Continual regeneration of the intestinal lining relies on stem and progenitor cells in sub-mucosal crypts, and intestinal disorders, including cancer, reflect dysfunction of these cells. Lgr5-expressing intestinal stem cells (ISC) replicate daily, producing bipotential progenitors (BP) that choose between alternative cell fates: enterocyte or secretory. This key cell fate decision is incompletely understood and the intestinal epithelium is a powerful model to investigate intermediate cell states. Therapeutic modulation of signaling pathways in the digestive tract also requires deeper appreciation of the underlying principles and mechanisms. Recent evidence reveals that crypt cells are considerably plastic, and in work supported by parent R01DK081113, we showed that broadly permissive chromatin underlies this plasticity.
Aim 1 of the parent R01 seeks to determine tissue-restricted enhancer elements and binding sites for the transcription factor ATOH1 in secretory progenitors because ATOH1 allocates cell fate by lateral inhibition. As mixed cell populations can and have addressed the question, this Aim does not need single-cell resolution.
The second Aim, however, investigates early ISC-derived BP, which are difficult to identify or characterize because they lack known molecular markers and information is lost in mixed-cell populations. Showing that single-cell analysis is well suited to advance project goals and address fundamental questions of broad interest, our preliminary single-cell gene expression analyses indicate that putative BP are a distinct subpopulation of Lgr5hi cells. Therefore, the first Specific Aim in this proposal is to identify and characterize these and other functionally distinct Lgr5+ ISC subpopulations. We will use single-cell RT-PCR to determine accurate transcript levels of ~275 genes carefully chosen to report cell lineage, replication status, surface markers, and differentiation states. We will alo measure these genes in specified secretory and enterocyte progenitors, purified by methods developed under the parent R01. We will build new analytical tools to determine cell relationships and hierarchies;preliminary data show that our experimental and computational approaches are feasible. Beyond gene expression, cells differ in surface markers and pathways for rapid intracellular signaling, and our second Specific Aim is to identify the basis for crypt cll heterogeneity in different signaling pathways using time-of-flight mass cytometry (CyTOF). Preliminary data reveal that AKT/S6 kinase signaling is confined to Lgr5hi ISC, quickly diminishing in BP and specified progenitors. We will substantively extend single-cell analysis by CyTOF to interrogate other signaling pathways and surface markers. For both Aims, we will rigorously test new hypotheses about cell subpopulations using crypt "organoid" cultures, and we propose new computational methods for integrated analysis of single-cell data from our two orthogonal approaches. This robust 2-tiered investigation of single cells will inform understanding of intestinal self- renewal and develop a strategy for broad application toward other tissues and biomedical problems.

Public Health Relevance

Continual, life-long renewal of the intestinal lining is essential for health and occurs through specialized stem and progenitor cells that reside in crypts and produce all intestinal cell types. Disturbances in these specialized cells lead to cancer and other digestive disorders, and better treatments for these illnesses require improved understanding of fundamental properties and circuits in distinct subpopulations of stem and progenitor cells. These properties and specific genes or signals escape detection in usual studies of mixed cell populations. We propose advanced single-cell analyses of intestinal crypt cells to determine when and how individual stem and progenitor cells acquire the ability to generate diverse intestinal cell types.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-R (50))
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Carrington, Jill L
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Dana-Farber Cancer Institute
United States
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Kim, Tae-Hee; Li, Fugen; Ferreiro-Neira, Isabel et al. (2014) Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506:511-5
San Roman, Adrianna K; Jayewickreme, Chenura D; Murtaugh, L Charles et al. (2014) Wnt secretion from epithelial cells and subepithelial myofibroblasts is not required in the mouse intestinal stem cell niche in vivo. Stem Cell Reports 2:127-34
Shivdasani, Ramesh A (2014) Radiation redux: reserve intestinal stem cells miss the call to duty. Cell Stem Cell 14:135-6
Kim, Tae-Hee; Escudero, Silvia; Shivdasani, Ramesh A (2012) Intact function of Lgr5 receptor-expressing intestinal stem cells in the absence of Paneth cells. Proc Natl Acad Sci U S A 109:3932-7
Woo, Janghee; Miletich, Isabelle; Kim, Byeong-Moo et al. (2011) Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation. PLoS One 6:e22493
Kim, Byeong-Moo; Woo, Janghee; Kanellopoulou, Chryssa et al. (2011) Regulation of mouse stomach development and Barx1 expression by specific microRNAs. Development 138:1081-6
Kim, Tae-Hee; Shivdasani, Ramesh A (2011) Notch signaling in stomach epithelial stem cell homeostasis. J Exp Med 208:677-88
Kim, Tae-Hee; Kim, Byeong-Moo; Mao, Junhao et al. (2011) Endodermal Hedgehog signals modulate Notch pathway activity in the developing digestive tract mesenchyme. Development 138:3225-33
San Roman, Adrianna K; Shivdasani, Ramesh A (2011) Boundaries, junctions and transitions in the gastrointestinal tract. Exp Cell Res 317:2711-8
Kim, Tae-Hee; Shivdasani, Ramesh A (2011) Genetic evidence that intestinal Notch functions vary regionally and operate through a common mechanism of Math1 repression. J Biol Chem 286:11427-33

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