Determining the role of the steroid hormone ecdysone in critical stages of Drosophila melanogaster early oogenesis

Abstract

Reproductive function in many organisms, including Drosophila melanogaster, is controlled by steroid hormones. The main steroid hormone in Drosophila is ecdysone, which is structurally and functionally similar to estrogen. Ecdysone is essential for many processes throughout oogenesis including germline stem cell (GSC) function, cell differentiation, encapsulation, and yolk uptake. Many studies have investigated the role and transcriptional targets of ecdysone during organismal development, but, despite its diverse roles in oogenesis, much less is known about transcriptional targets in the ovary. Reproductive capacity in the Drosophila ovary is maintained by the activity of germline stem cells (GSCs), which maintain an undifferentiated fate while creating daughters that will differentiate. A complex network influences stem cell fate, including local signaling and long-range endocrine signals. Female GSCs are directly regulated by ecdysone signaling. Using a reverse genetic screen, we identified Heterogeneous nuclear ribonucleoprotein at 27C (Hrb27C) as a putative target of ecdysone signaling. Hrb27C is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins, which function to bind mRNAs and regulate their splicing, maturation, and localization. We tested the hypothesis that specific hnRNPs function downstream of ecdysone signaling to promote GSC self-renewal. We identified four hnRNPs, squid (sqd), hephaestus (heph), Hrb27C, and Hrb87F whose expression was decreased in ecdysone mutants, suggesting that they are downstream targets of ecdysone in the ovary. Loss-of-function analyses confirmed that Hrb27C, sqd, and heph are required in GSCs for self-renewal. Our data suggest that GSC loss in the absence of Hrb27C, sqd, and heph is due, at least in part, to deregulation of BMP signaling, which represses differentiation in GSCs. Our data supports the model that specific hnRNPs help maintain stem cell fate in response to ecdysone signaling by stabilizing the expression of BMP signaling transcripts. To further determine what the transcriptional targets of ecdysone signaling are in early oogenesis we performed whole transcriptome sequencing (RNA-seq) comparing gene expression in ovaries isolated from Ecdysone Receptor (EcR) loss-of-function mutant females versus those from wild type females. Intriguingly, the differentiation factor bag of marbles (bam) was significantly over-expressed in the absence of ecdysone signaling. This suggests that ecdysone signaling may play a previously unappreciated role in early cyst divisions and differentiation and could provide insight into how GSC self-renewal is regulated. Loss of ecdysone signaling from early mitotically dividing cysts reduces the number of cells in M phase suggesting that ecdysone signaling regulates the cell cycle in differentiating cysts. We also observed increased expression of various cell cycle targets, including pimples (the Drosophila Securin homolog) and Nek2, a serine-threonine kinase, both required for mitosis. This suggests that ecdysone signaling may regulate the transition between mitotic divisions to meiosis and endocycling in differentiated germ cells. Moreover, transcripts encoding the meiotic cohesion protein, orientation disruptor (ord) were significantly reduced in the absence of ecdysone signaling. Ord stabilizes the synaptonemal complex in presumptive oocytes and is essential for the earliest events of oocyte specification. Interestingly Orb, an RNA binding protein specifically expressed in differentiated oocytes is also decreased in EcRts mutant ovaries. Taken together, these results suggest that ecdysone signaling may promote oocyte specification and differentiation. This data will give us further insight into cell type-specific interpretation of ecdysone signaling and increase our understanding of the functions of ecdysone signaling in oogenesis.