A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation

In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.     

Ngn3(+) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3^−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.     

Thyroid hormones promote endocrine differentiation at expenses of exocrine tissue

Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.     

Endocrine-committed progenitor cells retain their differentiation potential in the absence of neurogenin-3 expression

Neurogenin-3 (ngn-3) expression is critical for endocrine development in the developing pancreas. We found that when ngn-3 was inhibited in an E11.5 pancreas, using either morpholino antisense or siRNA, it led to a significant decrease in endocrine differentiation after seven days in culture. Endocrine differentiation was rescued when ngn-3 inhibition was withdrawn after three days of culture, suggesting that the embryonic pancreas retains progenitor cells with the ability to differentiate into endocrine cell types when ngn-3 expression recurs. To determine whether the rescue phenomenon observed after withdrawing ngn-3 antisense treatment was the result of the original endocrine-committed cells reinitiating endocrine differentiation, or was instead due to new recruitment of later progenitor cells, we blocked ngn-3 expression for only the last four days of a seven-day culture. Here, insulin-positive differentiation was slightly reduced, but there was a normal number of glucagon-positive cells. In addition, there was an increase in SOX9-positive cells in ngn-3 inhibited, as well as in ngn-3 rescued pancreata, with a significant proportion of these SOX9-positive cells co-localized with DBA, an early ductal marker. This increased number of cells with co-localization of SOX9 and DBA could indicate an increased number of endocrine progenitor cells.     

Distinct expression patterns of splicing isoforms of mNumb in the endocrine lineage of developing pancreas

The pancreas is composed of three tissues: endocrine, exocrine, and duct. The endocrine/exocrine lineages diverge from the ductal lineage before E12.5 in mice, and then further separate into endocrine and exocrine precursors. These processes are regulated by differential activation of Notch1-mediated signaling, which is required to repress the expression of the pro-endocrine gene neurogenin3 (ngn3) in the exocrine lineage. Mammalian Numb (mNumb) is an ortholog of Drosophila Numb (dNumb), which is likely to be an intracellular inhibitor of Notch signaling, and has four splicing isoforms: PTBS-PRRS, PTBL-PRRS, PTBS-PRRL, and PTBL-PRRL. Here we developed an anti-PRRL antibody, which recognizes only the PRRL forms of mNumb. We then performed immunohistochemical analyses using anti-PRRL together with anti-pan Numb, which recognizes all the isoforms of mNumb, antibodies that determine the spatio-temporal expression pattern of mNumb in the mouse fetal pancreas. mNumb PRRS and PRRL were first expressed in identical cells in the early stage of pancreatic development (i.e., E10.5), but gradually became biased. At the stage of endocrine and exocrine divergence, mNumb PRRS continued to be expressed in endocrine lineage cells, whereas PRRL was down-regulated during endocrine differentiation. Even after the endocrine/exocrine divergence, notch1 expression was sustained in endocrine lineage, where ngn3 was expressed. These results agree with the notion that mNumb PRRS has an inhibitory effect on Notch signaling, indicating its potential roles in the differentiation of pancreatic endocrine lineage. In addition, islet cells, which are produced from ductal tissue, were immunostained by the anti-panNb antibody. Our present results will contribute to the understanding of the mechanisms of islet development from ductal tissue.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

NRP1 and NRP2 cooperate to regulate gangliogenesis, axon guidance and target innervation in the sympathetic nervous system

During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities.