The Dynamic Regulation of Intestinal Stem Cells by Notch Signaling.

The intestinal epithelium has one of the fastest cellular turnover rates in the body, a process fueled by a highly active intestinal stem cell (ISC) compartment. Two ISC populations are thought to exist: the active crypt base columnar stem cell (CBCC) and quiescent stem cells (QSCs). The Notch signaling pathway is one of several pathways known to regulate ISC function. My thesis work has focused on understanding the specificity and kinetics underlying Notch regulation of CBCCs. First, I probed the specificity of Notch receptors in regulating intestinal homeostasis by conditionally deleting Notch1 (N1) and/or Notch2 (N2) in the intestinal epithelium in genetic mouse models. Deletion of N1 but not N2 led to increased numbers of secretory cells, demonstrating that N1 is the dominant receptor regulating cell fate decisions. Additionally, N1 deletion reduced the CBCC population by 50% and eliminated recovery after irradiation, discoveries that have clinical implications for using targeted anti-Notch drugs as cancer treatments. My thesis also investigated the cellular mechanism of decreased CBCCs and decreased transit-amplifying (TA) cell proliferation after Notch inhibition. I tracked the consequences of acute Notch inhibition on stem cells over time. Surprisingly, while acute inhibition resulted in decreased CBCC number it also led to increased TA proliferation rather than the decreased proliferation previously observed with chronic inhibition. I devised a compartmental mathematical model of the intestinal crypt to reconcile the proliferation differences observed with acute and chronic Notch inhibition. The model favored a mechanism where Notch signaling regulates both the symmetry of CBCC division into TA cells, as well as repopulation of the CBCC compartment, presumably by activation of QSCs. Further work investigating the role of Notch in QSCs suggests that Notch regulation of CBCC replacement is through regulation of the CBCC niche rather than direct regulation of QSCs. In summary, my thesis work has probed the role of Notch in intestinal epithelial homeostasis and CBCC maintenance. I show that loss of Notch signaling leads to a dynamic shift of CBCCs into the TA cell compartment and that N1 is the key receptor regulating these changes.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120757/1/acarulli_1.pd

Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5+ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5+ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.SynopsisThe Notch signaling pathway is required to maintain LGR5+ antral stem cells and epithelial cell homeostasis.Gastric antral stem cells display active Notch1 receptor signaling.Global Notch inhibition reduces stem and progenitor cell proliferation and increases differentiation of all lineages.Notch activation in LGR5+ stem cells increases stem and progenitor cell proliferation and inhibits differentiation.Notch activation enhances antral stem cell function, leading to tissue expansion via gland fission and tumor formation.The Notch signaling pathway is required to maintain LGR5+ antral stem cells and epithelial cell homeostasis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/1/embj201490583-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/2/embj201490583.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/3/embj201490583.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115949/4/embj201490583-sup-0001-Appendix.pd

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe