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Molecular mechanisms of cell fate specification

细胞命运规范的分子机制

基本信息

批准号：
7733927
负责人：
LYNNE M ANGERER
金额：
$ 107.47万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/7733927
关键词：
Achievement Agonist Andro-Diane Animals Bioinformatics Biological Assay Biological Models Cells Cilia Collaborations Development Developmental Biology Dopamine Dopamine Antagonists Eating Ecology Ectoderm Embryo Embryonic Development Endoderm Endomesoderm Event Evolution Food Forebrain Development G-Protein-Coupled Receptors Gene Components Gene Structure Genes Genetic Transcription Genome Germ Layers Growth Injection of therapeutic agent Intramural Research Program Introns Investigation Larva Lateral Length Manuscripts Mediating Mesoderm Methods Molecular Morphology Nerve Nervous system structure Neurons Non - mammalian blastula Numbers Operative Surgical Procedures Oral Pathway interactions Pattern Physiology Pigments Positioning Attribute Process Prosencephalon Refractory Regulator Genes Reporting Repression Research Rest Risk Role Sea Urchins Sensory Signal Pathway Signal Transduction Skeletal system Specific qualifier value Specificity Staging Structure System Tissues To specify Transforming Growth Factor beta Translations Upper arm Vertebrates Work animal tissue base blastocyst cell type density dopaminergic neuron embryo stage 2 gene function genetic regulatory protein improved insight interest knock-down loss of function nervous system development neurogenesis notch protein relating to nervous system research study response retinal rods

项目摘要

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.

我们关注的主要问题是：1）什么是上游调控组件，指定动物极域（APD）的海胆胚胎，其中包含神经和细胞轴承长，不动的纤毛？2)在早期卵裂和囊胚阶段，内中胚层特化和原肠胚形成所需的信号是什么？ 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理解。 Six3是APD规范（45%）的必要和充分条件（Zheng Wei，Junko Yaguchi，Shunsuke Yaguchi，Lynne Angerer）在过去的几年中，利用生物信息学方法和分子筛选，我们确定了许多基因编码的调节蛋白的海胆胚胎的主要神经原结构域中特异性表达。最早的两个，FoxQ2和Six3，控制外胚层模式的早期决定。 2008年进行的实验表明，Six3对于APD发育的所有已知特征都是必要和充分的，包括胚胎发育过程中形成的所有神经元，以及之前鉴定的大多数APD特异性调节蛋白，包括FoxQ2。我们确定了依赖于Six3的基因的完整调控库，并发现Six3还可以抑制典型的Wnt和TGF-β信号，这些信号对胚胎的其余部分起作用。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征。由于我们的研究已经确定了许多额外的调节基因，是Six3依赖性和APD中的表达，他们可能会促进脊椎动物前脑发育的理解。一份报告这项工作的手稿（魏等人）。正在审查中。 FoxQ 2是一个沿初级和次级发育轴（5%）协调细胞命运规范的检查点（沿着）（Shunsuke和Junko Yaguchi） FoxQ2负调节TGF-β信号传导，这是沿着除动物极外的外胚层所有区域的次级轴形成图案所必需的。为了使TGF-β信号传导发生，FoxQ2必须从外侧外胚层消除，这是一个依赖于典型Wnt（主轴信号传导系统）的事件。 FoxQ 2提供了沿主轴和次轴沿着协调图案化的检查点（Yaguchi等人，Developmental Cell 14，97 - 107，2008）。它还在APD中不同细胞类型的分化中起作用，并可能介导Six3s的作用。全基因组筛选显示，这是近500个基因表达所必需的。今年的成就为确定FoxQ2依赖性GRN以及Six3依赖性GRN在动物极神经源性结构域中的结构和细胞类型特异性提供了强大的动力。激活素B/ALK 4/5/7和Delta/Notch是诱导内中胚层和内胚层特化的早期信号（25%）（Adi Sethi，Radhika Wikramanayake）先前的实验表明，异位和正常的内中胚层诱导需要激活素B，并且该信号传导因子执行先前未知但长期寻求的早期微小信号的所有内中胚层诱导功能。他的工作是第一个将ActivinB信号传导与内中胚层基因调控（GRN）网络的特定组成部分联系起来的工作，该网络是任何胚胎中最大和最发达的网络，并导致对其运作的新见解。在这一年中，根据审查结果进行的其他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。以前的工作阻断Notch功能揭示了该途径在分离内中胚层领域内的两个初级胚层（内胚层和中胚层）中的新功能。在2008年期间，我们发现微粒Delta信号不仅激活gcm，主要色素细胞GRN调节器，而且还抑制早期内胚层特化所需的几个关键基因。 δ信号传导到内胚层比以前报道的更早，并且是早期内胚层特化基因表达所必需的。由于大单体Delta依赖于先前的小单体Delta信号，因此已经开发了在特定卵裂球中敲除它的新方法。这些方法包括注射阻断单个卵裂球中Delta翻译的吗啉代或使用反义内含子RNA诱导特定卵裂球中Delta转录的丧失。初步实验表明，这两种方法都是有用的。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臂结构以最大限度地摄取食物来感知和响应食物浓度的分子途径。这是一个高风险项目，它将幼虫发育生物学、幼虫生理学和幼虫扩散生态学联系起来。戴安发现，口后骨骼杆长度的变化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后臂的基部，以调节感觉反应。为了确定食物感应途径的组成部分，她正在使用微阵列分析整个基因组对食物浓度和多巴胺拮抗剂和激动剂的反应。她还与加州理工学院的安德鲁卡梅隆博士建立了合作关系，以寻找可能在幼虫阶段对食物的感知中起作用的G偶联蛋白受体。