Soma-germline communication in the Drosophila testes: Interplay between polarity, endocytosis, and EGFR signalling in a squamous epithelium

Tissue homeostasis relies on short-range communication between cells and their microenvironment. In the Drosophila testis, germ cells proliferate and differentiate encapsulated in a pair of squamous somatic cyst cells (CCs) that together form the functional unit of differentiation. During Drosophila spermatogenesis, the CCs support the differentiation of the germline by progressively increasing Epidermal Growth Factor Receptor (EGFR) signalling levels. Previously, it has been shown that loss of the polarity proteins Dlg, Scribble, Lgl, or components of clathrin-mediated endocytosis (CME) in CCs, results in cell autonomous EGFR upregulation and non-autonomous germ cell death. Therefore, under homeostatic conditions, Dlg, Scribble, Lgl and CME components act to downregulate EGFR signalling levels in CCs to facilitate the progressive differentiation of the neighbouring germ cells. However, the pathway mediating the non-autonomous germ cell death, the regulatory mechanisms of EGFR signalling levels in CCs, and the ultrastructure of squamous CCs, remain unclear. In this thesis, I analysed stress-induced signalling pathways to uncover the drivers of the non-autonomous germ cell death seen upon EGFR overactivation in CCs. The JNK signalling pathway was identified as the primary driver of the non-autonomous germ cell death, as JNK signalling levels significantly increased in CCs upon EGFR upregulation and mediated the apoptosis of germ cells. This work uncovered a bi-directional feedback mechanism between the JNK pathway in CCs and the production of reactive oxygen species (ROS) in germ cells. Moreover, a biotin-based proximity labelling assay was chosen to uncover how polarity regulates EGFR signalling. For this, I have made genetic constructs that can identify common and distinct interacting partners of Dlg and EGFR, as well as plasma membrane, endocytic, and cytoplasmic controls in the cyst cells. Finally, to reveal the CC ultrastructure, a protocol for employing Array Tomography Scanning Electron Microscopy (AT-SEM) in the Drosophila testis has been established. By integrating functional, proteomic, and ultrastructural data, this thesis has laid the foundation to uncover the mechanisms that underpin the function of squamous epithelia in the Drosophila testis, which may help to identify further the general principles of squamous cell function beyond Drosophila.