Identification and Functional Analysis of Genes Controlling Germ Cell Migration and Gonad Formation

Gonad formation in Drosophila involves the intricate movements of two cell types, the germ cells and the somatic gonadal precursor cells (SGPs), to generate a simple three-dimensional structure. The germ cells must firstly migrate to SGP clusters, which in turn fuse and ensheath the germ cells and the two cell types ultimately coalesce. Underlying this seemingly simple process is an array of genes required to guide germ cells to the SGPs. Additional complex transcriptional networks in SGPs are essential for their specification, maintenance and to initiate their ensheathment behavior. This system represents a good model to study organogenesis using essentially two cell types. In this thesis I have examined aspects of both germ cell migration and SGP behavior. Firstly I examined the role of the Drosophila lipid phosphate phosphatases, Wun and Wun2 (referred to collectively as Wunens), in guiding the migration of germ cells. These multi-pass transmembrane enzymes, expressed both in the germ cells and the embryonic somatic cells, are thought to create an extracellular gradient of lipid phosphate, which is required for both migration and survival of the germ cells. However in order to form this extracellular lipid gradient, Wunens would be expected to act as ecto-enzymes. To investigate the site of requirement of Wun2 activity, I examined the localization of endogenous and different tagged versions of Wun2. Although both the GFP and the Myc tagged Wun2 were functional, the GFP version replicated the endogenous Wun2 plasma membrane localization. In parallel I made a construct in which a TEV protease cleavage site was inserted in a predicted extracellular loop of Wun2. I demonstrated that secretion of TEV protease by neighboring cells reduced the ability of this construct, to rescue wunen loss of function in germ cells. Taken together these data support the notion that the cell surface localization and the ecto-phosphatase activity of Wunens is critical for germ cell migration and survival. Secondly I have characterized the role of three mutants in which gonad formation is defective. In these mutants, although early germ cell migration is unaffected, in later embryos germ cells are scattered and SGP clusters do not fuse or compact. Whole genome sequencing coupled to deficiency complementation was performed to determine the causative mutations in two of the mutants. I identified mutations in two transcription factors, Midline (Mid) and Longitudinals lacking (Lola) and showed that both genes are expressed by SGPs and are required cell autonomously. The mutation in lola affects only one (lola-R) of the 30 predicted lola isoforms. Whilst the SGP defects resulting from mutation of Lola-R could be rescued by expression of this Lola isoform, another functional Lola isoform (Lola-BC) was unable to do so. Therefore, this study identifies the first lola-R specific allele, and describes a role in gonad formation that cannot be substituted by other Lola isoforms. Additionally, I have shown that robust gonad expression of another transcription factor Traffic jam is lost in mid mutants, placing Mid into a SGP transcriptional cascade. Lastly, mid and lola interact genetically suggesting that they could have one or more overlapping downstream targets.

Author

• Tripathy, Ratna

Identifier

hdl:10900/49949

OAI record oai:identifier

https://publikationen.uni-tuebingen.de/oai/request?verb=GetRecord&metadataPrefix=mets&identifier=oai%3Apublikationen.uni-tuebingen.de%3A10900%2F49949