Alternative splicing provides a means of massively expanding the complexity of mammalian transcriptomes and nearly all human multi-exon gene transcripts are alternatively spliced. However, in contrast to studies of transcription factors and transcriptional gene expression programs, the role of alternative splicing in important developmental programs remains largely unstudied. Using a genome-wide cell-based screen my lab discovered two paralogous epithelial-specific splicing regulatory proteins, ESRP1 and ESRP2. The ESRPs regulate both alternative splicing as well as alternative polyadenylation as master regulators of an extensive post-transcriptional network. Our studies suggest that they play a key role in the epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). However, while our data in cell lines are consistent with this proposal, definitive evidence to support this contention requires analysis in more elegant in vivo genetic model systems. Towards this end we generated mice with conditional and complete knockout alleles for Esrp1 and Esrp2. Using these mice we will investigate the following hypotheses: First, we hypothesize that Esrp expression is required for proper ureteric bud branching and collecting duct formation. Second, we propose that the ability of metanephric cap mesenchyme cells to undergo MET and generate renal tubular epithelial cells requires Esrp expression. We further hypothesize that these requirements for ESRP involve critical ESRP target transcripts whose coordinated isoform switches are needed for proper kidney formation and function. We will address these hypotheses though the Following Specific Aims: 1) Define the developmental role of the ESRPs in ureteric bud branching and formation of the renal collecting system. We will conditionally ablate Esrp expression in the ureteric bud and derivatives in crosses with HoxB7 Cre mice. Potential defects in ureteric branching will be assessed after serial sectioning and 3D reconstructive imaging. 2) Determine whether the Esrps are required for the MET in cap mesenchyme and development of renal tubular epithelial cells. The role of Esrp induction during the MET and consequent formation of the glomerulus and renal tubular epithelium will be determined through conditional ablation in cap mesenchymal in breedings with Six2-Cre mouse lines. 3) Investigate the consequences of Esrp knockout in adult renal tubular and collecting duct epithelial cells and perform in vivo global profiling of ESRP target transcripts using high throughput sequencing. We hypothesize that ablation of Esrp expression in mature renal epithelial cells will induce dedifferentiation and cellular injury. In addition to determining the phenotypic consequences of Esrp knockout we will also use high throughput sequencing technologies to comprehensively determine the in vivo renal epithelial splicing and polyadenylation program that is enforced by the Esrps. The collective definition of Esrp regulated targets will reveal important gene transcripts and an extensive post-transcriptional network that function in renal organogenesis and function.
Alternative splicing allows the same gene transcript to be processed into proteins with different functions in different cell types, at different stages of development, and in numerous diseases. We identified essential master regulators of alternative splicing that control the expression of hundreds of genes, thereby resulting in a distinct set of proteins forms that are specific to epithelial cells. The kidney is a predominantly epithelial organ and as such the complete program of epithelial cell splicing controlled by these regulators is proposed to be essential for kidney development and function. Identification of the complete program of gene regulation controlled by these epithelial splicing proteins will provide fundamental insights with broad applications to our understanding of human health and diseases.
