The robust expression of Jag1
in the embryonic pancreas suggested that this gene plays a major role in pancreatogenesis. Of course, translational efficiency of the various Notch ligands differ, so although mRNA levels of Jag1
are higher than those of Dll1
, protein levels may not exactly reflect mRNA levels. Deletion of Jag1
in the foregut endoderm, which gives rise to the pancreatic epithelium, leads to a postnatal phenotype similar to early embryonic phenotypes of Hes1
mutants (Fujikura et al., 2006
; Jensen et al., 2000
). This similarity may be surprising since a recent report detailing a compound knockout of Notch1
describes a very mild pancreatic phenotype compared to those of Hes1
mutants; these Notch1/2
double knockout mice did not have any dysregulation of Ngn3.
The mild phenotype observed in the Notch1/2
compound mutant may, however, be due to expression of Notch3/4
in the pancreatic epithelium. Although one group has reported that only Notch1/2
are expressed in the pancreatic epithelium (Lammert et al., 2000
), others have demonstrated epithelial expression of Notch3/4
as well (Apelqvist et al., 1999
mice display important differences in comparison with previously reported Notch pathway mutants. Jag1
-deficient pancreata have normal total endocrine mass and a full complement of β-cells while other Notch signaling mutants have reduced endocrine mass despite an increased percentage of endocrine cells (Apelqvist et al., 1999
; Fujikura et al., 2006
; Jensen et al., 2000
mutants also have a reduced Ngn3+
population at E16.5 but have persistent Ngn3
expression after birth, while the pancreas-specific Rbpj
mutant has an increased Ngn3+
population at E10.5 but reduced thereafter (Fujikura et al., 2006
). Finally, Jag1
mutants have decreased exocrine mass due to acinar cell death, not due to a change in cell lineage allocation with precocious differentiation of common pancreatic progenitors.
This increase in acinar cell death may indicate that Notch signaling promotes acinar cell survival; Jag1loxP/loxP;Pdx1-Creearly
mice also exhibit acinar cell death, at even higher rates than Jag1loxP/loxP;Foxa3-Cre+
mice, with fatty replacement of the acinar tissue; the acinar cell death is probably due to ductal malformation (Golson et al., 2009
). The acinar cell death in Jag1loxP/loxP;Foxa3-Cre+
mice may also result from a ductal malformation that could be less severe due to the later deletion Jag1loxP/loxP;Foxa3-Cre+
Like many other Notch signaling pathway mutants, Jag1
mutants have an increased percentage of α-cells within the endocrine compartment. However, due to the timing of increased Ngn3
expression, the increase in α-cells is unexpected since the competency of the fetal pancreas to produce α-cells because of Ngn3
overexpression decreases dramatically with age (Johansson et al., 2007
). Differences observed in these two models could be explained in at least two ways: 1) misexpression of Ngn3 under control of the Pdx1-Cre promoter initially causes it to be expressed in pancreatic progenitor but later in β- and δ-cells, and this misexpression leads to the change in percent of differentiating α-cells; or 2) embryonic Notch upregulation in the Jag1
mutant pancreatic epithelium leads to competency alterations due to persistent general pancreatic precursors such, as in mice that overexpress Notch1 (Hald et al., 2003
). Differentiated cells of all types, including acinar, islet, and duct cells are observed in neonates, however, and no markers of general pancreatic precursors are available that do not also mark differentiated cell types; therefore, determining whether more pancreatic progenitors are present in the late embryonic and peritnatal Jag1
mutant pancreas has proven difficult.
Our most surprising finding is that during fetal life, Jag1
mutants mimic an increase in Notch signaling rather than the expected decrease. E16.5 Jag1
mutants have a reduced endocrine population, while direct transcriptional targets of Notch—Hes5 and Hey1—are upregulated, and Ngn3
expression is decreased compared to control littermates. This striking phenotype may be explained by the spatially and temporally restricted presence of the Notch glycosyltransferase, Mfng, which is expressed highly in endocrine progenitors but is absent or very low in the rest of the embryonic pancreas or in differentiated pancreatic cells. Mfng has previously been reported to induce endocrine cell development when overexpressed in chick endoderm and to overlap partially with Ngn3 in the developing murine pancreas (Svensson et al., 2009
; Xu et al., 2006
A recent report suggests that Mfng is unnecessary for pancreas development (Svensson et al., 2009
). Mfng null mice displayed no change in endocrine cell number or ratio between E9.5 and E14.5, and adults had normal pancreas morphology with no defects in blood glucose homeostasis. Endocrine cell counts after E14.5 were not reported, however, and endocrine cell number defects were not observed in Jag1
mutants until after E14.5. Therefore, this recent report does not preclude the possibility that Mfng causes Jag1 to act as an inhibitor of Notch signaling in the embryonic pancreas.
Genetic evidence in Drosophila
supports the hypothesis that Jag1 can act as an inhibitor of Notch signaling in the presence of Fringe (de Celis et al., 1998
; Klein and Arias, 1998
). Assays with Fringe glycosyltransferases in 3T3 cells reflect what is known of the function of Fringe in Drosophila
; specifically, cells expressing both Serrate and Fringe are unable to respond to Serrate ligand. In mammals, glycosylation of Notch receptors by Mfng decreases Jagged-induced Notch cleavage without preventing binding between Jagged and Notch (Hicks et al., 2000
; Yang et al., 2005
). In addition to the reduced signaling efficacy from Jag1 in the presence of Mfng, we show that Jag1 can act as a competitive inhibitor of Dll1-induced Notch signaling when Mfng is expressed ().
We present a model in which Jag1 binds to but does not activate glycosylated Notch and interferes with Dll1 binding, leading to relatively low levels of Notch activation (). Since endocrine precursors express high levels of Mfng, and genetic ablation of Jag1 leads to increased rather than decreased Notch signaling as assayed by Hes5, Hey1, and Ngn3 expression at E16.5, the function of Jag1 during pancreatogenesis may be to limit the number of cells with activated Notch, just like Serrate in the Drosophila wing margin. Increased Notch signaling observed in Jag1 deficient pancreata may be the result of more efficient binding between Dll1 and Notch receptors, leading to high levels of Notch activation and thus reduced Ngn3 expression.
Many modifiers of the Notch signaling pathway exist and our experiments do not unequivocally determine that Mfng is the reason that Jag1 mutants display increased Notch activation in the embryonic pancreas. However, we do show that Jag1 can act as an inhibitor of Dll1-induced Notch activation when Notch is glycosylated and that Mfng is highly expressed in endocrine progenitors. Our experiments also indicate that Jag1 normally inhibits Notch signaling embryonically but can stimulate it postnatally. In summary, we have shown that different Notch ligands have non-redundant roles in the developing pancreas and each serves to fine-tune the process of endocrine and exocrine cell differentiation from common precursors.