Abstract
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.
Highlights
The nature and identity of the pancreatic -cell precursor has remained elusive for many years
Using double immunofluorescence staining for glucagon and insulin together with the nuclear markers pancreatic duodenal homeobox factor-1 (PDX1), ISL1, paired box factor 6 (PAX6), and BRN4, we analyzed the development of the earliest endocrine cells in the mouse pancreas at the budding stage
We have shown that the early proliferating epithelium of the pancreas, which uniformly expressed the homeodomain protein PDX1, can be subdivided into Ngn3 + and Ngn3 – subpopulations
Summary
Ngn MARKS PANCREATIC ENDOCRINE PRECURSORS recent immunohistochemical analyses have suggested that -cells arise directly from the proto-differentiated epithelial cells, which express the -cell markers GLUT2, TrkA , PDX1, and Nkx6.1 [11,12,13,14,15]. We found evidence for a temporal sequence of gene activation and inactivation that differs between - and -cells Together, these findings suggest a molecular pathway where Ngn3 + precursor cells become committed to endocrine differentiation marked by activation of NeuroD and subsequently Isl , Pax , and the hormones, whereas Ngn expression is extinguished.
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