Alexis J. Carulli

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.

Blockade of individual Notch ligands and receptors controls graft-versus-host disease

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders.

Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration.

The Notch signaling pathway regulates intestinal epithelial cell homeostasis, including stem cell maintenance, progenitor cell proliferation and differentiation. Notch1 and Notch2 receptors are expressed in the epithelium, but individual contributions to these functions are unclear. We used genetic deletion to define receptor roles on stem cell function, cell proliferation/differentiation, and repair after injury. Loss of Notch1 induced a transient secretory cell hyperplasia that spontaneously resolved over time. In contrast, deletion of Notch2 had no secretory cell effect. Compound deletions of Notch1 and Notch2 resulted in a more severe secretory cell hyperplasia than deletion of Notch1 alone. Furthermore, only double deletion of Notch1 and Notch2 decreased cell proliferation, suggesting a low threshold for maintenance of proliferation compared to differentiation. Stem cells were affected by deletion of Notch1, with reduced expression of Olfm4 and fewer LGR5(+) stem cells. Deletion of Notch2 had no apparent affect on stem cell homeostasis. However, we observed impaired crypt regeneration after radiation in both Notch1- and Notch2-deleted intestine, suggesting that higher Notch activity is required post-injury. These findings suggest that Notch1 is the primary receptor regulating intestinal stem cell function and that Notch1 and Notch2 together regulate epithelial cell proliferation, cell fate determination, and post-injury regeneration.

Unraveling intestinal stem cell behavior with models of crypt dynamics

The definition, regulation and function of intestinal stem cells (ISCs) has been hotly debated. Recent discoveries have started to clarify the nature of ISCs, but many questions remain. This review discusses the current advances and controversies of ISC biology as well as theoretical compartmental models that have been coupled with in vivo experimentation to investigate the mechanisms of ISC dynamics during homeostasis, tumorigenesis, repair and development. We conclude our review by discussing the key lingering questions in the field and proposing how many of these questions can be addressed using both compartmental models and experimental techniques.

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.

Reduced funding and sequestration impact young biomedical researchers

The federal sequester has exacerbated an already grim research funding crisis. The continued budget cuts have had a widespread effect on all researchers in biomedical science, but the impact is especially keen on trainees who are deciding whether or not to stay in science. Steps must be taken to bolster young researcher morale, career options and funding to ensure the continued advancement of biomedical science in this country.

Sa1754 Isolation of Ribosome Associated mRNA As a Tool to Study Transcriptional Changes in Paneth Cells

or 120 μM). After 30 min loops were injected with purified toxin A (10ug in 50 μl PBS) or PBS alone in 200 μl volume (n=6 mice per group). After 4 hr ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion and edema) and cytokine measurements. Results: Toxin A significantly increased histology score (3 fold, p=0.0001), ileal TNFα protein (150%, p=0.01), TNFα mRNA (3 fold, p=0.03), IL1β protein (163%, p=0.04), IL-1β mRNA (3.6 fold, p=0.03), KC mRNA (6 fold, p=0.05) and neutrophil myeloperoxidase (MPO) protein (180%, p=0.03). Treatment with fidaxomicin (20 μM) or OPT-1118 (120 μM) significantly reduced toxin A mediated histologic damage (by 80% fidaxomicin, p=0.001; by 54% OPT-1118, p=0.01), ileal TNF α protein (by 20% fidaxomicin, p=0.049; by 34% OPT-1118, p=0.02), TNFα mRNA (by 70% fidaxomicin, p= 0.02; by 66% OPT-1118, p=0.01), IL-1β protein (by 47% fidaxomicin, p=0.007; by 48% OPT-1118, p=0.02), IL-1β mRNA (by 90% fidaxomicin, p=0.01; by 66% OPT-1118, p= 0.04), KC mRNA (by 93% fidaxomicin, p=0.04; by 77% OPT-1118, p=0.04) and MPO (by 40% fidaxomicin p=0.04; by 45% OPT-1118, 0.03). Treatment of lower dose of fidaxomicin (5 μM) and OPT1118 (60 μM) had a milder, statistically insignificant inhibition of histologic damage and proinflammatory cytokine expression in response to toxin A. Treatment with fidaxomicin or OPT-1118 without toxin A had no significant effects to histology score or basal cytokine of MPO levels. Conclusions: In addition to its well known anti-C. difficile bacteria effects, fidaxomicin may possess potent anti-inflammatory activity directed against the intestinal effects of C. difficile toxins. Grant support: Optimer Pharmaceuticals (CP); Crohns and Colitis Foundation of America Career Development Award (HWK); Student Research Fellowship (SH); NIH K01 DK084256 (HWK).