Jensen, J. et al. Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation. Diabetes 49, 163-176

Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Ángeles, California, United States
Diabetes (Impact Factor: 8.1). 03/2000; 49(2):163-76. DOI: 10.2337/diabetes.49.2.163
Source: PubMed


The nature and identity of the pancreatic beta-cell precursor has remained elusive for many years. One model envisions an early multihormonal precursor that gives rise to both alpha- and beta-cells and the other endocrine cell types. Alternatively, beta-cells have been suggested to arise late, directly from the GLUT2- and pancreatic duodenal homeobox factor-1 (PDX1)-expressing epithelium, which gives rise also to the acinar cells during this stage. In this study, we have identified a subset of the PDX1+ epithelial cells that are marked by expression of Neurogenin3 (Ngn3). Ngn3, a member of the basic helix-loop-helix (bHLH) family of transcription factors, is suggested to act upstream of NeuroD in a bHLH cascade. Detailed analysis of Ngn3/paired box factor 6 (PAX6) and NeuroD/PAX6 co-expression shows that the two bHLH factors are expressed in a largely nonoverlapping set of cells, but such analysis also suggests that the NeuroD+ cells arise from cells expressing Ngn3 transiently. NeuroD+ cells do not express Ki-67, a marker of proliferating cells, which shows that these cells are postmitotic. In contrast, Ki-67 is readily detected in Ngn3+ cells. Thus, Ngn3+ cells fulfill the criteria for an endocrine precursor cell. These expression patterns support the notion that both alpha- and beta-cells develop independently from PDX1+/Ngn3+ epithelial cells, rather than from GLU+/INS+ intermediate stages. The earliest sign of alpha-cell development appears to be Brain4 expression, which apparently precedes Islet-1 (ISL1) expression. Based on our expression analysis, we propose a temporal sequence of gene activation and inactivation for developing alpha- and beta-cells beginning with activation of NeuroD expression. Endocrine cells leave the cell cycle before NeuroD activation, but re-enter the cell cycle at perinatal stages. Dynamic expression of Notch1 in PDX+ epithelial cells suggests that Notch signaling could inhibit a Ngn-NeuroD cascade as seen in the nervous system and thus prevent premature differentiation of endocrine cells.

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    • "Several different signaling pathways are essential for pancreas development (Kimmel and Meyer, 2010; Serup, 2012). For instance, Notch signaling has long been known as central to both mammalian (Apelqvist et al., 1999; Esni et al., 2004; Hald et al., 2003; Jensen et al., 2000; Murtaugh et al., 2003) and zebrafish pancreas development (Esni et al., 2004; Lorent et al., 2004; Ninov et al., 2012; Parsons et al., 2009; Zecchin et al., 2007). Inhibition of Notch signaling leads to precocious differentiation of PNCs and the early appearance of endocrine cell types in the 21 islet position within the pancreatic tail (Parsons et al., 2009). "
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    ABSTRACT: As the developing zebrafish pancreas matures, hormone-producing endocrine cells differentiate from pancreatic Notch-responsive cells (PNCs) that reside within the ducts. These new endocrine cells form small clusters known as secondary (2°) islets. We use the formation of 2° islets in the pancreatic tail of the larval zebrafish as a model of β-cell neogenesis. Pharmacological inhibition of Notch signaling leads to precocious endocrine differentiation and the early appearance of 2° islets in the tail of the pancreas. Following a chemical screen, we discovered that blocking the retinoic acid (RA)-signaling pathway also leads to the induction of 2° islets. Conversely, the addition of exogenous RA blocks the differentiation caused by Notch inhibition. In this report we characterize the interaction of these two pathways. We first verified that signaling via both RA and Notch ligands act together to regulate pancreatic progenitor differentiation. We produced a transgenic RA reporter, which demonstrated that PNCs directly respond to RA signaling through the canonical transcriptional pathway. Next, using a genetic lineage tracing approach, we demonstrated these progenitors produce endocrine cells following inhibition of RA signaling. Lastly, inhibition of RA signaling using a cell-type specific inducible cre/lox system revealed that RA signaling acts cell-autonomously in PNCs to regulate their differentiation. Importantly, the action of RA inhibition on endocrine formation is evolutionarily conserved, as shown by the differentiation of human embryonic stem cells in a model of human pancreas development. Together, these results revealed a biphasic function for RA in pancreatogenesis. As previously shown by others, RA initially plays an essential role during embryogenesis as it patterns the endoderm and specifies the pancreatic field. We reveal here that later in development RA is involved in negatively regulating the further differentiation of pancreatic progenitors and expands upon the developmental mechanisms by which this occurs.
    Developmental Biology 08/2014; DOI:10.1016/j.ydbio.2014.07.021 · 3.55 Impact Factor
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    • "This is exemplified, in the pancreas, by recent studies of the Notch signaling pathway. Originally implicated specifically as a negative regulator of endocrine differentiation (Apelqvist et al., 1999; Jensen et al., 2000), Notch has since been shown to inhibit acinar development as well (Esni et al., 2004; Hald et al., 2003; Murtaugh et al., 2003), potentially through modulating proximal–distal patterning of the early organ (Afelik et al., 2012; Magenheim et al., 2011; Schaffer et al., 2010). Although much has been gleaned from studying Notch and other pathways individually , relatively little is known about these signals coalesce to pattern and specify progenitors in the developing pancreas. "
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    ABSTRACT: Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate β-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component β-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of β-catenin function to β-cell development remains controversial. To resolve the potential roles of β-catenin in development of MPCs and β-cells, we generated pancreas- and pre-endocrine-specific β-catenin knockout mice. Pancreas-specific loss of β-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in β-cell mass. The loss of β-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for β-catenin in maintaining proximal-distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent "trunk" fate when β-catenin is deleted. Moreover, β-catenin maintains proximal-distal patterning, in part, by inhibiting Notch signaling. Subsequently, β-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for β-catenin along the route of β-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.
    Developmental Biology 04/2014; 391(1). DOI:10.1016/j.ydbio.2014.03.019 · 3.55 Impact Factor
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    • "In addition to INS, NGN3 and PAX4 can be used to monitor the differentiation of INS+ cells from PDX1+ cells, because NGN3 is an essential transcription factor in the development of pancreatic endocrine tissue323334, and PAX4, a downstream gene of NGN3, plays a critical role in the development of β cells35. We found that the combination of forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide achieved the highest expression of NGN3, PAX4 and INS as reported previously (Figs. "
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    ABSTRACT: Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs.
    Scientific Reports 03/2014; 4:4488. DOI:10.1038/srep04488 · 5.58 Impact Factor
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