Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632).

Abstract

Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute to failure of VBW closure. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute to the development of VBWD. At 60h incubation, chick embryos were explanted into shell-less culture and treated with 50µL of vehicle for controls (n=33) or 50µL of 500µM of Y-27632 for the experimental group (Y-27, n=56). At 8h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E-cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A further cohort of embryos was treated in ovo by dropping 50µL of vehicle or 50µL of different concentrations of Y-27632 onto the embryo and allowing development to 12 and 14days for further assessment. Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly decreased in treated embryos compared with controls (p<0.05). Thickened actin filament bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were dissociated with reduced actin distribution in affected embryos. Clumping of vinculin expression was found in the neural tube and somites, whereas reduced expression of microtubules was observed in Y-27 embryos compared with controls. At 12 and 14days of development, affected embryos presented with an enlarged umbilical ring and herniation of abdominal contents through the defect. ROCK inhibition alters cytoskeletal arrangement during early chick embryogenesis, which may contribute to failure of anterior body wall closure causing VBWD at later stages of development.