The atypical Rho GTPase, RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

Abstract

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.

Regulation and functions of RhoU and RhoV

“…25 Apart from the regulation of cell adhesion and cell shape during development, RhoU plays a role in cell migration in developmental models. Over-expression of the C. elegans RhoU/RhoV ortholog CHW-1 disrupted migration of gonadal distal tip cells, 27 a well-characterized migration model in C. elegans. 31 In addition, RhoU is required for the migration of cranial neural crest cells in Xenopus laevis embryos.…”

“…[27][28][29] It contributes to the formation of cell-cell junctions between zebrafish cardiomyocytes through a pathway involving the GEF Arhgef7b and PAK1, thereby affecting cardiac morphogenesis. 27 It would be interesting to determine whether PAR6 is involved in RhoU signaling in zebrafish cardiomyocytes, similar to its role in MDCK epithelial cells. 25 In C. elegans, the RhoU/ RhoV ortholog CHW-1 (which is equally similar to RhoU and RhoV) contributes to establishing polarity in vulval precursor cells (VPCs) during vulval development.…”

Rho signaling: An historical and evolutionary perspective

“…Cnidarians also possess a neural net, and some of them, nerve cords and plexuses (Lanna, 2015). In mice, Rnd3 deficiency elicits neuromuscular defects with a reduced number of motor neurons (Mocholí et al, 2011) and RhoU is involved in heart development and failure (Dickover et al, 2014;Herrer et al, 2014). The use of a Cnidaria model is needed to address if functions of the ancestral Rnd and RhoUV GTPases also pertained to nerve and muscle biology.…”

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish

“…3). Double mutants lacking maternal pxna exhibited stringy, poorly looped hearts and pericardial edemas at 48 hpf, similar to zebrafish deficient in Rho GTPase signaling effectors (Dickover et al, 2014), while those without maternal pxnb developed visible cardiac defects by 3 dpf. Additionally, MZ pxna ;Z pxnb embryos had qualitatively more severe cardiac malformations than the majority of Z pxna ;MZ pxnb embryos.…”