586C Poster - 08. Patterning, morphogenesis and organogenesis
Saturday April 09, 1:30 PM - 3:30 PM

Authors:
Fionna Zhu; Michael Murray; Georgia Malloy

Affiliation: University of Melbourne

Keywords:
k. gut; b. cell polarity

Maintenance of epithelial barrier function is essential for human health. The adult Drosophila midgut provides a powerful model system for understanding mechanisms of epithelial homeostasis. It consists of a monolayered epithelium of absorptive enterocytes (ECs) and secretory enteroendocrine cells, underpinned by stem cells and intermediate progenitor enteroblasts (EBs). When EBs differentiate into ECs, they undergo a Mesenchymal-Epithelial Transition (MET) and intercalate into the epithelium.

While much is known about the transcription factors and signalling pathways that regulate EB identity and differentiation, the mechanical aspects of MET are less well studied. To identify novel EB MET regulators we conducted an RNAi screen using the lineage tracing tool, ReDDM. We screened genes likely to be involved in that process and identified three components of septate junctions (SJs): Discs Large (dlg), Mesh and Snakeskin.

Dlg is a member of the MAGUK family of scaffolding proteins and is known to regulate SJ formation, cell polarity and proliferation. EB-specific knockdown of dlg using Su(H)GBE-GAL4, resulted in a multilayered epithelium, a more rounded EB morphology, defective SJs and premature expression of the EC-marker Pdm1. dlg knockdown also led to a shortened lifespan and increased mortality due to chemical damage by DSS, likely as a result of impaired barrier function.

MARCM clones deficient for dlg, recapitulated the multilayering and SJ protein mislocalisation RNAi phenotypes. Interestingly, the extent of multilayering appears more severe in the anterior midgut compared to the posterior. Patches of Mesh and Ssk were mislocalised to the apical membranes of clones and were complementary to enriched F-actin, suggesting disruption of microvilli structure. We also observed intracellular actin-rich vacuoles (likely autophagosomes) and apical membrane disruption, features previously associated with delamination and cell death upon Ecc15 infection, coordinated by the EGFR pathway. Isolated clones were occasionally found detached from the basement membrane, supporting this hypothesis. Gaps in the epithelium were also found nearby clones, with enrichment of SJ proteins at their boundaries.

We are currently examining whether these phenotypes are caused by disruption to key signalling pathways. Together, our results highlight the role of Dlg in midgut homeostasis and MET and the utility of the Drosophila model to expand our understanding of epithelial homeostasis.