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）指定海胆胚胎动物极结构域（APD）的上游调控组件是什么，其中包含带有长而不动的纤毛的神经和细胞？ 2) 内中胚层规范和及时原肠胚形成所需的早期卵裂和囊胚阶段传输的信号是什么？ 3）胚胎改变其形态以适应食物浓度变化的能力的分子基础是什么？在每种情况下，我们都分析了基因组中所有预测基因的表达，以便全面了解这些过程背后的基因调控网络。 Six3 对于 APD 规范来说是必要且充分的（45%）（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，我们鉴定了许多编码调节蛋白的基因，这些蛋白在海胆胚胎的初级神经源性结构域中特异性表达。其中最早的两个，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（包括胚胎发生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）来说是必要且充分的。我们确定了依赖 Six3 的基因的完整调控库，并发现 Six3 还可以抑制规范胚胎其余部分的 Wnt 和 TGF-β 信号。我们的工作揭示了海胆胚胎 APD 中 Six3 的功能与脊椎动物前脑具有一些共同特征。因为我们的研究已经确定了许多其他依赖 Six3 并在 APD 中表达的调节基因，它们可能有助于了解脊椎动物前脑发育。报告这项工作的手稿（Wei 等人）正在审查中。 FoxQ2 是沿着初级和次级发育轴协调细胞命运规范的检查点 (5%)（Shunsuke 和 Junko Yaguchi） FoxQ2 负向调节 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轴形成图案所必需的。为了发生 TGf-β 信号传导，FoxQ2 必须从外侧外胚层中消除，这一事件取决于规范的 Wnt（主轴信号传导系统）。 FoxQ2 提供沿主轴和副轴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发挥作用，并可能介导 Six3s 的作用。全基因组筛选显示近 500 个基因的表达需要它。今年的成果为确定动物极神经源性域中 FoxQ2 依赖的 GRN 以及 Six3 依赖的 GRN 的结构和细胞类型特异性提供了强大的推动力。 ActivinB/ALK4/5/7 和 Delta/Notch 是诱导内中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，异位和正常内中胚层诱导都需要 ActivinB，并且该信号因子执行先前未知但长期寻找的早期微信号的所有内中胚层诱导功能。他的工作首次将 ActivinB 信号传导与内中胚层基因调控 (GRN) 网络的特定组件连接起来，该网络是任何胚胎中最大且发育最好的网络，并且引发了对其运作的新见解。今年，根据评论进行的额外实验证实了之前的工作，并且正在审查改进、修订的手稿。先前阻断Notch功能的工作揭示了该途径在内中胚层区域内分离两个初级胚层（内胚层和中胚层）的新功能。 2008 年，我们发现微粒 Delta 信号不仅使 gcm（主要色素细胞 GRN 调节因子）失活，而且还抑制早期内胚层规范所需的几个关键基因。内胚层的 Delta 信号传导比之前报道的要早，并且是早期内胚层规范基因表达所必需的。由于大粒 Delta 依赖于先前的微粒 Delta 信号，因此开发了在特定卵裂球中将其敲除的新方法。这些包括注射吗啉代阻断单个卵裂球中的 Delta 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是有用的。多巴胺能神经元调节胚胎对食物密度的反应 (25%) (Diane Adams) 一项新的研究旨在了解幼虫通过调整手臂结构以最大限度地提高食物摄入量来感知和响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。黛安发现，口后骨骼杆长度随食物密度的变化受到多巴胺的调节，多巴胺能神经元正确定位在口后臂的基部，以调节感觉反应。为了确定食物传感途径的组成部分，她正在使用微阵列分析整个基因对食物浓度以及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·卡梅伦博士建立了合作，寻找可能在幼虫阶段对食物的感知中发挥作用的 G 耦合蛋白受体。