Long term objectives: This proposal aims to train Dr. Rachel Stine under the mentorship of Dr. Patrick Seale of the University of Pennsylvania and co-sponsor Dr. Klaus Kaestner of the University of Pennsylvania. A thorough training plan has been designed to train Dr. Stine in the field of intestinal stem cells. She will learn and master te techniques required for the study of mammalian intestinal stem cells, gain experience in developing and communicating her science, develop a network of collaborators, and train in grantsmanship. This grant will prepare Dr. Stine for an academic job search and subsequently a career as an independent academic investigator studying mammalian stem cells and related diseases.
Specific aims : The challenging intestinal environment requires constant stem cell-mediated intestinal regeneration. Preliminary studies show that the transcription factor Prdm16 is required for intestinal regeneration. Acute deletion of Prdm16 in the adult mouse causes a severe intestinal phenotype within 5 days, ultimately leading to death within two weeks. While it is not expressed in the colon, Prdm16 is expressed in the crypts and villi of the small intestine. Consistent with a phenotype observed when Notch signaling is inhibited, preliminary analysis of the effects of Prdm16 deletion in the small intestine show downregulation of the stem cell marker and Notch target Olfm4 as well as the accumulation of Mucin-2 positive goblet cells and lysozyme positive Paneth cells in the intestinal crypt. This proposal will dissect the role of Prdm16 in intestinal maintenance.
Aim 1 A makes use of an intestine-specific inducible Cre recombinase to thoroughly characterize the effect of Prdm16 deletion specifically in the intestinal epithelium.
In Aim 1 B, intestinal stem cell-specific inducible Cre recombinase driven by the Lgr5 promoter will be used to characterize the effect of stem cell- specific deletion of Prdm16.
In Aim 2 A, cultured enteroids or mini-guts will be used to determine the effects of Prdm16 overexpression on the intestinal epithelium.
In Aim 2 B, enteroid culture will be used to test the requirement of specific domains of the Prdm16 protein.
In Aim 3, RNA-Seq in the villi and crypts of wild type and Prdm16 deleted small intestines and Prdm16 ChIP-Seq will be used to identify Prdm16 primary targets. Overall, this proposal will offer insight into the role of Prdm6 in the intestine as well as its specific mechanism of action and how it functions within the known signaling networks of the intestine. Disease Relevance: Intestinal epithelial cells are replaced every 4-5 days through stem cell-mediated intestinal regeneration. This process plays a critical role in mammalian nutrient absorption and defense against bacteria. Due to the large number of human diseases and environmental insults that cause intestinal problems, treatments to augment or replace intestinal stem cells will greatly impact regenerative medicine. To develop such treatments, a thorough understanding of the genes and pathways controlling intestinal regeneration is required. This proposal will provide insight into a key gene in intestinal regeneration.
While the constant regeneration of the intestine plays a key role in human health, further mechanistic understanding is required before intestinal stem cells can be manipulated to treat intestinal diseases. This proposal's preliminary data shows that Prdm16, which is known to play a key role in the maintenance of neuronal and hematopoietic stem cells, is required for intestinal maintenance. This proposal will provide fundamental insights into how the intestine is maintained and the general mechanisms by which Prdm16 controls stem cell maintenance and differentiation.
|Stine, Rachel R; Shapira, Suzanne N; Lim, Hee-Woong et al. (2016) EBF2 promotes the recruitment of beige adipocytes in white adipose tissue. Mol Metab 5:57-65|
|Brestoff, Jonathan R; Kim, Brian S; Saenz, Steven A et al. (2015) Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519:242-6|