The Drosophila adult midgut is a homeostatic tissue, in which cells constantly turn over and are replaced by the progeny of intestinal stem cells divisions. These progeny, called enteroblasts, are initially quiescent, lack an apical domain and lie on the basal side of the epithelium. In response to dying cells or signals that expand the gut, the enteroblasts integrate into the epithelium, polarise and form a new apical domain with a typical brush border. Two features of this process are very different from other epithelia in Drosophila. Firstly, enteroblast polarisation does not require any of the canonical epithelial polarity factors that have been identified in other tissues, and it depends instead on adhesion to the basement membrane, as is the case in mammalian epithelia (1). Secondly, a complete apical domain with brush border forms inside the epithelium before the enteroblasts reach the apical gut lumen, in a process that resembles lumen formation in mammalian epithelial cysts in 3D culture (2). The goal of the project is to identify the factors that initiate apical domain formation and to determine how these cooperate to direct the formation of the apical microvilli.
Objectives of the project include:
- Perform a targeted RNAi screen for factors required for normal apical domain formation using the MARCM technique to express the RNAi in GFP labelled clones. The screen will focus on apical transmembrane proteins that we have identified by proximity labelling or using bioinformatic approaches.
- Analyse the phenotypes of the most interesting lines using an expansion microscopy protocol that we have developed for the Drosophila
- Use CRISPR-mediated homologous recombination to insert fluorescent tags into the most promising candidate proteins and analyse their localisation in wild-type intestines and in mutants that affect apical domain formation.
Group and hosting institution
The St Johnston group is based in the Gurdon Institute at the University of Cambridge, one of the world’s oldest and most successful universities.
References
- Chen, J., Sayadian, A.-C., Lowe, N., Lovegrove, H.E., and St Johnston, D. (2018). An alternative mode of epithelial polarity in the Drosophila midgut. PLoS biology 16, e3000041. 10.1371/journal.pbio.3000041.
- Chen, J., and St Johnston, D. (2022). De novo apical domain formation inside the Drosophila adult midgut epithelium. Elife 11, e76366. 10.7554/elife.76366.
