The transcription factor grainy head regulates intestinal stem cells

Abstract

The constant renewal of the intestinal epithelium is achieved through the activity of a population of intestinal stem cells (ISC). These cells maintain tissue homeostasis by ensuring balance between cell division and the production of the differentiated cell types of the intestine. This process is under strict molecular control with ISC maintenance and function controlled by the expression of a particular subset of genes and their production of multiple protein isoforms. However, the mechanism by which different protein isoforms are able to regulate intestinal regeneration remains poorly understood. Using the adult intestine of Drosophila melanogaster, we identified grainy head (grh) as a candidate gene that is able to influence regeneration using different protein isoforms. The Drosophila genome contains a single grh gene which is alternatively spliced to produce 8 mRNA transcripts and two major isoform groups, the N-isoforms and the neural specific, O-isoforms. In this study, I show using droplet digital PCR that transcripts for both isoform groups including the neural specific isoforms are expressed in the midgut, albeit at low cellular levels. Moreover, cell lineage tracing studies using grh null mutants result in a reduction of progeny arising from a single ISC. Interestingly, cell lineage tracing using an O-specific mutant resulted in a more severe reduction in progeny number compared to grh null mutants. Additionally, lineage traced ISCs mutant for the O-isoforms were lost over time. In the opposite experiment, an O-isoform, GRH.O’ was ectopically expressed in ISCs and its immediate daughter, the enteroblast (EB). Over expression solely in ISCs did not lead to a change in ISC number while over expression in EBs led to a block in differentiation and an increase in EB number. Conversely, ectopic expression of two GRH N-isoforms, GRH.N and GRH.N’ led to a loss of ISC and EBs through forced differentiation. I therefore hypothesize that GRH O-isoforms are required for ISC maintenance while the N-isoforms are required for differentiation in the Drosophila midgut. In the final part of my study, I conducted expression analysis for GRHL-2 and GRHL-3, the mammalian orthologues of GRH in the mouse small intestine. Expression studies using qPCR and immunohistochemistry reveal that both GRHL-2 and GRHL-3 are expressed in intestinal crypt cells suggesting that the function of GRH is conserved the mammalian small intestine.