Experimental validation and characterization of putative targets of Escargot and STAT, two master regulators of the intestinal stem cells in Drosophila melanogaster

Abstract

Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. Here, we experimentally validate and characterize the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). Our results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis.

Keywords: Drosophila; Escargot; Intestinal stem cell; Master regulator; STAT.