Null mutations of NEUROG3 are associated with delayed-onset diabetes mellitus.

Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding ß cell mass sufficient to maintain euglycemia until early childhood.

The cellular regulators PTEN and BMI1 help mediate NEUROGENIN-3-induced cell cycle arrest.

Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.

Multiphoton Activation of Photo-Strain-Promoted Azide Alkyne Cycloaddition "Click" Reagents Enables in Situ Labeling with Submicrometer Resolution.

Irradiation of cyclopropenone-masked dibenzocyclooctynes with near-infrared pulses from a femtosecond laser triggers photodecarbonylation via nonresonant two- or three-photon excitation. Multiphoton-generated cyclooctynes undergo a SPAAC reaction with organic azides, yielding the expected triazoles. Multiphoton-triggered SPAAC (MP-SPAAC) enables high resolution 3-D photoclick derivatization of hydrogels and tissues.

Facile Quenching and Spatial Patterning of Cylooctynes via Strain-Promoted Alkyne-Azide Cycloaddition of Inorganic Azides.

Little is known about the reactivity of strain-promoted alkyne-azide cycloaddition (SPAAC) reagents with inorganic azides. We explore the reactions of a variety of popular SPAAC reagents with sodium azide and hydrozoic acid. We find that the reactions proceed in water at rates comparable to those with organic azides, yielding in all cases a triazole adduct. The azide ion's utility as a cyclooctyne quenching reagent is demonstrated by using it to spatially pattern uniformly doped hydrogels. The facile quenching of cyclooctynes demonstrated here should be useful in other bioorthogonal ligation techniques in which cyclooctynes are employed, including SPANC, Diels-Alder, and thiol-yne.

Origin of the brush cell lineage in the mouse intestinal epithelium.

Mix progenitors are short-lived multipotential cells formed as intestinal epithelial stem cells initiate a differentiation program. Clone dynamics indicates that various epithelial cell lineages arise from Mix via a sequence of progressively restricted progenitor states. Lateral inhibitory Notch signaling between the daughters of Mix (DOM) is thought to break their initial symmetry, thereby determining whether a DOM invokes a columnar (absorptive) or granulocytic (secretory) cell lineage program. This is supported by the absence of granulocytes following enforced Notch signaling or Atoh1 deletion. Conversely, granulocytes increase in frequency following inhibition of Notch signaling or Hes1 deletion. Thus reciprocal repression between Hes1 and Atoh1 is thought to implement a Notch signaling-driven cell-fate-determining binary switch in DOM. The brush (tuft) cells, a poorly understood chemosensory cell type, are not incorporated into this model. We report that brush cell numbers increase dramatically following conditional Atoh1-deletion, demonstrating that brush cell production, determination, differentiation and survival are Atoh1-independent. We also report that brush cells are derived from Gfi1b-expressing progenitors. These and related results suggest a model in which initially equivalent DOM progenitors have three metastable states defined by the transcription factors Hes1, Atoh1, and Gfi1b. Lateral inhibitory Notch signaling normally ensures that Hes1 dominates in one of the two DOMs, invoking a columnar lineage program, while either Atoh1 or Gfi1b dominates in the other DOM, invoking a granulocytic or brush cell lineage program, respectively, and thus implementing a cell fate-determining ternary switch.

Cell Lineage metastability in Gfi1-deficient mouse intestinal epithelium.

Elucidating the mechanisms determining multipotent progenitor cell fate remains a fundamental project of contemporary biology. Various tissues of mice and men with defects in the zinc-finger transcriptional repressor Gfi1 have dramatic perturbations in the proportions of their differentiated cell types. In Gfi1-deficient intestinal epithelium there is a shift from mucous and Paneth towards enteroendocrine cells, leading to the proposal that Gfi1 functions in the allocation of the progeny derived from a hypothetical common granulocytic progenitor. However, studies of clones have yielded no evidence of such a common progenitor prompting us to investigate alternate mechanisms explaining the Gfi1-deficient phenotype. We report that mucous and Paneth but not enteroendocrine lineage cells normally express Gfi1. Sporadic mucous and Paneth lineage cells in the crypts of Gfi1-deficient mice aberrantly express the pro-enteroendocrine transcription factor Neurog3, indicating that stable repression of Neurog3 in these lineages requires Gfi1. Importantly, we also find mucous and Paneth lineage cells in various stages of cellular reprogramming into the enteroendocrine lineage in Gfi1-deficient mice. We propose that mucous and Paneth cell lineage metastability, rather than reallocation at the level of a hypothetical common granulocytic progenitor, is responsible for the shifts in cell type proportions observed in Gfi1-deficient intestinal epithelium.

Intestinal epithelial stem cells and progenitors.

The adult intestinal epithelium contains a relatively simple, highly organized, and readily accessible stem cell system. Excellent methods exist for the isolation of intestinal epithelium from adults, and as a result collecting large quantities of intestinal stem and progenitor cells for study or culture and subsequent clinical applications should be routine. It is not, however, for two reasons: (1) adult intestinal epithelial cells rapidly initiate apoptosis on detachment from the basement membrane, and (2) in vitro conditions necessary for survival, proliferation, and differentiation are poorly understood. Thus to date the study of intestinal stem and progenitor cells has been largely dependent on in vivo approaches. We discuss existing in vivo assays for stem and progenitor cell behavior as well as current methods for isolating and culturing the intestinal epithelium.

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium.

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.

Gastrointestinal stem cells. II. Intestinal stem cells.

Current views of the identity, distribution, and regulation of small intestinal epithelial stem cells and their immediate progeny are discussed. Recent works implicating Wnt signaling in stem and progenitor proliferation, the involvement of Notch signaling in epithelial lineage specification, and the role of hedgehog and bone morphogenetic protein families in crypt formation are integrated. We had the good fortune that many of these papers came in pairs from independent groups. We attempt to identify points of agreement, reinterpret each in the context of the other, and indicate directions for continued progress.

Multipotential stem cells in adult mouse gastric epithelium.

Previous studies of chimeric animals demonstrate that multipotential stem cells play a role in the development of the gastric epithelium; however, despite much effort, it is not clear whether they persist into adulthood. Here, chemical mutagenesis was used to label random epithelial cells by loss of transgene function in adult hemizygous ROSA26 mice, a mouse strain expressing the transgene lacZ in all tissues. Many clones derived from such cells contained all the major epithelial cell types, thereby demonstrating existence of functional multipotential stem cells in adult mouse gastric epithelium. We also observed clones containing only a single mature cell type, indicating the presence of long-lived committed progenitors in the gastric epithelium. Similar results were obtained in duodenum and colon, showing that this mouse model is suitable for lineage tracing in all regions of the gastrointestinal tract and likely useful for cell lineage studies in other adult renewing tissues.