A transgenic system for targeted ablation of reproductive and maternal-effect genes

Abstract

Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually into an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including the essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, called maternal-effect genes. However, other maternal-effect genes are not accessible due to their essential zygotic functions during embryonic development. Identifying these regulators is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways contributing to fertility and to maternally regulated developmental processes. To identify these maternal factors, it is necessary to bypass the earlier requirement for these genes so that their potential later functions can be investigated. Here, we report reverse genetic systems to identify genes with essential roles in zebrafish reproductive and maternal-effect processes. As proof of principle and to assess the efficiency and robustness of mutagenesis, we used these transgenic systems to disrupt two genes with known maternal-effect functions: kif5ba and bucky ball.

Rbpms2 promotes female fate upstream of the nutrient sensing Gator2 complex component, Mios

“…Briefly, we used previously generated stable adult transgenic fish expressing mApple-Rbpms2 or mApple alone under the oocyte specific promoter, buckyball (Figure 1a) 2,23 . This promoter was selected because it is specifically expressed in oocytes but not ovarian somatic cells 23,24 . We identified 732 RNAs bound by Rbpms2 in oocytes of adult ovaries ( rboRNAs ) (Figure 1b).…”

Rbpms2 promotes female fate upstream of the nutrient sensing Gator2 complex component Mios

“…1a) 2,23 . This promoter was selected because it is specifically expressed in oocytes but not ovarian somatic cells 23,24 , buc is present in the same cell types of the early ovary as rbpms2a and rbpms2b (Supp. Figure 1), and previous work in our lab has shown that these proteins both localize to the Balbiani body of early oocytes and are not present in any cells of the testis 2,3 .…”