The factors that control intestinal development and stem cell homeostasis remain inadequately characterized. Kruppel like factor 5 (KLF5, also termed IKLF or BTEB2) is a zinc-finger transcription factor that is thought to regulate proliferation and differentiation of gastrointestinal epithelia. KLF5 is highly expressed in the early embryo and the primordial endoderm during gastrulation, with continued expression in the mid/hindgut and embryonic intestine during morphogenesis and cytodifferentiation. During postnatal emergence of intestinal progenitor do- mains (crypts of Lieberkuhn), KLF5 becomes restricted to the crypts and its expression is maintained in this pro- genitor zone throughout life. In this context, KLF5 is hypothesized to promote proliferation by regulating cell cycle machinery such as cyclins, and has a role in oncogenic transformation. However, KLF5's role in embryonic and adult intestinal stem cell self-renewal and differentiation remains untested. Here, we propose to test the hypothesis that KLF5 controls self-renewal and differentiation of both embryonic and adult intestinal stem cells. In preliminary studies, we developed transgenic mice in which KLF5 can be inducibly deleted or activated in the intestine, as a means to investigate the function of KLF5 in vivo. These data show that KLF5 has an essential role in regulating proliferation and differentiation of intestinal stem cell/progenitors. We have also established a novel method to direct development of human pluripotent stem cells (PSCs, embryonic stem cells and induced pluripotent stem cells) into intestinal organoids in vitro, and show that KLF5 is expressed at key developmental steps in this system. In this proposal, we will utilize these unique resources to define the role of KLF5 in regulating self-renewal and differentiation in the intestine.
Three aims will be pursued: 1.) Define the requirement and sufficiency for KLF5 to control intestinal development. Hypothesis: Loss of KLF5 will result in impaired intestinal morphogenesis and cytodifferentiation, whereas increased expression of KLF5 will direct embryonic intestinal progenitors to precociously differentiate. We will manipulate KLF5 expression in order to define KLF5's role in embryonic intestinal development, using human PSC-derived intestinal organoids and transgenic mice. 2.) Determine the requirement and sufficiency for KLF5 to control adult intestinal stem cell homeostasis. Hypothesis: Loss of KLF5 will block differentiation and promote expansion of stem cells, whereas pan-epithelial KLF5 will deplete the stem cell pool. Using adult transgenic mice and crypt-derived epithelial organoids, intestinal stem cell homeostasis will be defined under conditions of inducible KLF5 manipulation. 3.) Define key targets of KLF5 that regulate embryonic morphogenesis and adult intestinal homeostasis. We have identified putative downstream effectors of KLF5 using microarray and bioinformatic approaches. To gain insight into the mechanisms of KLF5 action, epistasis analysis will be performed with target genes to test their ability to control differentiation and homeostasis in KLF5-manipulatable embryonic and adult intestinal organ/oid culture. Direct KLF5 targets will be identified by DNA binding and promoter reporter assays.

Public Health Relevance

The proposed studies will determine whether KLF5 is a target for future therapies to regulate growth and maturation of the intestine as a means to prevent and treat intestinal diseases such as necrotizing enterocolitis and intestinal failure. The results of this project will expand scientific knowledge of key aspects of intestinal development and stem cells, and test a new model in which KLF5 is a master regulator of intestinal epithelial morphogenesis and homeostasis. Together, this project will contribute to our long-term goals to regulate growth and maturation of the intestine in infants and adults, and will demonstrate the feasibility of manipulating patient-derived PSCs to produce intestinal tissues for cell therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK092306-04
Application #
8905197
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Carrington, Jill L
Project Start
2011-07-05
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
4
Fiscal Year
2014
Total Cost
$402,220
Indirect Cost
$146,842
Name
Baylor College of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Bell, Kristin N; Shroyer, Noah F (2015) Krüpple-like factor 5 is required for proper maintenance of adult intestinal crypt cellular proliferation. Dig Dis Sci 60:86-100
Watson, Carey L; Mahe, Maxime M; Múnera, Jorge et al. (2014) An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20:1310-4
Moore, Sean R; Pruszka, Jill; Vallance, Jefferson et al. (2014) Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine. Dis Model Mech 7:1123-30
Noah, Taeko K; Shroyer, Noah F (2013) Notch in the intestine: regulation of homeostasis and pathogenesis. Annu Rev Physiol 75:263-88
Noah, Taeko K; Lo, Yuan-Hung; Price, Allison et al. (2013) SPDEF functions as a colorectal tumor suppressor by inhibiting ?-catenin activity. Gastroenterology 144:1012-1023.e6
Bell, Sheila M; Zhang, Liqian; Xu, Yan et al. (2013) Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium. Dev Biol 375:128-39
Wolfs, T G A M; Kallapur, S G; Knox, C L et al. (2013) Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner. Mucosal Immunol 6:547-56
Noah, Taeko K; Donahue, Bridgitte; Shroyer, Noah F (2011) Intestinal development and differentiation. Exp Cell Res 317:2702-10