500 genes. The achievements this year provide strong impetus for determining the structure and cell type specificity of the FoxQ2-dependent GRN, as well as the Six3-dependent GRN, in the neurogenic domain at the animal pole. ActivinB/ALK4/5/7 and Delta/Notch are early signals that induce specification of endomesoderm and endoderm (25%)(Adi Sethi, Radhika Wikramanayake) Previous experiments showed that both ectopic and normal endomesoderm induction requires ActivinB and that this signaling factor executes all of the endomesoderm inducing functions of the previously unknown, but long-sought, early micromere signal. His work is the first to connect ActivinB signaling to specific components of an endomesoderm gene regulatory (GRN) network, the largest and best developed in any embryo, and it has led to new insights into its operation. During this year additional experiments carried out in response to reviews have confirmed previous work and an improved, revised manuscript is in review. Previous work blocking Notch function reveal new functions for this pathway in separating two primary germ layers, the endoderm and mesoderm, within the endomesodermal field. During 2008, we found that micromere Delta signals not only aactivate gcm, the cardinal pigment cell GRN regulator, but also to repress several critical genes required for early endoderm specification. Delta signaling to endoderm is earlier than previously reported and necessary for expression of early endoderm specification genes. Because macromere Delta depends on prior micromere Delta signals, new methods to knock down it down in specific blastomeres have been developed. These include injection of a morpholino blocking Delta translation in single blastomeres or inducing loss of Delta transcription in specific blastomeres using antisense intron RNAs. Preliminary experiments suggest that both methods will be useful. Dopaminergic neurons regulate the embryos response to food density (25%) (Diane Adams) A new line of investigation aims to understand the molecular pathways by which larvae sense and respond to the concentration of food by adjusting arm structure to maximize food intake. It is a high-risk project that bridges larval developmental biology, larval physiology and the ecology of larval dispersal. Diane has found that changes in the post-oral skeletal rod length in response to food density is regulated by dopamine and that dopaminergic neurons are positioned correctly at the base of the post-oral arms to regulate the sensory response. To determine the components of the food sensing pathway, she is assaying the whole geneme reponse to food concentration and to dopamine antagonists and agonists using microarrays. She has also established a collaboration with Dr. Andrew Cameron at Caltech to search for G-coupled protein receptors that might function in larval sensing of food during larval stages.