Β-cell Heterogeneity Research Articles

β-Cell Heterogeneity and Plasticity.

The existence of functionally diverse and plastic β cells in islets of Langerhans has been reported since the 1980s. Recently, high-resolution technologies have advanced our understanding of β-cell heterogeneity and plasticity. Here, we define plasticity broadly as dynamic changes in cellular phenotypes and heterogeneity as differences in cellular behaviors. Individual β cells react differently to environmental challenges and act together to maintain β-cell mass and glucose homeostasis within a narrow range of 70-140mg/dL. During the progress of diabetes, this elaborate balance is disrupted, and a lack of β-cell compensation leads to dysregulated blood glucose. In this chapter, we assess β-cell stress that instigates increased β-cell heterogeneity and adaptive β-cell responses such as proliferation, dedifferentiation, maturity, and insulin secretion. We also discuss the maturity, electrical activity, and insulin secretion of well-characterized β-cell subgroups. Finally, we touch upon the plasticity of other non-β pancreatic cells and their cooperation with β cells to maintain homeostasis.

Single-Cell Transcriptome Profiling of Pancreatic Islets From Early Diabetic Mice Identifies Anxa10 for Ca2+ Allostasis Toward β-Cell Failure.

The transcriptome of single-islet cells from healthy, prediabetic, and diabetic mice was studied. Distinct β-cell heterogeneity and islet cell-cell network in prediabetes and diabetes were found. A new prediabetic β-cell marker, Anxa10, regulates intracellular Ca2+ and insulin secretion. Diabetes triggers β-cell to acinar cell transdifferentiation.

Single cell multiomic analysis reveals diabetes-associated β-cell heterogeneity driven by HNF1A

Broad heterogeneity in pancreatic β-cell function and morphology has been widely reported. However, determining which components of this cellular heterogeneity serve a diabetes-relevant function remains challenging. Here, we integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets and identify Type II Diabetes (T2D)-associated β-cell heterogeneity at both transcriptomic and epigenomic levels. We develop a computational method to explicitly dissect the intra-donor and inter-donor heterogeneity between single β-cells, which reflect distinct mechanisms of T2D pathogenesis. Integrative transcriptomic and epigenomic analysis identifies HNF1A as a principal driver of intra-donor heterogeneity between β-cells from the same donors; HNF1A expression is also reduced in β-cells from T2D donors. Interestingly, HNF1A activity in single β-cells is significantly associated with lower Na+ currents and we nominate a HNF1A target, FXYD2, as the primary mitigator. Our study demonstrates the value of investigating disease-associated single-cell heterogeneity and provides new insights into the pathogenesis of T2D.

1747-P: Islet Ca2+ Dynamics, Heterogeneity, and Consistency in Three Dimensions with Activators of Pyruvate Kinase

Pancreatic β-cell heterogeneity is important for Ca2+ triggering and insulin secretion and is affected in diabetes. Organization and consistency of β-cell subpopulations are poorly understood despite being key in understanding how individual cells contribute to islet function and diabetes. Existing studies analyze single 2D planes of the islet. However, subpopulation organization cannot be fully understood without examining β cell dynamics throughout the entire islet in 3D. Here, we imaged islets from β cell specific GCaMP6s Ca2+ reporter mice using high-speed light-sheet microscopy to analyze the 3D organization and consistency in high glucose and with pharmacologic activators of pyruvate kinase (PK), a glycolytic enzyme that controls βcell excitability. Phase analysis revealed that cells which initiated islet depolarization (leaders) are not highly consistent across oscillations, but the 3D region where the calcium wave originates is consistent, according to a metric based upon the KL-divergence (P<0.0001 over 11 oscillations). Using functional network analysis, β-cell hubs which drive islet synchrony are more consistent over an islet region than across individual cells (P<0.0001). The wave origin was more regionally dependent than the β-cell hubs (P<0.0001). We also found that network analysis is similar (P=0.04) and phase analysis is different between 2D and 3D (P<0.0001). Following quantification of heterogeneity in the 3D islet, certain sub-populations of β cells are similar throughout the islet, while others are not. Acute PK activation decreased the consistency of wave initiators (P<0.0001), but not the consistency of hub cells in the functional network (P=0.15), shedding new light onto the mechanism behind these subpopulations. Islet regions, rather than individual cells, are more consistent and thus likely responsible for initiating Ca2+ triggering and insulin secretion. These mechanisms help render the islet robust to single β cell failures. Disclosure J.Briggs: None. E.Jin: None. M.J.Merrins: None. R.K.Benninger: None. Funding William S. Middleton Memorial Veterans Hospital; U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01B005113); National Institutes of Health (R01DK113103, R01DK127637, R01DK102950, R01DK106412); National Science

Type 2 Diabetes: Is it Time to Target β-cell Heterogeneity?

Type 2 Diabetes: Is it Time to Target β-cell Heterogeneity?

Integrated transcriptomics contrasts fatty acid metabolism with hypoxia response in β-cell subpopulations associated with glycemic control

BackgroundUnderstanding how heterogeneous β-cell function impacts diabetes is imperative for therapy development. Standard single-cell RNA sequencing analysis illuminates some factors driving heterogeneity, but new strategies are required to enhance information capture.ResultsWe integrate pancreatic islet single-cell and bulk RNA sequencing data to identify β-cell subpopulations based on gene expression and characterize genetic networks associated with β-cell function in obese SM/J mice. We identify β-cell subpopulations associated with basal insulin secretion, hypoxia response, cell polarity, and stress response. Network analysis associates fatty acid metabolism and basal insulin secretion with hyperglycemic-obesity, while expression of Pdyn and hypoxia response is associated with normoglycemic-obesity.ConclusionsBy integrating single-cell and bulk islet transcriptomes, our study explores β-cell heterogeneity and identifies novel subpopulations and genetic pathways associated with β-cell function in obesity.

DNA Methylation-Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-Cell Heterogeneity.

The molecular and functional heterogeneity of pancreatic β-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of β-cells that co-express tyrosine hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ β-cells within islets is essential for appropriate function in mice, such that a higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of β-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ β-cells and show differences in their developmental restriction in mice and humans. We show that TH expression in β-cells is restricted by DNA methylation during β-cell differentiation. Ablation of de novo DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ β-cells, whereas β-cell-specific ablation of Dnmt3a does not. We demonstrate that maintenance of Th promoter methylation is essential for its continued restriction in postnatal β-cells. Loss of Th promoter methylation in response to chronic overnutrition increases the number of TH+ β-cells, corresponding to impaired β-cell function. These results reveal a regulatory role of DNA methylation in determining β-cell heterogeneity.

Generation of β-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids.

Since the identification of four different pancreatic β-cell subtypes and bi-hormomal cells playing a role in the diabetes pathogenesis, the search for in vitro models that mimics such cells heterogeneity became a key priority in experimental and clinical diabetology. We investigated the potential of human induced pluripotent stem cells to lead to the development of the different β-cells subtypes in honeycomb microwell-based 3D spheroids. The glucose-stimulated insulin secretion confirmed the spheroids functionality. Then, we performed a single cell RNA sequencing of the spheroids. Using a knowledge-based analysis with a stringency on the pancreatic markers, we extracted the β-cells INS+/UCN3+ subtype (11%; β1-like cells), the INS+/ST8SIA1+/CD9- subtype (3%, β3-like cells) and INS+/CD9+/ST8SIA1-subtype (1%; β2-like cells) consistently with literature findings. We did not detect the INS+/ST8SIA1+/CD9+ cells (β4-like cells). Then, we also identified four bi-hormonal cells subpopulations including δ-like cells (INS+/SST+, 6%), γ-like cells (INS+/PPY+, 3%), α-like-cells (INS+/GCG+, 6%) and ε-like-cells (INS+/GHRL+, 2%). Using data-driven clustering, we extracted four progenitors' subpopulations (with the lower level of INS gene) that included one population highly expressing inhibin genes (INHBA+/INHBB+), one population highly expressing KCNJ3+/TPH1+, one population expressing hepatocyte-like lineage markers (HNF1A+/AFP+), and one population expressing stem-like cell pancreatic progenitor markers (SOX2+/NEUROG3+). Furthermore, among the cycling population we found a large number of REST+ cells and CD9+ cells (CD9+/SPARC+/REST+). Our data confirm that our differentiation leads to large β-cell heterogeneity, which can be used for investigating β-cells plasticity under physiological and pathophysiological conditions.

Dynamic Ins2 Gene Activity Defines β-Cell Maturity States.

Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas. However, it is not clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in β-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of β-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single β-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH β-cells were enriched for markers of β-cell maturity. Ins2(GFP)HIGH β-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity.

A transcriptional cross species map of pancreatic islet cells

ObjectivePancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions. MethodsIn this study, we used single-cell RNA sequencing to profile more than 50′000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and β-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions. ResultsWe showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and β-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and β-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data. ConclusionsHere, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

Microtubules regulate pancreatic β-cell heterogeneity via spatiotemporal control of insulin secretion hot spots.

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the β-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single β-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant β-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional β-cell heterogeneity.

The physiological role of β-cell heterogeneity in pancreatic islet function.

Endocrine cells within the pancreatic islets of Langerhans are heterogeneous in terms of transcriptional profile, protein expression and the regulation of hormone release. Even though this heterogeneity has long been appreciated, only within the past 5 years have detailed molecular analyses led to an improved understanding of its basis. Although we are beginning to recognize why some subpopulations of endocrine cells are phenotypically different to others, arguably the most important consideration is how this heterogeneity affects the regulation of hormone release to control the homeostasis of glucose and other energy-rich nutrients. The focus of this Review is the description of how endocrine cell heterogeneity (and principally that of insulin-secreting β-cells) affects the regulation of hormone secretion within the islets of Langerhans. This discussion includes an overview of the functional characteristics of the different islet cell subpopulations and describes how they can communicate to influence islet function under basal and glucose-stimulated conditions. We further discuss how changes to the specific islet cell subpopulations or their numbers might underlie islet dysfunction in type 2 diabetes mellitus. We conclude with a discussion of several key open questions regarding the physiological role of islet cell heterogeneity.

Vertical sleeve gastrectomy triggers fast β-cell recovery upon overt diabetes

ObjectiveThe effectiveness of bariatric surgery in restoring β-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, whereas the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue far-progressed, clinically-relevant T2D and to promote β-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of β-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. MethodsWe performed VSG in late-stage diabetic db/db mice and analyzed the islet transcriptome using single-cell RNA sequencing (scRNA-seq). Immunohistochemical analyses and quantification of β-cell area and proliferation complement our findings from scRNA-seq. ResultsWe report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the β-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast β-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded β-cell area by means of redifferentiation and by creating a proliferation competent β-cell state. ConclusionCollectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of β-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.

Predisposition to Proinsulin Misfolding as a Genetic Risk to Diet-Induced Diabetes.

Throughout evolution, proinsulin has exhibited significant sequence variation in both C-peptide and insulin moieties. As the proinsulin coding sequence evolves, the gene product continues to be under selection pressure both for ultimate insulin bioactivity and for the ability of proinsulin to be folded for export through the secretory pathway of pancreatic β-cells. The substitution proinsulin-R(B22)E is known to yield a bioactive insulin, although R(B22)Q has been reported as a mutation that falls within the spectrum of mutant INS-gene-induced diabetes of youth. Here, we have studied mice expressing heterozygous (or homozygous) proinsulin-R(B22)E knocked into the Ins2 locus. Neither females nor males bearing the heterozygous mutation developed diabetes at any age examined, but subtle evidence of increased proinsulin misfolding in the endoplasmic reticulum is demonstrable in isolated islets from the heterozygotes. Moreover, males have indications of glucose intolerance, and within a few weeks of exposure to a high-fat diet, they developed frank diabetes. Diabetes was more severe in homozygotes, and the development of disease paralleled a progressive heterogeneity of β-cells with increasing fractions of proinsulin-rich/insulin-poor cells as well as glucagon-positive cells. Evidently, subthreshold predisposition to proinsulin misfolding can go undetected but provides genetic susceptibility to diet-induced β-cell failure.

Small subpopulations of β-cells do not drive islet oscillatory [Ca2+] dynamics via gap junction communication.

The islets of Langerhans exist as multicellular networks that regulate blood glucose levels. The majority of cells in the islet are excitable, insulin-producing β-cells that are electrically coupled via gap junction channels. β-cells are known to display heterogeneous functionality. However, due to gap junction coupling, β-cells show coordinated [Ca2+] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional β-cells have been suggested to control [Ca2+] dynamics across the islet. When these populations were targeted by optogenetic silencing or photoablation, [Ca2+] dynamics across the islet were largely disrupted. In this study, we investigated the theoretical basis of these experiments and how small populations can disproportionality control islet [Ca2+] dynamics. Using a multicellular islet model, we generated normal, skewed or bimodal distributions of β-cell heterogeneity. We examined how islet [Ca2+] dynamics were disrupted when cells were targeted via hyperpolarization or populations were removed; to mimic optogenetic silencing or photoablation, respectively. Targeted cell populations were chosen based on characteristics linked to functional subpopulation, including metabolic rate of glucose oxidation or [Ca2+] oscillation frequency. Islets were susceptible to marked suppression of [Ca2+] when ~10% of cells with high metabolic activity were hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when highly metabolic cells were removed from the model, [Ca2+] oscillations remained. Similarly, when ~10% of cells with either the highest frequency or earliest elevations in [Ca2+] were removed from the islet, the [Ca2+] oscillation frequency remained largely unchanged. Overall, these results indicate small populations of β-cells with either increased metabolic activity or increased frequency are unable to disproportionately control islet-wide [Ca2+] via gap junction coupling. Therefore, we need to reconsider the physiological basis for such small β-cell populations or the mechanism by which they may be acting to control normal islet function.

Harnessing the Endogenous Plasticity of Pancreatic Islets: A Feasible Regenerative Medicine Therapy for Diabetes?

Diabetes is a chronic metabolic disease caused by an absolute or relative deficiency in functional pancreatic β-cells that leads to defective control of blood glucose. Current treatments for diabetes, despite their great beneficial effects on clinical symptoms, are not curative treatments, leading to a chronic dependence on insulin throughout life that does not prevent the secondary complications associated with diabetes. The overwhelming increase in DM incidence has led to a search for novel antidiabetic therapies aiming at the regeneration of the lost functional β-cells to allow the re-establishment of the endogenous glucose homeostasis. Here we review several aspects that must be considered for the development of novel and successful regenerative therapies for diabetes: first, the need to maintain the heterogeneity of islet β-cells with several subpopulations of β-cells characterized by different transcriptomic profiles correlating with differences in functionality and in resistance/behavior under stress conditions; second, the existence of an intrinsic islet plasticity that allows stimulus-mediated transcriptome alterations that trigger the transdifferentiation of islet non-β-cells into β-cells; and finally, the possibility of using agents that promote a fully functional/mature β-cell phenotype to reduce and reverse the process of dedifferentiation of β-cells during diabetes.

Single Molecule-Based fliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet β-Cells.

Single-cell RNA-sequencing (scRNA-Seq) technologies have greatly enhanced our understanding of islet cell transcriptomes and have revealed the existence of β-cell heterogeneity. However, comparison of scRNA-Seq data sets from different groups have highlighted inconsistencies in gene expression patterns, primarily due to variable detection of lower abundance transcripts. Furthermore, such analyses are unable to uncover the spatial organization of heterogeneous gene expression. In this study, we used fluctuation localization imaging–based fluorescence in situ hybridization (fliFISH) to quantify transcripts in single cells in mouse pancreatic islet sections. We compared the expression patterns of Insulin 2 (Ins2) with Mafa and Ucn3, two genes expressed in β-cells as they mature, as well as Rgs4, a factor with variably reported expression in the islet. This approach accurately quantified transcripts across a wide range of expression levels, from single copies to >100 copies/cell in one islet. Importantly, fliFISH allowed evaluation of transcript heterogeneity in the spatial context of an intact islet. These studies confirm the existence of a high degree of heterogeneous gene expression levels within the islet and highlight relative and radial expression patterns that likely reflect distinct β-cell maturation states along the radial axis of the islet.

Pancreatic β-cell heterogeneity in health and diabetes: classes, sources, and subtypes

Pancreatic β-cells perform glucose-stimulated insulin secretion, a process at the center of type 2 diabetes etiology. Efforts to understand how β-cells behave in healthy and stressful conditions have revealed a wide degree of morphological, functional, and transcriptional heterogeneity. Sources of heterogeneity include β-cell topography, developmental origin, maturation state, and stress response. Advances in sequencing and imaging technologies have led to the identification of β-cell subtypes, which play distinct roles in the islet niche. This review examines β-cell heterogeneity from morphological, functional, and transcriptional perspectives, and considers the relevance of topography, maturation, development, and stress response. It also discusses how these factors have been used to identify β-cell subtypes, and how heterogeneity is impacted by diabetes. We examine open questions in the field and discuss recent technological innovations that could advance understanding of β-cell heterogeneity in health and disease.

CD81 marks immature and dedifferentiated pancreatic β-cells

ObjectiveIslets of Langerhans contain heterogeneous populations of insulin-producing β-cells. Surface markers and respective antibodies for isolation, tracking, and analysis are urgently needed to study β-cell heterogeneity and explore the mechanisms to harness the regenerative potential of immature β-cells. MethodsWe performed single-cell mRNA profiling of early postnatal mouse islets and re-analyzed several single-cell mRNA sequencing datasets from mouse and human pancreas and islets. We used mouse primary islets, iPSC-derived endocrine cells, Min6 insulinoma, and human EndoC-βH1 β-cell lines and performed FAC sorting, Western blotting, and imaging to support and complement the findings from the data analyses. ResultsWe found that all endocrine cell types expressed the cluster of differentiation 81 (CD81) during pancreas development, but the expression levels of this protein were gradually reduced in β-cells during postnatal maturation. Single-cell gene expression profiling and high-resolution imaging revealed an immature signature of β-cells expressing high levels of CD81 (CD81high) compared to a more mature population expressing no or low levels of this protein (CD81low/-). Analysis of β-cells from different diabetic mouse models and in vitro β-cell stress assays indicated an upregulation of CD81 expression levels in stressed and dedifferentiated β-cells. Similarly, CD81 was upregulated and marked stressed human β-cells in vitro. ConclusionsWe identified CD81 as a novel surface marker that labels immature, stressed, and dedifferentiated β-cells in the adult mouse and human islets. This novel surface marker will allow us to better study β-cell heterogeneity in healthy subjects and diabetes progression.

Single-Cell Transcriptome Analysis Dissects the Replicating Process of Pancreatic Beta Cells in Partial Pancreatectomy Model

SummaryHeterogeneity of gene expression and rarity of replication hamper molecular analysis of β-cell mass restoration in adult pancreas. Here, we show transcriptional dynamics in β-cell replication process by single-cell RNA sequencing of murine pancreas with or without partial pancreatectomy. We observed heterogeneity of Ins1-expressing β-cells and identified the one cluster as replicating β-cells with high expression of cell proliferation markers Pcna and Mki67. We also recapitulated cell cycle transition accompanied with switching expression of cyclins and E2F transcription factors. Both transient activation of endoplasmic reticulum stress responders like Atf6 and Hspa5 and elevated expression of tumor suppressors like Trp53, Rb1, and Brca1 and DNA damage responders like Atm, Atr, Rad51, Chek1, and Chek2 during the transition to replication associated fine balance of cell cycle progression and protection from DNA damage. Taken together, these results provide a high-resolution map depicting a sophisticated genetic circuit for replication of the β-cells.