CRISPR based screen for small GTPase regulators of morphogenesis in Xenopus

摘要 许多出生缺陷是由驱动正常组织的细胞行为中的缺陷引起的，包括 神经管缺陷和颅面部缺陷的常见病例。在……方面取得了相当大的进展 了解细胞行为的机制；相比之下，在细胞骨架水平上的上游控制 调节蛋白，以及转录控制的表达调节器已被较少的文献记载 案子。尽管如此，我们可以举出几个例子，其中特定的形态发生事件最终是 由编码小GTP酶，特别是Rho的调节器的基因的表达来调节。在这里我们 假设我们将通过直接筛选的方式来洞察形态发生的许多胚胎背景 对原肠形成和早期形态发生事件的亚群至关重要的小GTP酶的调节因子 神经形成。我们不是进行开放式筛选，而是专注于GTPase监管机构，我们在那里辩称 它们在形态发生中的普遍重要性是有先例的。有大约150个Rho缺口和 社区数据库Xenbase中列出的GEF及其时间表达的描述，以及 有时，他们的文字记录本地化。我们将优先考虑合子表达的基因，以避开监管机构 ，然后使用G0(又名F0)Crispr敲除目标，这足以识别 表型。使用高通量成像，我们将筛选与原肠形成和 神经发育缺陷。然后，将进一步描述它们的特征，以确定它们是在本地还是在全球范围内表达的。 这个项目最终将产生分子工具来操纵和分析力的产生和分析 决定细胞如何在胚胎中移动的细胞间相互作用。与对神经管的洞察一样 由于了解青蛙的神经运动而产生的缺陷，我们将建立原则来 检查导致出生缺陷的其他形态发生事件和失败。

Abstract Many birth defects arise from flaws in the cell behaviors that drive normal tissue organization, including the prevalent cases of neural tube defects, and craniofacial defects. There has been considerable progress in understanding the mechanics of cell behavior; in contrast, the upstream control, at the level of cytoskeletal regulatory proteins, and the transcriptional control of expression of the regulators has been documented in fewer cases. Nonetheless, we can point to several examples where a specific morphogenetic event is ultimately regulated by the expression of genes that encode regulators of small GTPases, particularly Rho. Here we hypothesize that we will gain insights into many embryonic contexts of morphogenesis by screening directly for regulators of small GTPases that are essential for subsets of the early morphogenetic events of gastrulation and neurulation. Rather than conduct open-ended screens, we focus on GTPase Regulators, where we have argued that there is a precedent for their general importance in morphogenesis. There are about 150 Rho GAPs and GEFs listed in the community database, Xenbase, with descriptions of their temporal expression, and sometimes, their transcript localization. We will prioritize genes that are zygotically expressed to avoid regulators of general cellular processes, and then knock out targets using G0 (aka F0) Crispr, which is sufficient to identify phenotypes. Using high-throughput imaging we will screen for phenotypes associated with gastrulation and neurulation defects. Hits will then be further characterized to determine if they are locally or globally expressed. This project will ultimately generate the molecular tools to manipulate and analyze the force generation and intercellular interactions that determine how cells move in the embryo. As with the insights into neural tube defects that have come from understanding neurulation movements in frogs, we will establish principles to examine other morphogenetic events and failures that lead to birth defects.