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形态发生过程，从而塑造了脊椎动物胚胎的发育。神经脊细胞(NCC)的迁移和神经管的关闭代表了两个中心的过程。两者都受Wnt/平面细胞极性(PCP)信号的调节。钙蛋白(CNN)是一个进化上高度保守但特性不佳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态的能力，但它们在体内的功能和调节仍然难以捉摸。我们最近展示了Cnn2在促进NCC在鸡和青蛙胚胎中迁移的新作用。Cnn2的功能是通过迁移细胞后端的Wnt/PCP介导物RhoA激酶(ROCK)的活性来调节的，从而导致Cnn2极化到细胞的前沿。我们的初步数据也支持Cnn3在非洲爪哇和鸡胚胎中促进NCC迁移和分化的作用。此外，还发现Cnn3功能是神经管关闭所必需的，因为它的功能丧失导致没有心尖收缩，导致神经管关闭缺陷和细胞的非极化形态。最近发表的但到目前为止尚未确定特征的条件性Cnn3基因敲除小鼠也表现出脑外畸形，证明了Cnn3功能在脊椎动物神经管闭合中的保守性质。基于这些发现，我们假设Cnn2和Cnn3作为Wnt/PCP/ROCK信号的中介，促进了NCC迁移和神经管关闭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：(I)确定Cnn2中的相关岩石靶序列；(Ii)研究Cnn2通过激活的ROCK和蛋白酶体途径在NCC中降解/极化的相关性；(Iii)确定Cnn3在NCC发生和分化中的作用；(Iv)揭示Wnt/PCP介导的Cnn3在神经管关闭中的功能。为了实现这些目标，将结合先进的发育、分子和细胞方法在体内和体外对鸡、青蛙和小鼠胚胎进行研究。基于我们大量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Wnt/PCP信号通路上游的肌动蛋白动力学的重要中介。这些预期的结果将为理解体内形态发生过程中的细胞骨架重塑提供重要的新见解。

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.