Affiliation: California State University, Northridge, Los Angeles, CA
Keywords: d. intestinal stem cells; n. networks
Many organs contain adult stem cells (ASCs) that replace cells lost to damage, disease or normal turnover. Like their embryonic counterparts, ASCs can divide asymmetrically, giving rise to a new copy of themselves (i.e. self-renewal) and a sister cell that commits to differentiation into a specific cell type. Previous work has led to the identification of so-called master regulators (MR) genes in ASCs, i.e. pleiotropic genes that directly or indirectly affect the expression and activity of hundreds of downstream effectors, and whose functional alteration simultaneously affects diverse aspects of normal stem cell homeostasis. Genome-wide screens around MR genes have identified hundreds of their putative targets. However, the experimental validation of these bioinformatic predictions and the further characterization of putative downstream effectors as true stem cell regulators is notoriously lacking.
To begin addressing this gap between bioinformatic predictions and experimental validation, we use intestinal stem cells (ISCs) of the Drosophila melanogaster posterior midgut. We first integrated genome-wide DNA mapping data for two known MR genes in these cells: the transcription factor Escargot (Esg) and the signal transducer STAT. We then used qPCR analysis to test the response of over 80 predicted targets to the genetic manipulation of either or both MR genes in vivo, which allowed us to validate several of them as true targets. This analysis revealed a noticeable false discovery rate (FDR) with regards to mode of regulation by the MR genes among the bioinformatic predictions, as reflected by a higher than predicted rate of co-regulation by both Esg and STAT. Furthermore, the genetic manipulation of nearly all confirmed targets that we have tested thus far has resulted in noticeable phenotypes in ISC homeostasis, including changes to their number and/or morphology. Therefore, while our data would indicate that bioinformatic prediction of downstream target regulation by these MR genes is rather noisy, it is nonetheless highly predictive of a true ISC regulatory role for those targets that have been validated experimentally by independent means.