Presenilins, Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window

The pancreas develops from Pdx1- and Ptf1a-expressing progenitors that emerge from the foregut endoderm to form ventral and dorsal buds. Shortly thereafter, these buds will fuse and form a complex organ composed of endocrine, acinar, and ductal cells. The steps involved in establishing the identity of the endocrine progenitors and the molecular steps involved in organogenesis have been the subjects of many studies, several identifying Notch signaling as a master regulator controlling the initial endocrine versus acinar decision (for review, see Jorgensen et al. 2007). The Notch genes encode conserved, membrane-bound receptors mediating short-range cell–cell communications during embryonic development and in the adult. Notch proteins are proteolytically processed in response to ligand binding (for review, see Kopan and Ilagan 2009). Intramembrane proteolysis of all Notch receptors is mediated by the enzyme γ-secretase with Presenilin proteins (Ps1 or Ps2) forming its catalytic core. Following γ-secretase cleavage, the released Notch intracellular domain (NICD) enters the nucleus, where it binds to RBP-Jκ, recruits Mastermind, and assembles an activator complex. Recent lineage studies with neurogenin3-Cre transgenic lines exclusively marked the endocrine tissue, suggesting that expression of Ngn3 identified committed endocrine progenitors (Gu et al. 2002, 2003). Deletion of Ngn3 compromised endocrine differentiation, indicating that this transcription factor is required (Gradwohl et al. 2000; Lee et al. 2001). Overexpression of Ngn3 in transgenic mice blocked exocrine development and accelerated endocrine differentiation (Apelqvist et al. 1999), demonstrating that Ngn3 by itself is sufficient to drive the endocrine fate at this stage. Ngn3 regulates endocrine differentiation in part through the activation of Pax6, a transcription factor that bears both a paired box and a homeodomain (for review, see Jorgensen et al. 2007). A few Ngn3+ progenitor cells are detected in the adult pancreas (Gu et al. 2002) where, under specific regeneration conditions, they can proliferate and differentiate into pancreatic β cells (Xu et al. 2008), further establishing Ngn3 as a marker of endocrine progenitors. Although Notch signaling is thought to antagonize Ngn3 expression through activation of HES1 (Esni et al. 2004), allowing the Ptf1a/p48 complex to direct the cells toward acinar differentiation (Obata et al. 2001; Beres et al. 2006; Fujikura et al. 2007; Masui et al. 2007), HES1 can also be activated by several other signaling pathways (Kageyama et al. 2007). Expression of receptors (Notch1–3), ligands (Jag-1, Jag-2, Dll-1, and Dll-3), and targets (HES1) was observed in cells that did not yet appear to be differentiated, but not in more mature endocrine cells within the embryonic pancreas (Jensen et al. 2000). Complicating matters are observations identifying an essential role for RBP-Jκ as a cofactor for Ptf1a/p48, thus directly promoting acinar differentiation independent of Notch (Obata et al. 2001; Beres et al. 2006; Fujikura et al. 2007; Masui et al. 2007). Since earlier studies were not aware of this dual function of RBP-Jκ, and thus did not account for it in the experimental design and interpretation of results, ambiguity surrounds the exact role of Notch signaling throughout pancreatic development. Disruption of Notch signaling by removing the ligand (Dll1) or RBP-Jκ resulted in decreased pancreatic bud size along with expansion of endocrine cells (Apelqvist et al. 1999); however, loss of p48 activity in RBP-Jκ-null cells was not considered. In contrast, Pdx1-controlled overexpression of NICD3, an ineffective HES1 activator that binds to RBP-Jκ with the same affinity as NICD1 (Lubman et al. 2007), led to reduction in HES1 and Ptf1a expression, with a subsequent increase in Ngn3 expression (Apelqvist et al. 1999). Again, the possible negative impact on Ptf1a function was not considered. Although recent studies have implicated Notch2 as a susceptibility locus for type 2 diabetes (Zeggini et al. 2008), confidence in an early role for Notch signaling during pancreas development has eroded further since removal of both Notch1 and Notch2 still permitted formation of a functional pancreatic organ, whereas removal of RBP-Jκ did not (Nakhai et al. 2008). Whatever their role, Notch receptors are required only transiently, as highlighted by the observation that the constitutively active form of Notch1 expressed under the control of the Pdx1 promoter caused marked reduction in both exocrine and endocrine differentiation (Hald et al. 2003; Murtaugh et al. 2003). To investigate whether Notch signaling was still involved after the initial endocrine/acinar decision, we inactivated Notch signaling by removal of presenilin1 and presenilin2 after the establishment of Ngn3 progenitors with an Ngn3-Cre line and, in parallel, traced the fate of presenilin-deficient cells with an EGFP. Unexpectedly, we discovered that Ngn3 progenitors, originally thought to be committed to the endocrine fate in the absence of Notch signals, selected instead the acinar fate even when presenilin dose was only reduced, not eliminated. The number of Ngn3 progenitors entering the acinar fate was inversely correlated with Presenilin activity. The phenotype associated with presenilin-nulls can be attributed in most cases to loss of Notch signaling (Struhl and Greenwald 2001; Pan et al. 2004, 2005); however, in addition to their role in Notch, ErbB, and Ryk proteolysis (Kopan and Ilagan 2004; Lyu et al. 2008), Presenilins have been implicated in multiple additional processes: transport vesicles (Sisodia and St George-Hyslop 2002; Pigino et al. 2003; Wood et al. 2005), scaffolds for AKT activation (Baki et al. 2004), β-catenin degradation (Kang et al. 1999), and Erk activation (Kim et al. 2005; Wines-Samuelson and Shen 2005). Since pharmacological inhibition of the γ-secretase function in vitro also diverted Ngn3 progenitors to the acinar fate, we concluded that γ-secretase activity was involved. Finally, genetic interaction with Notch2 indicates that Notch is a relevant substrate in this process. Once formed, we observed that Ngn3-derived, presenilin-deficient acinar cells undergo proper acinar differentiation, but display enhanced proliferation compared with control cells. Importantly, proper organ size is maintained by a corresponding increase in apoptosis. In addition, we found that whereas Ngn3+ cells are still bipotential, endocrine progenitors become irreversibly committed to the endocrine fate as soon as Pax6 is expressed and can no longer switch to acinar fates in the absence of presenilins. This study thus exposed a default (acinar) fate of Ngn3 progenitors that persists during a narrow developmental window. We propose that, within this window, the activity of γ-secretase generates NICD2 that sequesters RBP-Jκ away from p48 to promote, rather than inhibit, the endocrine fate. A similar noncanonical Notch function may also participate in controlling the glutaminergic/GABAergic decision within the CNS (Hori et al. 2008), and may be also involved in controlling horizontal/amacrine versus ganglion cell fate decisions in the retina (Fujitani et al. 2006).