Novel signals regulate cell fate patterning

线虫外阴细胞命运模式是研究信号转导机制的理想系统。 表皮生长因子诱导6个等价的外阴前体细胞(VPC)以3˚-3˚-2˚-1˚-2˚-3˚模式呈现细胞命运 99.8%的准确率。在外阴模式的“形态梯度”模型中，与EGF来源的距离 决定每个VPC的命运。在“序贯诱导”模型中，RAS-→-RAF-→-→-ERK-MAPK CASCADE通过LIN-12/Notch受体诱导1-˚的命运，诱导DSL配体的产生，从而诱导其2 相邻的˚将成为2个VPC。由于缺乏关键的分子细节，这两个模型 辩论了16年。我们解决了这一争论：为了解释表皮生长因子梯度，假定2个˚细胞使用 RAS→RAL→RAL而不是规范的RAS→RAF在1˚S中使用。突变的RAL或RAL(RAS的表亲) 不会产生强烈的图案缺陷，这表明顺序感应是主要的图案 机制。这些都是与临床相关的分子：RAS是突变最多的癌蛋白。RAS→→RAL 被认为与典型的RAS→RAF在肿瘤发生中同样重要。 我们的中心假设是，RAS→→的故事证明了在VPC的未开发区域进行探索是合理的 模式，因为临床上重要的信号被发现为积极和消极的调节因素。的目标 这一提议将揭示1˚/2˚命运选择及其高度重复性的分子基础的新方面 在活体中，利用这个系统的优势来剖析信号机制。我们的初步结果 支持三个目标的可行性，每个目标都有一个侧重于不同分子机制的假设。 目的1：我们的CRISPR在内源性RAP1(RAS近端)诱导的异位1˚中产生激活突变 细胞。缺乏RAP1可减少1˚细胞的诱导。我们将测试Rap1是两个节点的连接这一假设 相反的调节输入，分别促进和抑制假定的1˚和2˚细胞中ERK的激活。 目的2：我们发现了一种新的信号转导通路，GCK-2/MAP4，它可能通过p38 MAP激酶来传递信号 推动2个˚命运。我们将检验Ral→GCK-2触发p38级联的假设。我们还将测试 Ral→GCK-2信号是否调节CCCHRNA结合蛋白以稳定2个˚促进基因的3‘UTRs。 目的3：mIG-15/MAP4K是GCK-2/MAP4K的唯一同源基因，矛盾地抑制2-˚的命运。我们将测试 假设MIG-15触发JNK MAP激酶级联，这可能是抑制JNK的缺失信号 推测为1°细胞的Notch受体。MiG-15定义了一类新的外阴特异的Notch抑制因子。 我们将利用我们为类似米格-15的目标完成的筛查结果来识别新玩家。 对于所有这三个目标，我们将使用CRISPR工程的内源荧光记者来解开 嵌入到外阴信号网络中的特定信号。这些目标的成功实现将确定 外阴信令网络的组件和组织原则，从而实现卓越的保真度 模式，这很可能广泛地和专门地应用于其他发育系统。

Cell fate patterning of the C. elegans vulva is an ideal system for the study of signal transduction mechanisms. EGF induces six equipotent vulval precursor cells (VPCs) to assume cell fates in a 3˚-3˚-2˚-1˚-2˚-3˚ pattern with 99.8% accuracy. In the “Morphogen Gradient” model for vulval patterning, distance from the EGF source dictates the fate of each VPC. In the “Sequential Induction” model, the Ras→Raf→MEK→ERK MAP kinase cascade induces 1˚ fate, elicits DSL ligand production, and thus, via the LIN-12/Notch receptor, induces its two neighboring VPCs to become 2˚. Because of the absence of key molecular details, these two models were debated for 16 years. We resolved this debate: to interpret the EGF gradient, presumptive 2˚ cells use Ras→RalGEF→Ral rather than the canonical Ras→Raf used in 1˚s. Mutating RalGEF or Ral (a cousin of Ras) does not confer strong patterning defects, suggesting that sequential induction is the dominant patterning mechanism. These are clinically relevant molecules: Ras is the most mutated oncoprotein. Ras→RalGEF→Ral is thought to be equally important for oncogenesis as the canonical Ras→Raf. Our central hypothesis is that the Ras→RalGEF→Ral story justifies pursuing unexplored areas of VPC patterning because clinically important signals are found as positive and negative regulators. The objectives of this proposal are to unveil new facets of the molecular basis for the 1˚/2˚ fate choice and its high reproducibility in vivo, exploiting the strengths of this system for dissection of signaling mechanisms. Our preliminary results support the feasibility of three aims, each with a hypothesis focused on distinct molecular mechanisms. Aim 1: Our CRISPR-generated activating mutation in endogenous Rap1 (Ras proximal) induced ectopic 1˚ cells. Lack of Rap1 reduces 1˚ cell induction. We will test the hypothesis that Rap1 is the nexus of two opposed regulatory inputs that promote and repress ERK activation in presumptive 1˚ vs 2˚ cells, respectively. Aim 2: We identified a novel Ral effector, GCK-2/MAP4 kinase, that possibly signals via p38 MAP kinase to promote 2˚ fate. We will test the hypothesis that Ral→GCK-2 triggers a p38 cascade. We will also test whether Ral→GCK-2 signal regulates CCCH RNA binding proteins to stabilize 3’UTRs of 2˚-promoting genes. Aim 3: MIG-15/MAP4K, the sole paralog of GCK-2/MAP4K, paradoxically inhibits 2˚ fate. We will test the hypothesis that MIG-15 triggers a JNK MAP kinase cascade, which may be the missing signal that represses the Notch receptor in presumptive 1° cells. MIG-15 defines a novel class of vulval-specific Notch repressors. We will make use of the results of a screen we have completed for MIG-15-like targets to identify new players. For all three aims we will use CRISPR-engineered endogenous fluorescent reporters to deconvolute specific signals embedded within the vulval signaling network. Successful completion of these aims will define components and organizational principles of the vulval signaling network that leads to the exceptional fidelity of patterning, which are likely to apply both broadly and specifically to other developmental systems.