Mouse Pancreas Development Research Articles

M6A mRNA methylation by METTL14 regulates early pancreatic cell differentiation.

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported that m6A is involved in the postnatal control of β-cell function in physiological states and in type 1 and 2 diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells.

GRP94 is an IGF-1R chaperone and regulates beta cell death in diabetes

High workload-induced cellular stress can cause pancreatic islet β cell death and dysfunction, or β cell failure, a hallmark of type 2 diabetes mellitus. Thus, activation of molecular chaperones and other stress-response genes prevents β cell failure. To this end, we have shown that deletion of the glucose-regulated protein 94 (GRP94) in Pdx1+ pancreatic progenitor cells led to pancreas hypoplasia and reduced β cell mass during pancreas development in mice. Here, we show that GRP94 was involved in β cell adaption and compensation (or failure) in islets from leptin receptor-deficient (db/db) mice in an age-dependent manner. GRP94-deficient cells were more susceptible to cell death induced by various diabetogenic stress conditions. We also identified a new client of GRP94, insulin-like growth factor-1 receptor (IGF-1R), a critical factor for β cell survival and function that may mediate the effect of GRP94 in the pathogenesis of diabetes. This study has identified essential functions of GRP94 in β cell failure related to diabetes.

Abstract B095: Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract While inflammation is a known and well-validated risk factor for developing pancreatic ductal adenocarcinoma (PDA), it remains unclear how Leukemia inhibitory factor (LIF), a cytokine of the IL-6 class family, influences tumor initiation and progression in PDA. LIF is highly upregulated in PDA when compared to the normal healthy pancreas. Using a human single-cell RNAseq PDA data set, we identified pancreatic cancer cells as the main source of LIF, followed by fibroblasts and stellate cells. After confirming that Pdx1-expressing cells represent the majority of Lif-expressing cells in the pancreas, we crossed our newly developed Lif flox/flox allele into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. Lif flox/flox, Pdx1-Cre (LC) mice present with normal pancreas histology, mouse weight, and mouse size at 12 months, indicating that Lif is dispensable for mouse pancreas development and function. Lif flox/flox, Trp53flox/flox, Pdx1-Cre (LPC) mice also showed no differences in pancreas histology when compared to normal Lif-expressing PC mice. Lif flox/floxKPC (LKPC) mice develop PDA tumors. Cell lines from LKPC tumors (n=4) presented with undetectable Lif expression and no LIF protein secretion. Control KPC tumor cell lines (n=4) showed Lif expression and LIF secretion, confirming the successful deletion of Lif in our newly developed mouse model. LKPC mice (n=21) showed a significant increase in disease-specific overall survival at 72 days when compared to control KPC mice (n=26) at 62 days (p<0.05). When mice reached endpoint, LKPC mice presented with significantly smaller tumors with an average tumor weight of 0.7g compared to KPC control animals with an average tumor weight of 1.3g (p<0.05). In summary, we find that cancer-cell expressed LIF augments tumor growth and shortens disease-specific overall survival, nominating leukemia inhibitory factor depletion as a promising therapeutic target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Sarah Umetsu, Adam Olshen, Ritu Roy, Ashley Kiemen, Man-Tzu Wang, Eric Collisson. Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B095.

Cytocipher determines significantly different populations of cells in single-cell RNA-seq data.

Identification of cell types using single-cell RNA-seq is revolutionizing the study of multicellular organisms. However, typical single-cell RNA-seq analysis often involves post hoc manual curation to ensure clusters are transcriptionally distinct, which is time-consuming, error-prone, and irreproducible. To overcome these obstacles, we developed Cytocipher, a bioinformatics method and scverse compatible software package that statistically determines significant clusters. Application of Cytocipher to normal tissue, development, disease, and large-scale atlas data reveals the broad applicability and power of Cytocipher to generate biological insights in numerous contexts. This included the identification of cell types not previously described in the datasets analysed, such as CD8+ T cell subtypes in human peripheral blood mononuclear cells; cell lineage intermediate states during mouse pancreas development; and subpopulations of luminal epithelial cells over-represented in prostate cancer. Cytocipher also scales to large datasets with high-test performance, as shown by application to the Tabula Sapiens Atlas representing >480 000 cells. Cytocipher is a novel and generalizable method that statistically determines transcriptionally distinct and programmatically reproducible clusters from single-cell data. The software version used for this manuscript has been deposited on Zenodo (https://doi.org/10.5281/zenodo.8089546), and is also available via github (https://github.com/BradBalderson/Cytocipher).

Immunostaining patterns reveal potential morphogenetic role of Toll-like receptors 4 and 7 in the development of mouse respiratory system, liver and pancreas.

Toll-like receptors (TLRs) are the mammalian ortholog of Drosophila melanogaster protein Toll, originally identified for its involvement in embryonic development. In mammals, TLRs are mainly known for their ability to recognize pathogen- or damage-associated molecular patterns and, consequently, to initiate the immune response. However, it is becoming clear that TLRs can play a role also in mammal embryo development. We have previously described TLR4 and TLR7 expression in developing mouse peripheral nervous system and gastrointestinal tract. In the present study, we extended the investigation of TLR4 and TLR7 to the respiratory system and to the two main accessory organs of the digestive system, the liver and pancreas. TLR4 and TLR7 immunostaining was performed on mouse conceptuses collected at different stages, from E12 to E18. TLR4 and TLR7 immunoreactivity was evident in the embryo pancreas and liver at E12, while, in the respiratory apparatus, appeared at E14 and E17, respectively. Although further studies are required to elucidate the specific role of these TLRs in embryo development, the differential spatiotemporal TLR4 and TLR7 appearance may suggest that TLR expression in developing embryos is highly regulated for a possible their direct involvement in the formation of the organs and in the acquisition of immune-related features in preparation for the birth.

NEUROD1 Is Required for the Early α and β Endocrine Differentiation in the Pancreas.

Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting β cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, β-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of β cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and β-cell differentiation, β-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and β endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and β cells.

Immunohistochemical examination of p97/VCP expression in developing mouse pancreas and liver

Abstract
 Aim: The 97-kDa Valosin-containing protein has important functions in proteolysis. Although the expression of p97 / VCP has been studied in many types of cells and tissues, the localization of p97 / VCP at cellular level in developing mouse pancreas and liver has not been determined. Therefore, the aim of our study was investigate the immuolocalization of p97/VCP in the rat fetal and postnatal liver and pancreas. 
 Material Methods: The liver and pancreas from fetal, postnatal (1, 5, 15, 35 days old ) and adult (50 days old) mice were examined by using immunohistochemistry in order to determine the expression of p97/VCP. Furthermore the development of mouse pancreas and liver were histomorphologically analyzed under light microscope. 
 Results: The histological structures of the mouse pancreas and liver were maintained in postnatal period. The histological differences between endocrine and exocrine pancreas were significantly observed from the postnatal 5th day. The expression of p97/VCP in Langerhans islets was determined from day 5. p97/VCP expression was also seen in the exocrine pancreas in all postnatal days. p97/VCP was expressed in developing pancreatic and liver tissues and its expression was increased with the development.
 Conclusion: This study is the first to examine the distribution and the localization of p97/VCP in the fetal and postnatal rat liver and pancreas. This study forms the basis for clinical studies in order to determine the function of p97 / VCP in liver and pancreatic cells.

Establishment of a high-resolution 3D modeling system for studying pancreatic epithelial cell biology in vitro

ObjectiveTranslation of basic research from bench-to-bedside relies on a better understanding of similarities and differences between mouse and human cell biology, tissue formation, and organogenesis. Thus, establishing ex vivo modeling systems of mouse and human pancreas development will help not only to understand evolutionary conserved mechanisms of differentiation and morphogenesis but also to understand pathomechanisms of disease and design strategies for tissue engineering. MethodsHere, we established a simple and reproducible Matrigel-based three-dimensional (3D) cyst culture model system of mouse and human pancreatic progenitors (PPs) to study pancreatic epithelialization and endocrinogenesis ex vivo. In addition, we reanalyzed previously reported single-cell RNA sequencing (scRNA-seq) of mouse and human pancreatic lineages to obtain a comprehensive picture of differential expression of key transcription factors (TFs), cell–cell adhesion molecules and cell polarity components in PPs during endocrinogenesis. ResultsWe generated mouse and human polarized pancreatic epithelial cysts derived from PPs. This system allowed to monitor establishment of pancreatic epithelial polarity and lumen formation in cellular and sub-cellular resolution in a dynamic time-resolved fashion. Furthermore, both mouse and human pancreatic cysts were able to differentiate towards the endocrine fate. This differentiation system together with scRNA-seq analysis revealed how apical-basal polarity and tight and adherens junctions change during endocrine differentiation. ConclusionsWe have established a simple 3D pancreatic cyst culture system that allows to tempo-spatial resolve cellular and subcellular processes on the mechanistical level, which is otherwise not possible in vivo.

SIRT1 Activation Promotes β-Cell Regeneration by Activating Endocrine Progenitor Cells via AMPK Signaling-Mediated Fatty Acid Oxidation.

Induction of β-cell regeneration from endogenous cells represents a highly promising strategy in stem cell-based treatment for patients with diabetes. Recently, calorie restriction has been shown to affect the regulation of tissue and cell regeneration, including β cells, via metabolic related mechanisms. Here, we examined the potential utility of sirtuin 1 (SIRT1), a calorie restriction mimetic, for stimulating β-cell regeneration and the underlying mechanisms of such stimulation. The present results showed that SIRT1 activation with SRT1720 promoted β-cell regeneration in streptozotocin (STZ)-induced β-cell-deficient neonatal rats. This beneficial effect involved enhanced activation of neurogenin3 (NGN3)-positive endocrine progenitors from pancreatic ductal cells, rather than an expansion of residual β cells. A dynamic expression profile of SIRT1 was observed in endocrine progenitors both during β-cell regeneration in neonatal rats and in the second transition phase of mouse pancreas development. Consistently, SRT1720 treatment upregulated endocrine progenitor differentiation in cultured pancreatic rudiments. Upregulation of NGN3 by SIRT1 activation was through stimulating AMP-activated protein kinase (AMPK) signaling-mediated fatty acid oxidation (FAO) in human pancreatic progenitor cells; AMPK inhibition abolished these effects. The present findings demonstrate a promotional effect of SIRT1 activation on β-cell restoration and endocrine progenitor differentiation that involves regulation of AMPK signaling-mediated FAO. Stem Cells 2019;37:1416-1428.

Protein Methyltransferase Inhibition Decreases Endocrine Specification Through the Upregulation of Aldh1b1 Expression.

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self‐renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into β‐cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient β‐cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI‐5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640–651

Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development

Constitutive depletion of p21-activated kinase 4 (PAK4) in the mouse causes embryonic lethality associated with heart and brain defects. Given that conventional gene depletion of PAK1 or PAK3 caused functional deficits in the mouse pancreas, while gene depletion of PAK5 or PAK6 did not, we asked if PAK4 might have a functional role in pancreas development. We therefore introduced conditional, Pdx1-Cre-mediated, pancreatic PAK4 gene depletion in the mouse, verified by loss of PAK4 protein expression in the pancreas. PAK4 knock-out (KO) mice were born at Mendelian ratios in both genders. Further, morphological and immunohistochemical examinations and quantifications indicated that exocrine, endocrine and ductal compartments retained the normal proportions and distributions upon PAK4 gene depletion. In addition, body weight records and a glucose tolerance test revealed no differences between WT and PAK4 KO mice. Together, this suggests that PAK4 is dispensable for mouse pancreas development. This will facilitate future use of our Pdx1-Cre-driven conditional PAK4 KO mouse model for testing in vivo potential functions of PAK4 in pancreatic disease models such as for pancreatitis and different pancreatic cancer forms.

Phosphorylation of NEUROG3 Links Endocrine Differentiation to the Cell Cycle in Pancreatic Progenitors

During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G1 lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis.

New ideas connecting the cell cycle and pancreatic endocrine-lineage specification

Neurogenin 3 (Neurog3) is a pro-endocrine transcription factor required for endocrine-lineage specification during mouse pancreas development. It was a long-standing notion that Neurog3 activation ...

GATA4 and GATA6 regulate pancreatic endoderm identity through inhibition of hedgehog signaling.

GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4(fl/fl); Gata6(fl/fl); Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube.

Taming the young and restless—epigenetic gene regulation in pancreas and beta‐cell precursors

The in vivo assessment of epigenetic changes during mouse pancreatic beta‐cell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell-based therapies.

Role of BMP Signaling in Pancreatic Progenitor Differentiation from Human Embryonic Stem Cells

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.

Cx36 Is a Target of Beta2/NeuroD1, Which Associates with Prenatal Differentiation of Insulin-producing β Cells

The insulin-producing β cells of pancreatic islets are coupled by connexin36 (Cx36) channels. To investigate what controls the expression of this connexin, we have investigated its pattern during mouse pancreas development, and the influence of three transcription factors that are critical for β-cell development and differentiation. We show that (1) the Cx36 gene (Gjd2) is activated early in pancreas development and is markedly induced at the time of the surge of the transcription factors that determine β-cell differentiation; (2) the cognate protein is detected about a week later and is selectively expressed by β cells throughout the prenatal development of mouse pancreas; (3) a 2-kbp fragment of the Gjd2 promoter, which contains three E boxes for the binding of the bHLH factor Beta2/NeuroD1, ensures the expression of Cx36 by β cells; and (4) Beta2/NeuroD1 binds to these E boxes and, in the presence of the E47 ubiquitous cofactor, transactivates the Gjd2 promoter. The data identify Cx36 as a novel early marker of β cells and as a target of Beta2/NeuroD1, which is essential for β-cell development and differentiation.

Rbms3, an RNA-Binding Protein, Mediates the Expression ofPtf1aby Binding to Its 3′UTR During Mouse Pancreas Development

The development of the pancreas is a complicated process that is regulated on several levels. Pancreas transcription factor 1, alpha subunit (Ptf1a), also known as p48, is a pancreas-specific basic helix-loop-helix transcription factor that is critical for both exocrine pancreas development and maintenance of acinar cell differentiation. Based on a differential screening assay, we identified Rbms3, a gene encoding a glycine-rich RNA-binding protein, to be specifically expressed in the neural tube and the pancreatic rudiment of e10.5 embryos. The presence of Rbms3 in the early developing pancreas suggests that specific post-transcriptional regulation mechanisms play an important role in controlling pancreas development. In this study, we show that Rbms3 binds to the 3'UTR of Ptf1a mRNA, but not the 3'UTR of Pdx1, which is another pancreatic transcription factor. The ectopic expression of Rbms3 stimulates the translation of a reporter gene carrying the Ptf1a 3'UTR. In addition, when Rbms3 expression is suppressed in the AR42J-B13 pancreatic exocrine cell line, the expression of Ptf1a is also down-regulated. These results suggest that binding of Rbms3 to the 3'UTR of Ptf1a regulates the production of the Ptf1a protein and, thereby, indirectly regulates the expression of the Ptf1a downstream target genes.

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.

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1(+) pancreatic progenitor cells. Expression of activated Cdk4(R24C) kinase leads to increased Nkx2.2(+) and Nkx6.1(+) cells and a rise in the number and proliferation of Ngn3(+) endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors.