rbpms2 functions in Balbiani body architecture and ovary fate

Abstract

The most prominent developmental regulators in oocytes are RNA-binding proteins (RNAbps) that assemble their targets into ribonucleoprotein granules where they are stored, transported and translationally regulated. RNA-binding protein of multiple splice forms 2, or Rbpms2, interacts with molecules that are essential to reproduction and egg patterning, including bucky ball, a key factor for Bb formation. Rbpms2 is localized to germ granules in primordial germ cells (PGCs) and to the Balbiani body (Bb) of oocytes, although the mechanisms regulating Rbpms2 localization to these structures are unknown. Using mutant Rbpms2 proteins, we show that Rbpms2 requires distinct protein domains to localize within germ cells and somatic cells. Accumulation and localization to subcellular compartments in the germline requires an intact RNA binding domain. Whereas in zebrafish somatic blastula cells, the conserved C-terminal domain promotes localization to the bipolar centrosomes/spindle. To investigate Rbpms2 functions, we mutated the duplicated and functionally redundant zebrafish rbpms2 genes. The gonads of rbpms2a;2b (rbpms2) mutants initially contain early oocytes, however definitive oogenesis ultimately fails during sexual differentiation and, rbpms2 mutants develop as fertile males. Unlike other genes that promote oogenesis, failure to maintain oocytes in rbpms2 mutants was not suppressed by mutation of Tp53. These findings reveal a novel and essential role for rbpms2 in oogenesis. Ultrastructural and immunohistochemical analyses revealed that rbpms2 is not required for the asymmetric accumulation of mitochondria and Buc protein in oocytes, however its absence resulted in formation of abnormal Buc aggregates and atypical electron-dense cytoplasmic inclusions. Our findings reveal novel and essential roles for rbpms2 in Buc organization and oocyte differentiation.

Loss ofdmrt1restores female fates in the absence ofcyp19a1abut notrbpms2

“…2C), or were testis-like ( Fig. 2D) as we previously reported [27]. All dmrt1 single mutant gonads examined had early oocytes ( Fig.…”

“…rbpms2 and dmrt1 are both expressed as RNAs in stage I oocytes ( Fig. 2) [11,27]. Because female development initiates but ultimately fails in rbpms2DMs, and because we identified Rbpms2 binding sites in the 3'utr of dmrt1 transcripts, it is possible that Rbpms2 promotes female differentiation by repressing dmrt1 directly, and/or by antagonizing the male specific program mediated by Dmrt1.…”

“…We previously showed that cellular features of rbpms2DM juvenile gonads are comparable to wild-type [27]. To determine if rbpms2DM juvenile gonads resemble wild-type molecularly, we prepared cDNA from day 21 post fertilization (d21) trunks, prior to sex-determination, and used RTPCR to examine markers associated with female or male-specific somatic gonad or germ cell fates.…”

“…Because rbpms2 double mutants can develop as fertile males that complete meiosis and spermatogenesis but fail to complete female differentiation [27], we reasoned that rbpms2 is not required for meiosis per se, but instead is required to prevent the germline from adopting a male fate. This could occur either by promoting expression of female factors, by repressing male factors, or a combination of both.…”

“…Recent work from our lab identified an RNA binding protein, RNA-binding protein of multiple splice forms 2 (Rbpms2), as a critical germline expressed [27] factor for female sex differentiation in zebrafish [27]. Simultaneous loss of both redundant copies of rbpms2 (rbpms2a and rbpms2b; rbpms2 double mutants (DMs)) results in recovery of exclusively fertile males [27]; however, how Rbpms2 promotes female fates is not clear. It has also been shown that both the somatic gonad and meiotic oocytes contribute to establishing and maintaining the female gonad in domesticated zebrafish (reviewed by [1]).…”