Understanding proper β-cell formation and function are imperative to finding treatments and cures for β-cell pathologies such as diabetes. Currently, it is accepted that Shh expression must be excluded from the budding mouse pancreatic epithelium. However the subsequent expression of Hh signaling components later during pancreas formation suggest that there may be a functional requirement for Hh signaling during pancreas morphogenesis. In this study, we examined the requirement for epithelial Hh signaling in the developing pancreas. Apelqvist et al. (9
) showed that Ptch1
is not expressed in e9.5 pancreatic epithelium. Through Ptch1-LacZ staining, we show that Ptch1
expression is detectable by e10.5, indicating that Hh-active cells reside in the pancreatic epithelium soon after pancreas specification. While the number of Ptch1-LacZ–positive cells expands during development, the expression levels are low and positive cells are restricted to the ductal and endocrine compartments. In contrast, after birth, Ptch1-LacZ–positive cells stain robustly in the islets and ducts, suggesting that the level of Hh signaling after birth is higher than in utero.
During early stages, we also find some Ptch1-LacZ–positive cells in the neighboring pancreatic mesenchyme. Epithelial-mesenchymal crosstalk is important for proper organ formation, and Hh signaling may function differently in either compartments. To address the functional requirement of Hh signaling in the pancreas epithelium, we generated Pdx1-Creearly;Smoflox/null
mice, which sufficiently lose Smo expression and Hh signaling specifically in the epithelial compartment of the organ. The finding that Hh signaling is required for proper early epithelial expansion and branching in developing pancreata is contrary to previous studies (9
) that suggested that Hh signaling inhibits mammalian pancreas growth. However, prior studies focused on ectopic activation of Hh ligand expression and the negative effects observed on pancreas development resulted from perturbing the epithelial-mesenchymal crosstalk required for proper organ formation. Moreover, previous studies emphasize that the inhibitory effects of Hh signaling is primarily responsible for establishing the pancreas organ boundaries in the foregut. Our results extend this model by demonstrating an additional role for low-level Hh signaling that promotes the early expansion of pancreatic epithelium by e12.5. Evidence for Hh signaling as mediator of cell proliferation has been broadly noted (4
). Surprisingly, Hh activity appears to block proliferation of other pancreatic epithelial cells at midgestation, suggesting temporally distinct roles during pancreas formation. Thus, while our data point to Hh signaling as a mediator of cell proliferation during pancreas development, the exact mechanisms by which the pathway regulates epithelial proliferation in a transient and dynamic manner need to be explored further.
In addition to impaired pancreas morphogenesis, Pdx1-Creearly;Smoflox/null mice have delayed β-cell development. This conclusion is based on the results that the insulin-positive β-cell area was reduced, but β-cell progenitor numbers were increased at e15.5, and the full complement of β-cells was established at the end of gestation. Moreover, albeit not conclusive, temporal analysis of Ngn3 expression indicated a trend whereby peak expression of Ngn3/endocrine neogenesis was delayed. This suggests a temporary delay in general endocrine cell development in Pdx1-Creearly;Smoflox/null mice that is overcome with time. In support of this notion is the observation that the number of α-cells, which form earlier than β-cells during normal pancreas organogenesis, was unchanged at e15.5 compared with controls.
Our studies in mice complement Zebrafish work that has demonstrated the requirement for Hh signaling during gastrulation, a developmental stage that precedes the onset of organ formation. In Zebrafish, inhibition of Hh signaling at early gastrulation stages blocks the formation of pancreatic endocrine cells (37
). Interestingly, this requirement for Smo function is non-cell autonomous (37
). Later during gastrulation, inhibition of Hh signaling increases the formation of insulin-producing cells (39
). While the relationship of Hh signaling during mouse gastrulation and subsequent pancreatic endocrine cell formation has not been elucidated, our present work and the studies in Zebrafish support the notion that the level and timing of Hh signaling need to be closely regulated to allow proper endocrine cell development.
mice possess normal pancreas morphology, while the β-cells are dysfunctional. Although these mice are sensitive to exogenous insulin and have an increased β-cell mass, their β-cells produce less insulin, resulting in reduced insulin secretion and a glucose intolerance phenotype. Importantly, our data indicate that the primary defect lies within the production of insulin whereas secretion appears intact. Thomas and colleagues (16
) showed that β-cell lines respond to Hh activity by increasing insulin production and secretion through regulation of Pdx1
expression. Although we did not detect significant changes in Pdx1
transcription by RT-PCR or in Pdx1 protein levels by qualitative staining in mutant mice, Insulin
expression was reduced and isolated islets recapitulated decreased insulin secretion in culture. While further studies are needed, this suggests a mechanism independent of Pdx1 regulation on Insulin
transcription for Hh-regulated insulin production. The differences observed between the previous cell culture experiments and our in vivo analysis maybe due to the inherent altered state of insulinoma cell lines versus the native β-cell. Nevertheless, both studies emphasize the role of Hh signaling in maintaining proper insulin production. Further support comes from the finding that Ptch1-LacZ expression, indicative of Hh signaling activity, becomes dramatically stronger in postnatal islets at a time when the demand for β-cell functionality and insulin activity begins.
Interestingly, Pdx1-Creearly;Smoflox/null mice are more insulin sensitive. While we do not understand the mechanisms that result in this change, these data rule out impaired insulin sensitivity as a cause for glucose intolerance phenotypes in Pdx1-Creearly;Smoflox/null mice. Coupled with data demonstrating that insulin secretion is intact, these results emphasize that the primary defect in Pdx1-Creearly;Smoflox/null mice lies in insulin production in β-cells.
It should be noted that previous studies have linked Hh signaling to the formation and progression of pancreatic adenocarcinoma (40
). During neoplastic transformation, excessive Hh ligands secreted from the tumorigenic epithelium (41
) are likely to act predominately on the surrounding stroma in a paracrine manner (40
). In the endocrine compartment, previous work (13
) has shown expression of Hh ligands, Ihh and Dhh, in pancreatic islet cells. Unfortunately, due to the absence of agents suitable for cell type–specific expression analysis, the exact complement of those cells within the islets that produce and secrete ligands is currently missing. Thus, while our work and work from others indicate that epithelial-derived β-cells respond to Hh ligands in a juxtacrine or autocrine fashion, unequivocal resolution of this question awaits additional experiments with improved reagents.
As we struggle to find potential treatments and cures for diabetes, the need to generate or expand more functional β-cells remains unrequited. Understanding the mechanisms that will allow us to generate and sustain β-cell populations and function is imperative. Data presented here demonstrate that epithelial Hh signaling is important during pancreas development and in maintaining insulin production in the adult β-cell, thus adding another layer to the current perspective that views Hh inhibition as important for the generation of functional pancreas endocrine cells.