Novel signals regulate cell fate patterning

C. Elegans vulva是研究信号转导机制的理想系统。 EGF诱导6个等能的外阴前体细胞（VPC）以3 × 3 × 2 × 1 × 2 × 3 × 3的模式承担细胞命运， 准确率99.8%在外阴图案化的“形态梯度”模型中， 决定每个VPC的命运在“序贯诱导”模型中，Ras→Raf→MEK→ERK MAP激酶 级联反应诱导1 β-fate，ele-fate DSL配体产生，因此，通过LIN-12/Notch受体，诱导其两个 相邻的VPC变成2个。由于缺乏关键的分子细节，这两个模型被 争论了16年。我们解决了这个争论：为了解释EGF梯度，假定使用2 μ g细胞， Ras→RalGEF→Ral，而不是在1 π s中使用的规范Ras→Raf。突变型RalGEF或Ral（Ras的表亲） 并不赋予强烈的图案缺陷，这表明顺序诱导是占主导地位的图案 机制这些是临床相关的分子：Ras是突变最多的癌蛋白。Ras→RalGEF→Ral 被认为与经典的Ras→Raf对肿瘤发生同样重要。 我们的中心假设是Ras→RalGEF→Ral的故事证明了追求VPC未开发领域的合理性 因为临床上重要的信号被发现为正和负调节器。的目标 这一建议揭示了1 β/2 β酯选择的分子基础及其高重现性的新方面 在体内，利用该系统的优势解剖信号机制。我们的初步结果 支持三个目标的可行性，每个目标都有一个专注于不同分子机制的假设。 目的1：我们的CRISPR在内源性Rap 1（Ras近端）中产生的激活突变诱导了异位1型胶原的表达。 细胞Rap 1的缺乏减少了1-HT细胞的诱导。我们将测试Rap 1是两个连接点的假设 分别在假定的1 μ细胞和2 μ细胞中，反对促进和抑制ERK激活的调节输入。 目的2：我们鉴定了一种新的Ral效应子，GCK-2/MAP 4激酶，其可能通过p38 MAP激酶进行信号传导 以促进2-羟基丁酸酯。我们将检验Ral→GCK-2触发p38级联反应的假设。我们还将测试 Ral→GCK-2信号是否调节CCCH RNA结合蛋白以稳定2个启动子基因的3 'UTR。 目的3：GCK-2/MAP 4K的唯一拮抗剂β-15/MAP 4K，矛盾地抑制2 β命运。我们将测试 假设JNK-15触发JNK MAP激酶级联反应，这可能是抑制JNK的缺失信号。 Notch受体在假定的1°细胞中。Notch-15定义了一类新的外阴特异性Notch阻遏物。 我们将利用我们已经完成的一个屏幕的结果，为类似的目标，以确定新的球员。 对于所有这三个目标，我们将使用CRISPR工程化的内源性荧光报告基因去卷积， 嵌入外阴信号网络的特定信号。这些目标的成功实现将决定 外阴信号网络的组成部分和组织原则，导致异常的保真度， 模式，这是可能适用于广泛和具体的其他发展系统。

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.