Mesodermal cell fate specification in C. elegans

描述(申请人提供)：中胚层胚层产生多种功能重要的细胞类型，包括横纹肌和非横纹肌以及非肌肉细胞。了解中胚层分化的调控机制在基础生物学、干细胞生物学和临床研究中具有广泛的意义。线虫胚胎后中胚层谱系，即M谱系，为在单细胞分辨率下研究中胚层的多样化提供了独特的优势。M系来源于单一的多能前体细胞，即M中胚细胞，在两性人胚胎后发育过程中，M中胚细胞增殖并产生六种细胞类型：有条纹的体壁肌肉(BWM)、非肌肉体腔细胞(CC)和四种类型的非条纹性肌。M及其后代都以可复制的方式分裂，这既受发育控制，也受细胞周期控制。因此，M谱系非常适合于研究不同的中胚层命运如何从单个祖细胞多样化，位置信息如何与谱系内在信息整合，以及不对称图案、细胞分裂时机和方向以及细胞命运指定的不同程序如何整合。我们的长期目标是从机械细节上理解M血统多样化的监管逻辑。在之前和现在的资助期间，我们已经成功地对涉及M谱系发育的信号和转录调控机制进行了深入的分子遗传学研究。我们的结果使我们能够开始组装BWM、CC和非横纹肌的前体--性成肌细胞(SM)的适当规范所涉及的调控网络。我们对参与这些命运指定过程的几个转录因子的鉴定和鉴定也为这些因子如何在脊椎动物的类似过程中发挥作用以及它们的功能障碍如何与某些人类疾病有关提供了关键的见解。更重要的是，这些研究为我们提供了试剂和令人兴奋的机会来解决发育生物学中的其他基本问题。在这项提案中，我们将利用M谱系来剖析与多能祖细胞的规范和增殖有关的机制(目标1)和不同类型的非条纹/平滑肌肉的规范的潜在机制(目标2)。因为我们已经确定的许多因子在脊椎动物中是保守的，我们对它们在M谱系中的研究有助于从机制上理解它们的一般功能，我们建议从分子上识别和表征两个对M谱系发育至关重要的“新”因子(目标3)。最后，我们将利用M谱系来剖析细胞类型和阶段特异性染色质特征与转录因子在体内细胞命运指定和细胞分化过程中的作用之间的联系，这是发育生物学中我们知之甚少的一个重要领域。我们建议使用新开发的完整(在特定细胞类型中标记的核的分离)方法(目标4)来解决这个问题。

DESCRIPTION (provided by applicant): The mesodermal germ layer gives rise to a variety of functionally important cell types, including striated and non-striated muscles as well as non-muscle cells. Understanding the regulatory mechanisms underlying mesoderm diversification has widespread implications in basic biology, stem cell biology and clinical research. The C. elegans postembryonic mesodermal lineage, the M lineage, provides unique advantages for studying mesoderm diversification at single cell resolution. The M lineage is derived from a single pluripotent precursor cell, the M mesoblast, which during hermaphrodite postembryonic development proliferates and produces six cell types: striated bodywall muscles (BWM), non-muscle coelomocytes (CC), and four classes of non-striated sex muscles. Both M and its descendants divide in a reproducible pattern, which is under both developmental and cell cycle control. The M lineage is thus ideally suited to investigating how different mesodermal fates are diversified from a single progenitor cell, how positional information is integrated with lineage-intrinsic information, and how diverse programs of asymmetric patterning, cell division timing and orientation, and cell fate specification are integrated. Our long-term goal is to understand the regulatory logic of M lineage diversification in mechanistic detail. During the previous and current funding periods, we have successfully conducted in-depth molecular genetic studies on signaling and transcriptional regulatory mechanisms involved in M lineage development. Our results have allowed us to begin to assemble the regulatory networks involved in the proper specification of BWM, CC and the precursor of the non-striated muscles, the sex myoblast (SM). Our identification and characterization of several transcription factors involved in these fate specification processes have also provided key insights into how these factors may function in similar processes in vertebrates and how their malfunction may be linked to certain human diseases. More importantly, these studies have provided us with reagents and exciting opportunities to address additional fundamental questions in developmental biology. In this proposal, we will exploit the M lineage to dissect mechanisms involved in the specification and proliferation of multi-potent progenitors (Aim 1) and mechanisms underlying the specification of different types of non- striated/smooth muscles (Aim 2). Because many of the factors that we have identified are conserved in vertebrates and our studies of them in the M lineage have contributed to the mechanistic understanding of their functions in general, we propose to molecularly identify and characterize two "new" factors critical for M lineage development (Aim 3). Finally, we are at the point to exploit the M lineage to dissect the connection between cell type- and stage-specific chromatin features and transcription factor action during cell fate specification and cell differentiation in vivo, an important area in developmental biology that we know very little about. We propose to address this question using the newly developed INTACT (isolation of nuclei tagged in specific cell types) method (Aim 4).