Expression Of Neurogenin Research Articles

Electrical stimulation enhances neurogenin2 expression through β-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury

Bone marrow stromal cells (BMSCs) have received significant attention for its use in neural regeneration. However, neural replacement by transplanted BMSCs was not very effective. Recently, the gene transfection method has improved the capability of cell transplantation; however, this method results in canceration and immune rejection. We induced the differentiation of mouse BMSCs into neural cells using electrical stimulation and transplanted the cells into traumatic brain injury (TBI) model mice. We found that the electrically stimulated cells have good potential to differentiate into neural cells and contribute to recovery from TBI without differentiating into astrocytes. In addition, we found that electrical stimulation enhanced neurogenin2 (Ngn2) expression. Ngn2 is involved in neural differentiation and inhibits astrocytic differentiation during cell growth. Furthermore, we found that this enhancement of Ngn2 expression occurred through β-catenin signaling pathway. This study may contribute to the use of BMSCs for neural replacement in central nervous system diseases.

Neurogenin2 expression together with NeuroM regulates GDNF family neurotrophic factor receptor α1 (GFRα1) expression in the embryonic spinal cord

In many regions of the nervous system, the combinatorial action of transcriptional factors specifies the individual fate of neuronal subtypes. Contrary to this, we report that a single transcriptional factor controls a phenotype shared by different subtypes of neurons, namely the expression of a neurotrophic factor receptor in the spinal cord. Along the dorsoventral axis of the chick embryo spinal cord, the expression pattern of a specific receptor for glial cell line derived-neurotrophic factor (GDNF family of receptors α1: GFRα1) was related to that of two basic helix-loop-helix (bHLH) transcriptional factors (NeuroM and Neurogenin2: Ngn2). In ovo electroporation in the chick embryo revealed that the overexpression of NeuroM alone was sufficient to induce ectopic GFRα1 expression without overt neuronal differentiation, whereas the suppression of NeuroM activity resulted in the specific loss of GFRα1 expression, indicating that NeuroM may act as a differentiation factor for GFRα1 expression. Ngn2 overexpression was also sufficient to induce precocious GFRα1 expression. However, the forced expression of both obligate suppressor and activator forms of Ngn2 also induced aberrant GFRα1 expression. Thus, any deviation from an optimum level of Ngn2 expression resulted in aberrant GFRα1 expression. Consistent with this, manipulation of Ngn2 expression levels by other bHLH factors also resulted in ectopic GFRα1 expression. For example, the downregulation by Ascl1 and the upregulation by Ptf1a induced ectopic GFRα1 expression, irrespective of endogenous expression patterns of Ascl1 and Ptf1a (Ascl1/Ptf1) in the spinal cord. The suppression of Ascl1/Ptf1a activities abolished Ngn2 and GFRα1 expression, even in Ascl1/Ptf1a-negative regions. These data indicate the presence of a distinct regulatory sequence for a determinant of GFRα1 expression, in which Ascl1/Ptf1a may competitively intervene to stochastically modulate default Ngn2 expression levels. Thus, Ngn2 together with NeuroM serves as readout to regulate GFRα1 expression, which occurs in multiple subtypes of spinal neurons.

Establishment of a new method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells.

To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells. Mouse embryos at day 12.5 were used and embryonic pancreata were isolated. The isolated embryonic pancreata were cultured on the filter for 7 days, which floated in the dish containing medium. During culture, the expression of pancreas duodenum homeobox-1 (PDX-1), a pancreatic stem cell marker, was examined at day 1. The expression of neurogenin 3 (Ngn3), a pancreatic progenitor cell marker, was examined at day 3. The expressions of endocrine and exocrine markers, insulin, glucagon, and carboxypeptidase (CPA) were examined at day 7 by immunohistochemistry. The kinetics of pancreatic marker expression during culture was assayed by real-time PCR. Many pancreatic stem cells still existed in embryonic pancreata cultured for 1 day; meanwhile, these pancreatic stem cells proliferated in high rate. A large amount of pancreatic progenitor cells were found in embryonic pancreata cultured for 3 days.Pancreatic stem/progenitor cells differentiated into mature endocrine and exocrine cells in embryonic pancreata after having been cultured for 7 days. Furthermore, the expression pattern of pancreatic marker is consistent with that in vivo. We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.

Ectopic Expression of Neurogenin 2 Alone is Sufficient to Induce Differentiation of Embryonic Stem Cells into Mature Neurons

Recent studies show that combinations of defined key developmental transcription factors (TFs) can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2) is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs) into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.

Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development

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 (Neurog3CreERT/+) and Neurog3-deficient (Neurog3CreERT/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.

Neurogenin2 expression regulates GDNF family neurotrophic factor receptor α1 (GFR α1) expression in the embryonic spinal cord

Neurogenin2 expression regulates GDNF family neurotrophic factor receptor α1 (GFR α1) expression in the embryonic spinal cord

RESOURCE IMPACT

Insulin deficiency underlies both major forms of diabetes. In type 1 diabetes, the immune system attacks and destroys the insulin-producing β-cells in the pancreatic islets of Langerhans, leaving few, if any, of these crucial cells. Patients with type 2 diabetes have reduced numbers of β-cells, which are not sufficient to overcome the insulin resistance that comes with weight gain and a sedentary lifestyle. Insulin production declines further in type 2 diabetics as the disease progresses, eventually causing the failure of all currently available therapies for normalizing blood glucose except for insulin replacement. Therapies that enable replacement of β-cells are needed for both type 1 and type 2 diabetes, and we need animal models for identifying and testing these therapies.As islet cells develop from progenitor cells, they transiently express the transcription factor neurogenin-3. The authors have made transgenic mice in which developing islet cells can be detected in living tissues, by using the regulatory regions of the human neurogenin-3 gene to drive expression of secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) in these developing cells. They show in both intact animals and pancreatic organ cultures that EGFP fluorescence and secretion of SeAP accurately mark neurogenin-3-expressing cells, thus serving as indicators of islet cell genesis. Using cells from the transgenic mice, the authors then look at regulation of neurogenin-3 expression in fetal pancreatic organ cultures: neurogenin-3 expression can be inhibited by growth and differentiation factor 11 (GDF11), and is induced by blocking expression of a known inhibitor of neurogenin-3 signaling, the Notch protein. Interestingly, blocking Notch only induces neurogenin-3 expression during a narrow developmental time window corresponding to the normal peak in neurogenin-3 expression. In adult mice, partial pancreatectomy decreases circulating SeAP levels, whereas duct ligation increases them.This model provides a convenient tool for following the differentiation of new islet cells in living cells, tissues and animals, and for testing the impact of differentiation interventions. It can be used to dissect the pathways that regulate islet cell generation, both during the critical fetal period that sets the initial size of the β-cell population, and during β-cell regeneration in the adult. An understanding of these processes will provide a basis for explaining why βcell genesis fails in type 2 diabetes. In addition, this mouse can be used to screen and test potential methods for generating β-cells for the treatment of both type 1 and type 2 diabetes.

Sox3 Functions in a Cell-Autonomous Manner to Regulate Spermatogonial Differentiation in Mice

The X-linked Sox3 gene encodes a member of the Sry high-mobility group box proteins, which play a role in many developmental processes including neurogenesis and testis development. This study further examined the role of Sox3 in spermatogenesis. Males without Sox3 expression exhibited a similar number of germ cell nuclear antigen-positive germ cells at 1, 5, and 10 d postpartum (dpp) compared to their wild-type littermates, but there was significant germ cell depletion by 20 dpp. However, spermatogenesis later resumed and postmeiotic germ cells were observed by 56 dpp. The VasaCre transgene was used to generate a germ cell-specific deletion of Sox3. The phenotype of the germ cell-specific Sox3 knockout was similar to the ubiquitous knockout, indicating an intrinsic role for Sox3 in germ cells. The residual germ cells in 20 dpp Sox3(-/Y) males were spermatogonia as indicated by their expression of neurogenin3 but not synaptonemal complex protein 3, which is expressed within cells undergoing meiosis. RNA expression analyses corroborated the histological analyses and revealed a gradual transition from relatively increased expression of spermatogonia genes at 20 dpp to near normal expression of genes characteristic of undifferentiated and meiotic germ cells by 84 dpp. Fluorescent-activated cell sorting of undifferentiated (ret tyrosine kinase receptor positive) and differentiated (kit receptor tyrosine kinase-positive) spermatogonia revealed depletion of differentiated spermatogonia in Sox3(-/Y) tubules. These results indicate that Sox3 functions in an intrinsic manner to promote differentiation of spermatogonia in prepubertal mice but it is not required for ongoing spermatogenesis in adults. The Sox3(-/Y) males provide a unique model for studying the mechanism of germ cell differentiation in prepubertal testes.

Effect of EGCG on Expression of Neurogenin 3 via the MAP Kinase Signaling Pathway in AR42J Cells, a Rat Pancreatic Tumor Cell Line

Epigallocatechin gallate (EGCG), or epigallocatechin 3-gallate. is the ester of epigallocatcchin and gallic acid, and is a type of catec hin. EGCG may be therapeutic for many disorders including diabetic s and some types of cancer. Howev-er it is unknown whet her EGCG ca n induce transdifferentiation of pancreatic cells in pancreatitis. The aim of this study was to investigate the effects of EGCG on the expression of pancreatic regenerating related markers in pancreatic AR42J cells, a model of pancreatic progenitor cells. AR42J cells. differentiated with betacellulin and activin A, were cultured with/without EGCG in a time-dependent manner. Cell growth rate, levels of mRNA. and protein expression were examined with the MTT assay, quantitative PCR, and Western blots, respectively. The results showed that AR42J cell growth rates were inhibited by EGCG in a dose-dependent manner. mRNA and protein expression of amylase, insulin and neurogenin 3 (ngn 3) increased in AR42J cells treated with EGCG. Additionally, we demonstrated that the signal transduction pathway of mitogen-activated protein (MA P) kinase is active in EGCG-treated AR42J cells. ERK and JNK phosphorylation decreased in cells treated with EGCG but not p38 phosphorylation. Activation of the p38 MAP kinase pathway was confirmed by specific MAP kinase pathways inhibitors: U0126 for ERK, SP600126 for INK, and SB203580 for p38. Activated p38 phosphorylation was inhibited by the specific p38 inhibitor SB203580 but p38 phosphorylation was inhibit ed with increased EGCG treatment. The ERK and JNK MAP kinase pathways were not affected by EGCG treatment. Although further studies are needed, these result s suggest that EGCG affects the induction of pancreatic cell regeneration by increasing the ngn 3 protein and mRNA expression and activating the p38 MAP kinase pathway.

Neurogenin3 inhibits proliferation in endocrine progenitors by inducing Cdkn1a

During organogenesis, the final size of mature cell populations depends on their rates of differentiation and expansion. Because transient expression of Neurogenin3 (Neurog3) in progenitor cells in the developing pancreas initiates their differentiation to mature islet cells, we examined the role of Neurog3 in cell cycle control during this process. We found that mitotically active pancreatic progenitor cells in mouse embryos exited the cell cycle after the initiation of Neurog3 expression. Transcriptome analysis demonstrated that the Neurog3-expressing cells dramatically up-regulated the mRNA encoding cyclin-dependent kinase inhibitor 1a (Cdkn1a). In Neurog3 null mice, the islet progenitor cells failed to activate Cdkn1a expression and continued to proliferate, showing that their exit from the cell cycle requires Neurog3. Furthermore, induced transgenic expression of Neurog3 in mouse β-cells in vivo markedly decreased their proliferation, increased Cdkn1a levels, and eventually caused profound hyperglycemia. In contrast, in Cdkn1a null mice, proliferation was incompletely suppressed in the Neurog3-expressing cells. These studies reveal a crucial role for Neurog3 in regulating the cell cycle during the differentiation of islet cells and demonstrate that the subsequent down-regulation of Neurog3 allows the mature islet cell population to expand.

A mouse model for monitoring islet cell genesis and developing therapies for diabetes

SUMMARYTransient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

In vitro expression of NGN3 identifies RAB3B as the predominant Ras-associated GTP-binding protein 3 family member in human islets.

Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.

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.

Glial cell line-derived neurotrophic factor enhances neurogenin3 gene expression and beta-cell proliferation in the developing mouse pancreas.

Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that is required for the development of the enteric nervous system. In transgenic mice that overexpress GDNF in the pancreas, GDNF has been shown to enhance beta-cell mass and improve glucose control, but the transcriptional and cellular processes involved are not known. In this study we examined the influence of GDNF on the expression of neurogenin3 (Ngn3) and other transcription factors implicated in early beta-cell development, as well as on beta-cell proliferation during embryonic and early postnatal mouse pancreas development. Embryonic day 15.5 (E15.5) mouse pancreatic tissue when exposed to GDNF for 24 h showed higher Ngn3, pancreatic and duodenal homeobox gene 1 (Pdx1), neuroD1/beta(2), paired homeobox gene 4 (Pax4), and insulin mRNA expression than tissue exposed to vehicle only. Transgenic expression of GDNF in mouse pancreata was associated with increased numbers of Ngn3-expressing pancreatic cells and higher beta-cell mass at embryonic day 18 (E18), as well as higher beta-cell proliferation and Pdx1 expression in beta-cells at E18 and postnatal day 1. In the HIT-T15 beta-cell line, GDNF enhanced the expression of Pax6. This response was, however, blocked in the presence of Pdx1 small interfering RNA (siRNA). Chromatin immunoprecipitation studies using the HIT-T15 beta-cell line demonstrated that GDNF can influence Pdx1 gene expression by enhancing the binding of Sox9 and neuroD1/beta(2) to the Pdx1 promoter. Our data provide evidence of a mechanism by which GDNF influences beta-cell development. GDNF could be a potential therapeutic target for the treatment and prevention of diabetes.

This Month in Gastroenterology

This Month in Gastroenterology

Neurogenin 3 and Neurogenic Differentiation 1 are Retained in the Cytoplasm of Multiple Endocrine Neoplasia Type 1 Islet and Pancreatic Endocrine Tumor Cells

To investigate if transcription factors involved in pancreatic differentiation and regeneration are present in pancreatic endocrine tumors and if they are differentially expressed in normal pancreas compared with multiple endocrine neoplasia type 1 (MEN1) nontumorous pancreas. The expression of neurogenin 3 (NEUROG3), neurogenic differentiation 1 (NEUROD1), POU class 3 homeobox 4 (POU3F4), pancreatic duodenal homeobox factor 1 (PDX1), ribosomal protein L10 (RPL10), delta-like 1 homolog (Drosophila; DLK1), and menin was analyzed by immunohistochemistry in normal pancreas and pancreatic endocrine tumors from 6 patients with MEN1 and 16 patients with sporadic tumors, as well as pancreatic specimens from Men1 heterozygous and wild type mice. Quantitative polymerase chain reaction was performed in a subset of human tumors. Tumors and MEN1 nontumorous endocrine cells showed a prominent cytoplasmatic NEUROG3 and NEUROD1 expression. These factors were significantly more expressed in the cytoplasm of Men1 heterozygous mouse islet cells compared with wild type islets; the latter showed an exclusively nuclear reactivity. The degree of Pou3f4, Rpl10, and Dlk1 immunoreactivities differed significantly between islets of heterozygous and wild type mice. The expressions of RPL10 and NEUROD1 were prominent in the MEN1 human and heterozygous mouse exocrine pancreas. Insulinomas had significantly higher PDX1 and DLK1 messenger RNA levels compared with other tumor types. Transcription factors involved in pancreatic development show altered expression and subcellular localization in MEN1 nontumorous pancreas and pancreatic endocrine tumors.

β-Cell Proliferation, but Not Neogenesis, Following 60% Partial Pancreatectomy Is Impaired in the Absence of FoxM1

OBJECTIVE—This study was designed to determine whether the transcription factor FoxM1 was required for regeneration of β-cell mass via proliferation and/or neogenesis in the adult after 60% partial pancreatectomy (PPx).RESEARCH DESIGN AND METHODS—Adult mice with a pancreas-wide deletion of Foxm1 (Foxm1flox/flox;Pdx1-Cre [FoxM1Δpanc]) and their control littermates (Foxm1flox/flox) were subjected to PPx or a sham operation, after which islet expression of Foxm1 and several target genes, β-cell mass, proliferation, β-cell size, islet size, islet density, and neurogenin-3 expression were analyzed.RESULTS—In control mice, PPx stimulated β-cell proliferation and neogenesis and upregulated Foxm1 and several of its known targets (Plk1, Cenp-a, Birc5/Survivin, and Ccnb1) in islets. Within 1 week post-PPx, control mice underwent significant regeneration of β-cell mass, and average islet size within the regenerating lobe was similar to that after a sham operation. However, FoxM1Δpanc mice exhibited specific impairments in β-cell mass regeneration and islet growth after PPx, with reduced proliferation of α- and β-cells but no impairments in acinar or ductal cell proliferation. Interestingly, FoxM1 was not required for proliferation of β-cells within small endocrine cell clusters located in the regenerating portion of the pancreas but was specifically required for proliferation of β-cells within larger islets. Additionally, FoxM1 was not required for β-cell neogenesis following PPx.CONCLUSIONS—Our results indicate that FoxM1 is partially required for increased β-cell proliferation, but not β-cell neogenesis, stimulated by PPx. Furthermore, FoxM1 seems to be dispensable for proliferation of β-cells following neogenesis but is required for proliferation of preexisting β-cells.

Combinatorial signalling controls Neurogenin2 expression at the onset of spinal neurogenesis

A central issue during embryonic development is to define how different signals cooperate in generating unique cell types. To address this issue, we focused on the function and the regulation of the proneural gene Neurogenin2 ( Neurog2) during early mouse spinal neurogenesis. We showed that Neurog2 is first expressed in cells within the neural plate anterior to the node from the 5 somite-stage. The analysis of Neurog2 mutants established a role for this gene in triggering neural differentiation during spinal cord elongation. We identified a 798 base pair enhancer element ( Neurog2-798) upstream of the Neurog2 coding sequence that directs the early caudal expression of Neurog2. Embryo culture experiments showed that Retinoic Acid (RA), Sonic hedgehog (Shh) and Fibroblast Growth Factor signals act in concert on this enhancer to control the spatial and temporal induction of Neurog2. We further demonstrated by transgenesis that two RA response elements and a Gli binding site within the Neurog2-798 element are absolutely required for its activity, strongly suggesting that the regulation of Neurog2 early expression by RA and Shh signals is direct. Our data thus support a model where signal integration at the level of a single enhancer constitutes a key mechanism to control the onset of neurogenesis.

β Cells Can Be Generated from Endogenous Progenitors in Injured Adult Mouse Pancreas

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Increased islet neogenesis without increased islet mass precedes autoimmune attack in diabetes-prone rats

We reported previously that young BioBreeding diabetes-prone (BBdp) rats display increased neogenic extra-islet insulin+ clusters (EICs, <4 insulin+ cells) without an increase in β-cell mass. Therefore, we investigated the possibility that abnormal islet expansion occurs in BBdp rats before the appearance of islet inflammation. Islet expansion was analyzed in pancreata from 14 to 45 day BBdp and control (BioBreeding control, BBc) rats using immunohistochemistry, morphometry, laser capture microdissection and reverse transcriptase-PCR. mRNA expression for Neurogenin-3, a developmental marker of endocrine progenitors, was three-fold greater in EIC of weanling BBdp and BBc rats compared with islet cells. With increasing age (14–30 days), Neurogenin-3 expression decreased in EIC and increased in islets. In BBdp rats, EIC number and β-cell proliferation within EIC was greater compared with BBc animals; apoptosis did not differ. The area of small and medium islets in BBdp rats was greater than BBc rats between 14 and 30 days, but this did not result in increased total islet area or β-cell mass. In addition, the number and area of very large islets was low at 45 days. The frequency of proliferating β-cells decreased with increasing islet size in BBdp but was constant in BBc rats. Cell cycle analysis of islets revealed more G1 cells and fewer G2 cells in BBdp rats. The ratio of cyclinD2/Cdkn1a, genes that respectively promote or inhibit cell cycle progression, was decreased in BBdp islets. These results suggest that despite increased islet neogenesis, the capacity for islet expansion in diabetes-prone rats is compromised possibly due to decreased proliferative capacity with increasing islet size associated with a partial block at the G1/S cell cycle boundary in islet cells.