Molecular mechanisms of cell fate specification in the s

细胞命运规范的分子机制

• 批准号: 7146131
• 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/7146131
• 关键词: Internet; activins; cadherins; cell differentiation; cell growth regulation; developmental genetics; ectoderm; endoderm; gene expression profiling; genetic regulatory element; growth factor receptors; immunocytochemistry; in situ hybridization; invertebrate embryology; metalloendopeptidases; microarray technology; molecular biology information system; neurogenesis; polymerase chain reaction; protein protein interaction; sea urchins; serine threonine protein kinase; transcription factor; transforming growth factors

Our laboratory investigates mechanisms of cell fate specification along the animal?vegetal (A?V) axis of the sea urchin (Strongylocentrotus purpuratus) embryo. Our major focus is to understand the gene regulatory networks (GRNs) and signaling pathways that specify ectodermal domains, beginning with pre-ectoderm and the subsequent aboral, oral, neural and ciliagenic territories. SoxB1 and nuclear beta-catenin cross-regulatory mechanisms. Previously we showed that the SoxB1transcription factor is a key regulator of mesoderm and endoderm because it antagonizes function of nuclear beta-catenin, which is the initial activator of the endomesoderm (EM) GRN. Thus, tight regulation of the Sox B1/beta-catenin ratio is critical for allocating different cell fates to early blastomeres. Regulation of SoxB1 levels is achieved by transcriptional repression and, surprisingly, spatially regulated turnover of SoxB1 protein (Angerer et al., 2005; experimental work was completed before arriving at NIDCR and the publication was finished shortly after). The aims of this subproject are to understand the molecular mechanisms by which SoxB1 antagonizes nuclear beta-catenin and, conversely, the pathways that lead from nuclear beta-catenin to SoxB1 transcriptional repression and turnover. Mechanisms of specification of primitive neurons at the embryo?s animal pole. A second subproject seeks to define the GRN underlying specification of cells at the animal pole of the sea urchin embryo [Animal Pole Domain (APD)], which will form part of the larval nervous system and ciliated band. This region of presumptive ectoderm is unique in that it is the most resistant to alteration of cell fates by mis-expression of genes that promote EM differentiation. In contrast, in the absence of beta-catenin-dependent EM signals, almost the entire ectoderm differentiates with an APD-like fate. Thus, APD-specifying genes are repressed by EM signals in all ectodermal cells, except those at the animal pole. This, and other evidence suggest that some APD cells may initially be specified by maternal molecules. We plan to define the core GRN of these primitive neurons, especially maternal and zygotic factors that initiate neuronal specification. Two different approaches have been used to attack this problem. The first, under extramural funding (GM2553-25) at the University of Rochester, was a cDNA subtractive hybridization screen to select mRNAs up regulated when the EM GRN was completely inactivated. Selected sequences are depleted of those involved in housekeeping functions and differentiation of most tissues and enriched in mRNAs involved in APD specification and differentiation. Selected and normal embryo cDNAs were used to identify, in macroarrays of ~ 100,000 cDNA clones, those that represent mRNAs strongly enriched in the selected cDNA population. Analysis of these continued this year at the NIH. From the initial screen, the most interesting genes expressed in the APD are: 1) FoxQ2, a novel winged helix transcription factor, 2) at least one new tolloid/BMP1-like astacin protease, 3) a matrix metalloprotease, most closely related to a neural metalloprotease, 4) a neural-specific molecular chaperone, PACRG. Unexpectedly, we identified some mRNAs expressed exclusively at both poles of the embryo. The protease genes are expressed in a few cells scattered throughout the APD at very early stages, suggesting that they identify neural precursors. The second approach initiated this year at NIDCR, was to survey the expression of genes whose homologs in other systems have neurogenic gene regulatory functions. We used the newly completed S. purpuratus genome sequence and computational tools (see below) to rapidly identify homologs of genes encoding neurogenic transcription factors. Of 16 such genes, 11 are expressed in early development. Most interesting are 10 expressed in the absence of intercellular signals and 7 expressed in the absence of nuclear beta-catenin function, suggesting that they are activated cell-autonomously by maternal factors. mRNAs for at least 6 of these accumulate in the APD and 4 (FoxQ2, retinal anterior homeobox, achaete-scute and SoxC) appear very early, consistent with their functioning at the top of the neurogenic GRN. Their functions will be established by morpholino antisense knockdowns. TGF-beta signaling in endoderm development. In a third sub-project, Aditya Sethi showed that specific inhibition of the Alk4 receptor, which transduces a subset of TGF-beta signals, blocks normal development of early ectoderm and endoderm. In addition to confirming that signaling from the TGF-beta family member, nodal, specifies oral ectoderm, he found that an additional signal(s) is required for timely endoderm specification and correct gastrulation movements. By analyzing endodermal and ectodermal marker gene expression using in situ hybridization, immunohistochemistry and real time PCR assays, he found that the new signal may correspond to the long-sought, but still undefined ?early signal? required for EM development. After mining the genome with the new computational tools developed in our lab (see below), he identified 6 TGF-beta candidates. The expression of only one of these, activin B, is appropriate for its being the identified signal. He will test whether activin B promotes endoderm development by eliminating it with a morpholino. If it does, he will examine whether it signals through Alk4 by testing whether endoderm development can be rescued with constitutively active Alk4 receptor. Development of resources and tools for mining and annotation of the sea urchin genome. This year Zheng Wei developed computational tools for analyzing the sea urchin genome. He converted whole S. purpuratus genome sequence into a database of predicted peptides and then constructed a relational database that linked individual peptides (approximately 40,000) to gene names, expressed sequence tags and cDNA sequences available in the public databases. This effort produced a gene list, one of three currently being used to annotate the sea urchin genome. Dr. Wei also created, and made publicly available on our web site, a set of very user-friendly computational tools for identifying genes, which are being used by the international consortium of laboratories working on the annotation project. We will continue our annotation efforts by creating a whole-genome microarray with information obtained from our gene list. Dr. Wei has identified oligonucleotides representing more than 20,000 individual genes for this microarray, the first in this system. We will use this microarray initially for temporal expression profiling and then create a second smaller microarray of developmentally regulated embryonic genes, which will be made publicly available for experimental analyses of development.

我们的实验室调查了沿着动物的植物（A？v）轴的细胞命运规范的机制，海胆（purvuratus purpuratus）胚胎。我们的主要重点是了解指定外胚层结构域的基因调节网络（GRN）和信号通路，从外胚层和随后的原住民，口腔，神经和纤毛型领土开始。 SOXB1和核β-catenin交叉调节机制。以前我们表明SOXB1转录因子是中胚层和内胚层的关键调节剂，因为它拮抗了核β-catenin的功能，核β-catenin是内胚层（EM）GRN的最初激活剂。因此，SOX B1/β-catenin比率的严格调节对于将不同的细胞命运分配给早期胚泡至关重要。 SOXB1水平的调节是通过转录抑制来实现的，而且令人惊讶的是，Soxb1蛋白的空间调节营业额（Angerer等，2005；实验性工作在到达NIDCR之前完成，并在到达NIDCR之前完成，并在不久之后完成。该副本的目的是了解SOXB1拮抗核β-catenin的分子机制，相反，从核β-catenin到Soxb1转录抑制和周转率的途径。 胚胎杆上原始神经元规范的规范机制。第二个副本试图定义海胆胚胎动物极[动物极结构域（APD）]的细胞的基础规范，该细胞将构成幼虫神经系统和纤毛带的一部分。该假定外胚层的区域是独特的，因为它是通过促进EM分化的基因表达的错误表达来对细胞命运的改变。相反，在没有β-catenin依赖性EM信号的情况下，几乎整个外胚层与APD样命运区分开。因此，除在动物极处的外胚层细胞中，所有外胚层细胞中的EM信号抑制了APD指定的基因。这和其他证据表明，某些APD细胞最初可能由母体分子指定。我们计划定义这些原始神经元的核心GRN，尤其是启动神经元规范的母体和合子因子。已经使用了两种不同的方法来攻击这个问题。第一个在罗切斯特大学的校外资金（GM2553-25）下，是cDNA减法杂交屏幕，当EM GRN完全灭活时，可以选择受到调节的mRNA。选定的序列耗尽了参与管家功能和大多数组织的分化的序列，并富含参与APD规范和分化的mRNA。在约100,000个cDNA克隆的宏观阵列中，选择和正常的胚胎cDNA被用来识别那些代表MRNA在所选cDNA群体中的mRNA的界面。对这些分析的分析持续了今年的NIH。从初始屏幕开始，在APD中表达的最有趣的基因是：1）FOXQ2，一种新型的有翼螺旋转录因子，2）至少一个新的Toloid/bmp1类抗曲霉蛋白酶，3）基质金属蛋白酶，与神经金属蛋白酶最封闭相关，与神经金属蛋白酶最封闭，4）Neural Mellecarecolocel Chaperole chaperlg，pacrg。出乎意料的是，我们确定了一些在胚胎的两极中仅表达的mRNA。蛋白酶基因在很早就散布在整个APD中的一些细胞中表达，这表明它们鉴定了神经前体。今年在NIDCR上启动的第二种方法是调查其在其他系统中具有神经源基因调控功能的基因的表达。我们使用了新完成的紫癜链球菌基因组序列和计算工具（见下文）来迅速识别编码神经源性转录因子的基因的同源物。在16个这样的基因中，有11个在早期发展中表达。最有趣的是在没有细胞间信号的情况下表达的10个，在没有核β-catenin功能的情况下表达了7个，表明它们被母体因子激活了细胞自主。其中至少6个积聚在APD和4（FOXQ2，视网膜前同源物，Achaete-Scute和Soxc）中的mRNA出现很早就出现，这与它们在神经发生的GRN顶部的功能一致。它们的功能将由形态反义敲低确定。 内胚层发育中的TGF-β信号传导。在第三次亚项目中，Aditya sethi表明，转导TGF-β信号子集的ALK4受体的特异性抑制会阻止早期外胚层和内胚层的正常发育。除了确认来自TGF-beta家族成员Nodal的信号指定口服外胚层外，他还发现，及时的内胚层规范和正确的胃分解需要附加信号。通过使用原位杂交，免疫组织化学和实时PCR分析来分析内皮细胞和外胚层标记基因表达，他发现新信号可能与长期探索但仍然不确定的早期信号相对应？ EM开发所必需的。在我们实验室中开发的新计算工具（见下文）中挖掘基因组后，他确定了6个TGF-beta候选者。其中一种，即激活素B的表达适用于其作为识别信号。他将通过使用形态蛋白消除活化素B来测试活化素B是否促进内胚层发育。如果是这样，他将通过测试是否可以用组成性活跃的ALK4受体来挽救内胚层发育来检查它是否通过ALK4发出信号。 开发用于采矿和注释海胆基因组的资源和工具。今年，Zheng Wei开发了用于分析海胆基因组的计算工具。他将整个S. purpuratus基因组序列转换为预测肽的数据库，然后构建了一个关系数据库，该数据库将单个肽（约40,000个）与基因名称联系起来，表达的序列标签和公共数据库中可用的cDNA序列。这项工作产生了一个基因清单，这是目前用于注释海胆基因组的三个基因清单之一。 Wei博士还创建了网站，并在我们的网站上公开可用，这是一组非常用户友好的计算工具，用于识别基因，这些基因正在由国际实验室在注释项目中使用。我们将通过创建一个从我们的基因列表中获得的信息来创建全基因组微阵列来继续我们的注释工作。 WEI博士已经确定了该微阵列的20,000多个单个基因的寡核苷酸，这是该系统中的第一个基因。我们最初将使用此微阵列进行时间表达谱分析，然后创建第二个较小的开发调节胚胎基因的微阵列，该微阵列将公开用于开发实验分析。