New ideas connecting the cell cycle and pancreatic endocrine-lineage specification

Abstract

Neurogenin 3 (Neurog3) is a pro-endocrine transcription factor required for endocrine-lineage specification during mouse pancreas development. It was a long-standing notion that Neurog3 activation in Sox9 C epithelial cells, a subset of early multipotent progenitors that produce ductal and endocrine lineages, triggers a rapid progression to a post-mitotic, Neurog3 HI endocrine-committed precursor state. We challenged this model by demonstrating that a mitotic progenitor state, in which Neurog3 is transcriptionally active but at a low level (Neurog3 TA.LO ), pre-empts endocrine-commitment. 1 We postulated a new model in which this Neurog3 TA.LO progenitor state is a mitotic, metastable condition in which low Neurog3 levels regulate decisions regarding progenitor maintenance and endocrine-lineage specification. 1 Our work showed that Neurog3 TA.LO progenitors resemble other progenitor populations, such as intestinal and haematopoietic progenitors, in their ability to undergo multiple rounds of division that seem to be symmetric and produce either more Neurog3 TA.LO progenitors, or two endocrine-committed daughters. 1 This modification of the endocrine-lineage specification model focuses attention on the Neurog3 TA.LO progenitor state as being the stage wherein the critical decision is made as to which endocrine cell fate will be established within committed cells emerging from Neurog3 TA.LO progenitors. Below, we discuss potential connections between the cell cycle and Neurog3 protein levels in Neurog3 TA.LO progenitors, and the implications of bringing this model together with a systems-level understanding of the 3-dimensional and dynamic nature of an epithelial plexus state that represents the endocrine-birth niche. Understanding how cycling Neurog3 TA.LO progenitors integrate their behavior within the surrounding dynamics of a developing epithelial endocrine birth niche will be important. For example, what determines progenitor-maintaining or symmetric endocrinebirth divisions, and the end-fate endocrine potential of Neurog3 TA.LO progenitors, could inform on how to generate multilineage islets rather than simply populations of b cells.Gain and loss of function studies show the power of Neurog3 in instructing endocrine differentiation, but other studies had already begun to suggest correlations between a more finely adjusted Neurog3 expression level and the potential of the Neurog3 TA.LO population to produce endocrine cells (Fig. 1). Wang et al. (2010) reported that a strong Neurog3 hypomorphic condition significantly decreased the degree of endocrine biasing, diverting many cells to non-endocrine acinar or ductal fates (Fig. 1). 2 Our analysis of the same hypomorphic condition showed a doubling of the mitotic index of Neurog3 TA.LO progenitors, but not of the surrounding nonendocrine-biased cells, and expansion of the Neurog3 TA.LO pool at the expense of endocrine-committed cells.

FUCCI tracking shows cell‐cycle‐dependent Neurog3 variation in pancreatic progenitors

“…These findings support our model that, in actively cycling Neurog3 TA.LO progenitors, Neurog3 protein levels vary according to the cell-cycle phase (Bechard and Wright, 2017). To address this issue, we used Neurog3 P2A.FUCCI reporter expression in Neurog3 TA.LO progenitors to track cell-cycle progression in relation to Neurog3 protein levels.…”

“…These findings support our model that, in actively cycling Neurog3 TA.LO progenitors, Neurog3 protein levels vary according to the cell-cycle phase (Bechard & Wright, 2017 (30/120) (Figure 3a).…”

“…Moreover, time-lapse observations show that the transition from the low level of Neurog3 observed in mitotic Neurog3 TA.LO progenitors to the high level necessary for endocrine-commitment occurs ~3–6 hours after division of the parental Neurog3 TA.LO cell, during what is likely the early part of an extended G 1 or long-term cell-cycle exit (Bechard et al, 2016). These findings led to our proposal that the level and stability of Neurog3 in mitotic Sox9 + Neurog3 TA.LO progenitors is regulated by the cell cycle and that G 1 extension promotes Neurog3 stabilization, accumulation, and endocrine commitment (Bechard and Wright, 2017). Two recent reports support this model, demonstrating that Neurog3 is targeted and destabilized by Cdks (Azzarelli et al, 2017) and that G 1 lengthening, by reducing Cdk activity, causes the accumulation of a more stable un(der)phosphorylated form of Neurog3 (Krentz et al, 2017).…”

FUCCI tracking shows that Neurog3 levels vary with cell-cycle phase in endocrine-biased pancreatic progenitors

“…Recent studies showed that during S-G 2 -M, Neurog3 is kept in a hyperphosphorylated unstable state via Cdk phosphorylation, and that decreased Cdk activity associated with entrance into G 1 result in stabilization and accumulation ( 14 , 15 ). These findings support our model that, in actively cycling Neurog3 TA.LO progenitors, Neurog3 protein levels vary according to the cell-cycle phase ( 13 ). To address this issue, we used Neurog3 P2A.FUCCI reporter expression in Neurog3 TA.LO progenitors to track cell-cycle progression in relation to Neurog3 protein levels.…”

“…Moreover, time-lapse observations show that the transition from the low level of Neurog3 observed in mitotic Neurog3 TA.LO progenitors to the high level necessary for endocrine-commitment occurs ~3-6 hours after division of the parental Neurog3 TA.LO cell, during G 1 ( 6 ). These findings led to our proposal that the level and stability of Neurog3 in mitotic Sox9 + Neurog3 TA.LO progenitors is regulated by the cell cycle and that G 1 extension promotes Neurog3 stabilization, accumulation, and endocrine commitment ( 13 ). Two recent reports support this model, demonstrating that Neurog3 is targeted and destabilized by Cdks and that G 1 lengthening, by reducing Cdk activity, causes the accumulation of a more stable un(der)phosphorylated form of Neurog3 ( 14 , 15 ).…”