Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.     

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.     

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.