The actin binding proteins Calponin 2 and 3: effectors of Wnt/PCP-mediated cell migration and neural tube closure

肌动蛋白细胞骨架的动态变化驱动了脊椎动物胚胎发育过程中的形态发生过程。神经嵴细胞（NCCs）的迁移和神经管的闭合代表了两个主要的过程。两者都受Wnt/平面细胞极性（PCP）信号调节。钙钙蛋白（Cnn）代表了一个进化上高度保守但特征不明显的三种肌动蛋白结合蛋白家族。虽然一些研究已经证明了Cnn蛋白在体外调节肌动蛋白细胞骨架动力学的能力，但它们在体内的功能和调节因子仍然难以捉摸。我们最近证明了Cnn2在促进小鸡和青蛙胚胎中NCCs迁移中的新作用。研究发现，Cnn2的功能通过迁移细胞后端Wnt/PCP介质RhoA激酶（ROCK）的活性受到调节，从而导致Cnn2向细胞前沿极化。我们的初步数据还支持Cnn3在爪蟾和鸡胚胎中促进NCC迁移和分化的作用。此外，我们还发现Cnn3的功能是神经管闭合所必需的，因为Cnn3的功能缺失导致缺乏根尖收缩，从而导致神经管闭合缺陷和细胞形态非极化。最近发表的但迄今尚未表征的条件Cnn3敲除小鼠也显示出异常，证明Cnn3在脊椎动物神经管闭合中的功能是保守的。基于这些发现，我们假设Cnn2和Cnn3作为Wnt/PCP/ROCK信号的介质，促进NCC迁移和神经管闭合所必需的肌动蛋白动力学。为了确定Cnn2和Cnn3的功能和调控，我们的目标是(1)确定Cnn2中相关的ROCK靶序列；（II）通过ROCK和在此过程中活跃的蛋白酶体途径研究Cnn2降解/极化在NCCs中的相关性；（三）确定Cnn3在NCC发育分化中的作用；（四）揭示Wnt/ pcp介导的Cnn3功能在神经管闭合中的作用。为了实现这些目标，将结合先进的发育、分子和细胞方法，在鸡、青蛙和老鼠的体内和体外胚胎中使用。基于我们广泛的初步和已发表的数据以及实验计划，我们希望揭示Cnn2和Cnn3在形态发生过程中作为肌动蛋白动力学上游Wnt/PCP信号传导的重要介质。预期的结果将有助于对体内形态发生过程中细胞骨架重塑的理解提供重要的新见解。

Dynamic changes of the actin cytoskeleton drive the morphogenetic processes which shape the vertebrate embryo during development. Migration of neural crest cells (NCCs) and closure of the neural tube represent two central such processes. Both are regulated by Wnt/planar cell polarity (PCP) signaling. The calponins (Cnn) represent an evolutionarily highly conserved but poorly characterized family of three actin-binding proteins. While several studies have demonstrated the ability of Cnn proteins to modulate the dynamics of the actin cytoskeleton in vitro, their in vivo functions and regulators have remained elusive. We have recently demonstrated a novel role for Cnn2 in promoting the migration of NCCs in chick and frog embryos. Cnn2 function was found to be regulated via the activity of the Wnt/PCP mediator RhoA kinase (ROCK) at the rear end of the emigrating cell, which resulted in the polarization of Cnn2 to the cell's leading edge. Our preliminary data also support a role of Cnn3 in promoting NCC migration and differentiation in Xenopus and chick embryos. Moreover, Cnn3 function was found to be required for neural tube closure, as its loss-of-function resulted in lack of apical constriction resulting in neural tube closure defects and non-polarized morphology of the cells. A recently published but so far uncharacterized conditional Cnn3 knockout mouse displays exencephaly as well, demonstrating the conserved nature of Cnn3 function in vertebrate neural tube closure. Based on these findings, we hypothesize that Cnn2 and Cnn3 act as mediators of Wnt/PCP/ROCK signaling to promote actin-dynamics essential for NCC migration and neural tube closure. To determine the function and regulation of Cnn2 and Cnn3 we aim at (I) identifying the relevant ROCK target sequences in Cnn2; (II) studying the relevance of Cnn2 degradation/polarization in NCCs via ROCK and proteasomal pathways active in this process; (III) determining the role of Cnn3 in NCC development and differentiation; (IV) revealing the function of Wnt/PCP-mediated Cnn3 function in neural tube closure. To achieve these goals, a combination of advanced developmental, molecular and cellular approaches will be utilized in chick, frog and mouse embryos in vivo and ex vivo. Based on our extensive preliminary and published data and the experimental plan, we expect to reveal that Cnn2 and Cnn3 act as essential mediators of Wnt/PCP signaling upstream of actin dynamics during morphogenesis. The anticipated results will contribute important new insights into the understanding of cytoskeletal remodeling during morphogenetic processes in vivo.