FOXO1 regulates developmental lymphangiogenesis by upregulating CXCR4 in the mouse-tail dermis.

Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.

Glucotoxicity-induced suppression of Cox6a2 expression provokes ß-cell dysfunction via augmented ROS production.

Oxidative stress is a deteriorating factor for pancreatic ß-cells under chronic hyperglycemia in diabetes. However, the molecular mechanism underlying the increase in oxidative stress in ß-cells under diabetic conditions remains unclear. We demonstrated previously that the selective alleviation of glucotoxicity ameliorated the downregulation of several ß-cell factors, including Cox6a2. Cox6a2 encodes a subunit of the respiratory chain complex IV in mitochondria. In this study, we analyzed the role of Cox6a2 in pancreatic ß-cell function and its pathophysiological significance in diabetes mellitus. Cox6a2-knockdown experiments in MIN6-CB4 cells indicated an increased production of reactive oxygen species as detected by CellROX Deep Red reagent using flow cytometry. In systemic Cox6a2-knockout mice, impaired glucose tolerance was observed under a high-fat high-sucrose diet. However, insulin resistance was reduced when compared with control littermates. This indicates a relative insufficiency of ß-cell function. To examine the transcriptional regulation of Cox6a2, ATAC-seq with islet DNA was performed and an open-chromatin area within the Cox6a2 enhancer region was detected. Reporter gene analysis using this area revealed that MafA directly regulates Cox6a2 expression. These findings suggest that the decreased expression of Cox6a2 increases the levels of reactive oxygen species and that Mafa is associated with decreased Cox6a2 expression under glucotoxic conditions.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis.

The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.

Tip-cell behavior is regulated by transcription factor FoxO1 under hypoxic conditions in developing mouse retinas.

Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.

ATF4-mediated induction of 4E-BP1 contributes to pancreatic beta cell survival under endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress-mediated apoptosis may play a crucial role in loss of pancreatic beta cell mass, contributing to the development of diabetes. Here we show that induction of 4E-BP1, the suppressor of the mRNA 5' cap-binding protein eukaryotic initiation factor 4E (eIF4E), is involved in beta cell survival under ER stress. 4E-BP1 expression was increased in islets under ER stress in several mouse models of diabetes. The Eif4ebp1 gene encoding 4E-BP1 was revealed to be a direct target of the transcription factor ATF4. Deletion of the Eif4ebp1 gene increased susceptibility to ER stress-mediated apoptosis in MIN6 beta cells and mouse islets, which was accompanied by deregulated translational control. Furthermore, Eif4ebp1 deletion accelerated beta cell loss and exacerbated hyperglycemia in mouse models of diabetes. Thus, 4E-BP1 induction contributes to the maintenance of beta cell homeostasis during ER stress and is a potential therapeutic target for diabetes.

Improvement in protocol to generate homogeneous glutamatergic neurons from mouse embryonic stem cells reduced apoptosis.

Obtaining a homogenous population of central nervous system neurons has been a significant challenge in neuroscience research; however, a recent study established a retinoic acid-treated embryoid bodies-based differentiation protocol that permits the effective generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem cells. We were able to reproduce this protocol regarding the purity of glutamatergic neurons, but these neurons were not sufficiently healthy for long-term observation under the same conditions that were originally described. Here, we achieved a substantial improvement in cell survival by applying a simple technique: We changed the medium for glutamatergic neurons from the original complete medium to commercially available SBM (the Nerve-Cell Culture Medium manufactured by Sumitomo Bakelite Co. Ltd.) and finally succeeded in maintaining healthy neurons for at least 3 weeks without decreasing their purity. Because SBM contains glial conditioned medium, we postulated that brain-derived neurotrophic factor or basic fibroblast growth factor is the key components responsible for pro-survival effect of SBM on neurons, and examined their effects by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population.

Analysis of Foxo1-regulated genes using Foxo1-deficient pancreatic ß cells.

Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic ß-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient ß cells. To elucidate Foxo1's function in ß cells, we produced a ß-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a ß-cell line from an insulinoma induced in this knockout mouse by the ß-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced ß-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to ß-cell function. These cells should be useful for further studies on Foxo1's roles in ß-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus.

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy.

Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Maternal-effect gene Ces5/Ooep/Moep19/Floped is essential for oocyte cytoplasmic lattice formation and embryonic development at the maternal-zygotic stage transition.

In a search for genes specifically expressed in mouse embryonic stem cells, we identified one we called Ces5. We found that it corresponded to the Ooep gene, which was recently reported to be expressed specifically in oocytes. Mouse Ces5/Ooep, also called Moep19 or Floped, encoded a 164-amino acid protein, which was detected in the cytoplasm of developing and mature oocytes and in embryos throughout the preimplantation period. To examine its function, we carried out targeted disruption of this gene. The Ces5/Ooep-null mice were grossly normal, but the females were infertile. Although the ovaries and ovulation appeared normal, the embryos from Ces5/Ooep-null females mated with wild-type males showed developmental arrest at the two- or four-cell stage. In addition, their first cleavage was considerably delayed and often asymmetrical. Thus, Ces5/Ooep is a maternal-effect gene. By electron microscopy, we found that the eggs from Ces5/Ooep-null females lacked oocyte cytoplasmic lattices (CPLs), which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo. Further analysis showed that CES5/OOEP was directly associated with the CPLs. These results indicate that CES5/OOEP is an essential component of the CPLs and is required for embryonic development at the maternal-zygotic stage transition.

Establishment of a long-term stable ß-cell line and its application to analyze the effect of Gcg expression on insulin secretion.

A pancreatic ß-cell line MIN6 was previously established in our lab from an insulinoma developed in an IT6 transgenic mouse expressing the SV40 T antigen in ß-cells. This cell line has been widely used for in vitro analysis of ß-cell function, but tends to lose the mature ß-cell features, including glucose-stimulated insulin secretion (GSIS), in long-term culture. The aim of this study was to develop a stable ß-cell line that retains the characteristics of mature ß-cells. Considering that mice derived from a cross between C3H and C57BL/6 strains are known to exhibit higher insulin secretory capacity than C57BL/6 mice, an IT6 male mouse of this hybrid background was used to isolate insulinomas, which were independently cultured. After 7 months of continuous culturing, we obtained the MIN6-CB4 ß-cell line, which stably maintains its GSIS. It has been noted that ß-cell lines express the glucagon (Gcg) gene at certain levels. MIN6-CB4 cells were utilized to assess the effects of differential Gcg expression on ß-cell function. Our data show the functional importance of Gcg expression and resulting basal activation of the GLP-1 receptor in ß-cells. MIN6-CB4 cells can serve as an invaluable tool for studying the regulatory mechanisms of insulin secretion, such as the GLP-1/cAMP signaling, in ß-cells.

Gtsf1/Cue110, a gene encoding a protein with two copies of a CHHC Zn-finger motif, is involved in spermatogenesis and retrotransposon suppression in murine testes.

We recently reported that the Gtsf1/Cue110 gene, a member of the evolutionarily conserved UPF0224 family, is expressed predominantly in male germ cells, and that the GTSF1/CUE110 protein is localized to the cytoplasm of these cells in the adult testis. Here, to analyze the roles of the Gtsf1/Cue110 gene in spermatogenesis, we produced Gtsf1/Cue110-null mice by gene targeting. The Gtsf1/Cue110-null mice grew normally and appeared healthy; however, the males were sterile due to massive apoptotic death of their germ cells after postnatal day 14. In contrast, the null females were fertile. Detailed analyses revealed that the Gtsf1/Cue110-null male meiocytes ceased meiotic progression before the zygotene stage. Thus, the Gtsf1/Cue110 gene is essential for spermatogenesis beyond the early meiotic phase. Furthermore, the loss of the Gtsf1/Cue110 gene caused increased transcription of the long interspersed nucleotide element (Line-1) and the intracisternal A-particle (IAP) retrotransposons, accompanied by demethylation of their promoter regions. These observations indicate that Gtsf1/Cue110 is required for spermatogenesis and involved in retrotransposon suppression in male germ cells.

Sohlh2 affects differentiation of KIT positive oocytes and spermatogonia.

The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

Sema4D deficiency results in an increase in the number of oligodendrocytes in healthy and injured mouse brains.

Semaphorins, a family of secreted and membrane-bound proteins, are known to function as repulsive axon guidance molecules. Sema4D, a class 4 transmembrane-type semaphorin, is expressed by oligodendrocytes in the central nervous system, but its role is unknown. In this study, the effects of Sema4D deficiency on oligodendrocytes were studied in intact and ischemic brains of adult mice. As observed in previous studies, Sema4D marked by beta-galactosidase in Sema4D mutant mice was localized exclusively on myelin-associated glycoprotein (MAG)-positive oligodendrocytes but not on NG2-positive oligodendrocyte progenitor cells (OPCs). Although there was no difference in the number of the latter cells between Sema4D-deficient and wild-type mice, the number of MAG-positive cells was significantly increased in the cerebral cortex of both nonischemic and postischemic brains of Sema4D-deficient mice. Cell proliferation, observed by using bromodeoxyuridine incorporation, was evident in the MAG-positive cells that developed after cerebral ischemia. These data indicate that Sema4D is involved in oligodendrogenesis during development and during recovery from ischemic injury.

Transgenic expression of antioxidant protein thioredoxin in pancreatic beta cells prevents progression of type 2 diabetes mellitus.

The authors previously established a transgenic mouse line in the type 1 diabetes model, NOD mouse, in which thioredoxin (TRX), a redox protein, is overexpressed in pancreatic beta cells, and found that TRX overexpression slows the progression of type 1 diabetes. Recent reports on type 2 diabetes suggest that oxidative stress also degrades the function of beta cells. To elucidate whether TRX overexpression can prevent progressive beta cell failure from oxidative stress in type 2 diabetes, the authors transferred the TRX transgene from the NOD mouse onto a mouse model of type 2 diabetes, the db/db mouse. The progression of hyperglycemia and the reduction of body weight gain and insulin content of the db/db mouse were significantly suppressed by the TRX expression. Furthermore, TRX suppressed the reduction of Pdx-1 and MafA expression in the beta cells, which may be one of the cellular mechanisms for protecting beta cells from losing their insulin-secreting capacity. These results showed that TRX can protect beta cells from destruction not only in type 1 but also in type 2 diabetes, and that they provide evidence that oxidative stress plays a crucial role in the deterioration of beta cell function during the progression of type 2 diabetes.

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization.

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c-/- mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c-/- cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c-/- cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.

IRE1-XBP1 pathway regulates oxidative proinsulin folding in pancreatic ß cells.

In mammalian pancreatic ß cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed ß cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic ß cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic ß cells.

Gene expression pattern of Cue110: a member of the uncharacterized UPF0224 gene family preferentially expressed in germ cells.

The large number of expressed sequence tags (ESTs) now available in databases has enabled the analysis of gene expression profiles in silico. We searched public databases for uncharacterized transcripts specifically expressed in germ cells, in an attempt to identify genes involved in gametogenesis. We found a transcript that is expressed in unfertilized eggs, ovaries, and testes of the mouse. It has an open reading frame (ORF) encoding a 167-amino acid protein belonging to the UPF0224 (unknown protein family 0224) family. We called the novel gene Cue110. We examined the Pfam database for other members of the UPF0224 family, and found a conserved N-terminal portion among members of various species. To study the cellular localization of the Cue110 transcript and protein, we performed in situ hybridization and immunohistochemical analysis of the adult mouse ovary and testis. In the testis, specific hybridization signals were observed weakly in preleptotene spermatocytes but maximally in late round spermatids. Immunostaining showed that Cue110 protein was present predominantly in the cytoplasm of pachytene spermatocytes and round spermatids. In the ovary, weak hybridization signals were observed in primary oocytes in the primordial, primary, and secondary follicles, but Cue110 protein was not detected in oocytes by immunostaining. We next examined the developmental expression pattern of the Cue110 gene using RT-PCR and western blotting, and found its increasing expression coincided with the appearance of spermatocytes. Thus, the Cue110 gene is expressed predominantly in male germ cells at stages from the pachytene spermatocytes to round spermatids.

Zfp296 negatively regulates H3K9 methylation in embryonic development as a component of heterochromatin.

The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296 -/- mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296 -/- embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.

Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice.

A promising approach to new diabetes therapies is to generate ß cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into ß cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into ß cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.