Research Article: Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Date Published: November 5, 2009

Publisher: Public Library of Science

Author(s): Nicolae Adrian Leu, Satoshi Kurosaka, Anna Kashina, Andreas Bergmann.

Abstract: Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.

Partial Text: Protein arginylation is a posttranslational modification that constitutes addition of arginine to proteins and is mediated by arginyltransferase (Ate1) [1]–[3]. Mice lacking Ate1 die between embryonic days E12.5 and E17.5 with severe cardiovascular defects, including abnormal heart development and angiogenesis [4], [5]. While Ate1 knockout (KO) embryos initially develop normal blood vessels in the process of vasculogenesis, the capillary network formation during subsequent angiogenesis is impaired, leading to defective capillary branching and their premature termination. These defects result in paleness, thin blood vessels, frequent skin edemas, and hemorrhages in the Ate1 KO embryos, and have been previously proposed to underlie the lethality in Ate1 KO mice, however the mechanisms of these defects, and the cells and tissues responsible for the Ate1−/− angiogenic phenotypes, are unknown.

In this study, we used a Tek-driven conditional mouse knockout of Ate1 to show that Ate1-mediated protein arginylation plays a previously unknown role in germ cell development. Our results suggest that Tek-Cre-induced deletion of Ate1 in premeiotic germ cells affects gametogenesis, resulting in the formation of defective gametes that cannot support normal embryogenesis. Unlike the complete Ate1 knockout mice that die on or after E12.5 [4], a significant percentage of Tek-Ate1 embryos die at early post-implantation stages, before E7.5, likely due to the defects in gamete formation from Ate1 knockout premeiotic germ cells (Figure 6). These defects are partially masked by the early lethality observed in the commercial Tek-Cre mouse line; according to our data all animals carrying homozygous Tek transgene die early in development, providing a ‘background’ lethality rate of approximately 25%. Since expression of Cre recombinase does not result in lethality in other conditional mouse strains, and since the Jackson Laboratory currently has another Tek-Cre mouse strain in development, which is stated to be viable as Tek homozygotes, we suggest that this lethality originates from the site of insertion of the Tek-Cre transgene, which may disrupt the function of an essential gene.



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