Genetic identification of a novel NeuroD1 function in the early differentiation of islet alpha, PP and epsilon cells

Nkx2.2 and NeuroD1 are vital for proper differentiation of pancreatic islet cell types. Nkx2.2-null mice fail to form β cells, have reduced numbers of α and PP cells and display an increase in ghrelin-producing ε cells. NeuroD1-null mice display a reduction of α and β cells after embryonic day (e) 17.5. To begin to determine the relative contributions of Nkx2.2 and NeuroD1 in islet development, we generated Nkx2.2−/−;NeuroD1−/− double knockout (DKO) mice. As expected, the DKO mice fail to form β cells, similar to the Nkx2.2-null mice, suggesting that the Nkx2.2 phenotype may be dominant over the NeuroD1 phenotype in the β cells. Surprisingly, however, the α, PP and ε phenotypes of the Nkx2.2-null mice are partially rescued by the simultaneous elimination of NeuroD1, even at early developmental time points when NeuroD1 null mice alone do not display a phenotype. Our results indicate that Nkx2.2 and NeuroD1 interact to regulate pancreatic islet cell fates, and this epistatic relationship is cell-type dependent. Furthermore, this study reveals a previously unappreciated early function of NeuroD1 in regulating the specification of α, PP and ε cells.     

Function and regulation of zebrafish nkx2.2a during development of pancreatic islet and ducts

In the mouse Nkx2.2 is expressed in the entire pancreatic anlage. Nevertheless, absence of Nkx2.2 only perturbs the development of endocrine cell types, notably beta-cells which are completely absent. In order to test the possibility that Nkx2.2 might fulfil additional functions during pancreas development we analysed its zebrafish homologue nkx2.2a using gene targeting and GFP-transgenic fish lines. Our results suggest similar roles for nkx2.2a and Nkx2.2 during the development of the endocrine pancreas. Morpholino-based knock-down of nkx2.2a leads to a reduction of alpha- and beta-cell number and an increase of ghrelin-producing cells but, as in mice, does not affect delta-cells. Moreover, like in the mouse, two spatially distinct promoters regulate expression of nkx2.2a in precursors and differentiated islet cells. In addition we found that in zebrafish nkx2.2a is also expressed in the anterior pancreatic bud and, later, in the differentiated pancreatic ducts. A nkx2.2a-transgenic line in which pancreatic GFP expression is restricted to the pancreatic ducts revealed that single GFP-positive cells leave the anterior pancreatic bud and move towards the islet where they form intercellular connections between each other. Subsequently, these cells generate the branched network of the larval pancreatic ducts. Morpholinos that block nkx2.2a function also lead to the absence of the pancreatic ducts. We observed the same phenotype in ptf1a-morphants that are additionally characterized by a reduced number of nkx2.2a-positive duct precursors. Whereas important details of the molecular program leading to the differentiation of endocrine cell types are conserved between mammals and zebrafish, our results reveal a new function for nkx2.2a in the development of the pancreatic ducts.     

Rac1 regulates pancreatic islet morphogenesis

Background  

Pancreatic islets of Langerhans originate from endocrine progenitors within the pancreatic ductal epithelium. Concomitant with differentiation of these progenitors into hormone-producing cells such cells delaminate, aggregate and migrate away from the ductal epithelium. The cellular and molecular mechanisms regulating islet cell delamination and cell migration are poorly understood. Extensive biochemical and cell biological studies using cultured cells demonstrated that Rac1, a member of the Rho family of small GTPases, acts as a key regulator of cell migration.     

Distinct Cell Clusters Touching Islet Cells Induce Islet Cell Replication in Association with Over-Expression of Regenerating Gene (REG) Protein in Fulminant Type 1 Diabetes

Background

Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear.

Procedures

The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans.

Result

Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells.

Conclusion

The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.     

Islet cell antigens and autoantigen(s): Monoclonal antibodies and further characterization

A novel series of murine monoclonal antibodies to islet cells (1–45, 1–51, 1–52 and 1–39) have been generated using human insulinoma homogenate as the immunogen in order to characterize pathogenetically relevant islet cell autoantigen(s). Differentiation antigens recognized by these islet cell monoclonal antibodies displayed varied cytological distribution (pan-islet or peripheral mantle only). Monoclonal antibody 1–45 reacted with all endocrine subsets of the pancreatic islet, similar to the reactivity of islet cell autoantibody positive sera from type I diabetes subjects. Preexposure to pH2 abolished the immunoreactivity of the autoantigen; 1–45 antigen was also sensitive to low pH. Preexposure to 100° C for 1 h did not significantly alter the immunoreactivity of islet antigens recognized by ICAb positive patient sera and monoclonal antibody 1–39, thus demonstrating the extraordinary heat stability of the corresponding epitopes; those recognized by 1–45 were less heat stable. Islet cells were found to share 1–45 differentiation antigen(s)/epitope(s) with other neuroendocrine cells,viz. amerior pituitary, adrenal medulla and gut endocrine cells.     

NCI-H716 cells as a model for endocrine differentiation in colorectal cancer

In colonic neoplasms, endocrine differentiation is encountered not only in carcinoid tumors but also in adenocarcinomas, where endocrine cells may represent a distinct line of differentiation in the tumor. The significance of endocrine differentiation in colorectal cancer is not well established, partly because of the paucity of tumor cell lines which can serve as a model for studying endocrine differentiation. In this report we describe the properties of NCI-H716 cells, a cell line derived from a poorly differentiated adenocarcinoma of the caecum, under various in vitro conditions and as xenografts in athymic mice. Phenotypical properties were immunohistochemically assessed using a panel of differentiation related antibodies, and also by Northern blot analysis and by electron microscopy. Receptors for biogenic amines and peptide hormones were analyzed by ligand binding assay. These studies show that:

Beta cells within single human islets originate from multiple progenitors

Background

In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and Findings

In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion

The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.     

Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas

Most insulin-producing beta-cells in the fetal mouse pancreas arise during the secondary transition, a wave of differentiation starting at embryonic day 13. Here, we show that disruption of homeobox gene Nkx6.1 in mice leads to loss of beta-cell precursors and blocks beta-cell neogenesis specifically during the secondary transition. In contrast, islet development in Nkx6. 1/Nkx2.2 double mutant embryos is identical to Nkx2.2 single mutant islet development: beta-cell precursors survive but fail to differentiate into beta-cells throughout development. Together, these experiments reveal two independently controlled pathways for beta-cell differentiation, and place Nkx6.1 downstream of Nkx2.2 in the major pathway of beta-cell differentiation.