Conservation of a GAP independent function of the DLC3/Cv-c RhoGAP proteins required for male gonadogenesis

Abstract

HIGHLIGHTS: - Drosophila Cv-c RhoGAP null mutations experiment testis dysgenesis akin to human patients with mutations in the homologous DLC3 RhoGAP protein. -The DLC3/Cv-c male gonadogenesis function is independent of their Rho GAP function and requires a functional START domain. -Cv-c is required to maintain the germ cell ensheathment in Drosophila testis. - DLC3 rescues testis dysgenesis in cv-c null mutants suggesting a deep functional evolutionary conservation. The DLC3 RhoGAP human protein has been implicated in a case of 46,XY gonadal dysgenesis where two patients inherited a mutation in the START domain, however, no definitive confirmation has been provided yet linking this mutation with male gonadal dysgenesis. DLC3 belongs to a subfamily of RhoGAP proteins containing three conserved domains: a SAM, a GAP and a START domain. The three domains are also present in the homologous Drosophila Cv-c RhoGAP88C protein and DLC3 can functionally substitute for Cv-c. We have previously analysed Cv-c activity in the ectoderm where the GAP domain is absolutely required for its function. However, Cv-c mesodermal requirement has not been analysed yet. We show Cv-c is specifically expressed and required in the male gonadal mesoderm. In vivo analysis of cv-c null mutants deleting the GAP and START domains shows normal testis development up to gonad coalescence at st15. However, after st15 the germ cells become extruded from the testis due to their defective ensheathment by mesodermal interstitial gonadal cells, which express lower levels of E-Cad and Neurotactin in the mutants. Surprisingly,mutantsforthecv-c7 allele,apointmutationonlylackingafunctionalGAPdomain,havenormaltestis.Wealso find Rho1 mutations do not normalize cv-c null mutations indicating a novel Rho GAP independent function in the gonad. Expression of Cv-c protein variants lacking a functional GAP domain can rescue testis development but not mutants lacking the START domain. Interestingly, human DLC3 can partially rescue cv-c null gonad defects but not the DLC3 START mutant allele present in the human patients. Our results show a new Rho GAP independent specific function for this protein family that is required for testis development and has a deep evolutionary conservation.