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

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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.