The Puoti laboratory investigates the molecular mechanisms that regulate differentiation of germ cells in the hermaphroditic nematode Caenorhabditis elegans. The C. elegans hermaphrodite is a perfect model for this study because its germ line harbors two fundamental cell fate decisions: germ cells first divide mitotically to amplify the pool of germ cells then enter meiosis and differentiate into mature gametes. At this point, gametes can either become sperm or oocytes, thus undergoing to another important cell fate decision.
During the last decade, numerous contributions have demonstrated that both aspects of germ cell development are governed by post-transcriptional events. These include processing on the pre-mRNA, regulation of translation initiation or elongation and stabilization of decay of mRNAs.
The first germ cells in the hermaphrodite differentiate into sperm. Later in the adult, spermatogenesis is arrested and all subsequent germ cells differentiate into oocytes. This switch is dependent on the post-transcriptional repression of the fem-3 mRNA. In the absence of fem-3, hermaphrodites develop into oocyte-producing females. In contrast, abnormally high levels of fem-3 lead to masculinized germlines. Using genetic and biochemical approaches, we have identified several genes involved the repression of the fem-3 mRNA.
Mitotic proliferation of germ cell precursors occurs in the distal portion of the germline, under the control of Notch/GLP signaling. In the absence of Notch/GLP signaling, germ cells do not divide mitotically, thus leading to mutants that hardly contain germ cells. In contrast, failure in meiosis leads to tumorous germlines. Surprisingly, many of the genes that are required for germline sex determination also function in the decision between mitotic proliferation and meitoic differentiation.
C. elegans is a protandric hermaphrodite, which means that it first produces a limited amount of sperm cells and then switches to oogenesis. This specific pattern of germ cell has been disrupted in a number of mutants that either produce only oocytes or sperm. We focus on the sperm/oocyte switch, which appears to be regulated post-transcriptionally in that the fem-3 mRNA is repressed for oogenesis. A number of proteins that repress fem-3 have been identified. These can roughly be put into two separate categories. On one side are FBF, GLD-3 and NOS-3 that are expressed in the cytoplasm and directly bind to the fem-3 target mRNA. In the nucleus, we have found the MOG proteins that do not bind the fem-3 RNA, but nevertheless bind to each other and to MEP-1, a nuclear zinc finger protein that plays multiple roles in gametogenesis and soma versus germline distinctions.
We investigate the genetic and molecular interactions between regulators of fem-3 and their implications in germline proliferation. Our studies aim at gaining a better insight into the molecular mechanisms through which they exert post-transcriptional regulation on fem-3 and other RNA targets. Since all genes that are involved in fem-3 regulation are conserved in other species, our investigations help understanding similar regulatory processes in more complex organisms.
Forward and reverse genetics; cloning of genes; molecular biology; analysis of transcripts; RNA interference; protein-protein interactions; antibody techniques (blotting, in-situ staining, precipitation); microscopy, microinjection.