Mark A. Magnuson

SRY-box transcription factor 17 (Sox17) is essential for hepato-pancreato-biliary development. We identified two distributed enhancers of Sox17, Sox17e-13 and Sox17e-231, within the likely murine Sox17 topologically associating domain. Individual CRISPR-mediated enhancer deletions temporally reduce endodermal Sox17 expression, delaying the transition of bipotent ventral pancreato-biliary progenitor cells to a lineage-unipotent ventral pancreatic fate, reducing pancreas size and increasing gallbladder size. Deleting both enhancers further reduces Sox17 expression and delays conversion of bipotent progenitor cells to ventral pancreatic or biliary fates, reducing both pancreas and gallbladder size. Crossing each enhancer mutation to a Sox17 null allele revealed differential and profound sensitivities of the extrahepatic biliary system to alterations of Sox17 expression, as demonstrated by gallbladder hypoplasia, gallbladder agenesis with a cystic duct, and biliary atresia. These studies indicate that Sox17e-13 and Sox17e-231 additively modulate Sox17 expression, that the differentiation of bipotential ventral pancreato-biliary cells to unipotent fates is Sox17 dosage dependent, and that thresholds for Sox17 action during pancreato-biliary formation vary by developmental stage, further defining the role of Sox17 in hepato-pancreato-biliary formation and function.

Electron microscopy (EM) is essential for resolving cellular ultrastructure, yet quantitative analysis remains limited by labor-intensive segmentation and the scarcity of generalizable models. Here we present QuantEM, an open-source platform for segmentation and analysis of EM data across imaging modalities, tissues, and species. We assembled the largest curated collection of intracellular EM datasets to date, comprising over 15,000 two-dimensional images and 1,700 three-dimensional acquisitions from more than 600 datasets, including nearly 4,000 newly released acquisitions. Using this resource, we trained an EM-specific vision transformer foundation model and systematically optimized adaptation strategies for organelle segmentation. QuantEM provides pretrained models for mitochondria, endoplasmic reticulum, nuclei, and lipid droplets, integrated with interactive proofreading and downstream quantitative analyses through standalone and napari interfaces. Across diverse naive datasets, QuantEM consistently matches or exceeds existing models on zero-shot segmentation while requiring less data for finetuning. We further demonstrate its utility by revealing previously unrecognized subcellular compartmentation of hepatic glucokinase using immuno-electron microscopy.

Endocrine islet β cells comprise heterogenous subtypes with different gene expression and function levels. Here we study when/how this heterogeneity is induced and how long each subtype maintains its characteristic properties. We show that islet progenitors with distinct gene expression and DNA methylation patterns produce β-cell subtypes of different secretory function, proliferation rate, and viability in male and female mice. These subtypes have differential gene expression that regulates insulin vesicle production or stimulation-secretion coupling and differential DNA methylation in the putative enhancers of these genes. Maternal obesity, a major diabetes risk factor, reduces the proportion of the β-cell subtype with higher levels of glucose responsiveness. The gene signature that defines mouse β-cell subtypes can reliably divide human cells into two sub-populations, with the one having higher predicted glucose responsiveness reduced in diabetic donors. These results suggest that β-cell subtypes can be derived from islet progenitor subsets modulated by maternal nutrition.

By augmenting ATP-sensitive K+ channel–induced membrane depolarization, chronic metabolic stress in prediabetes may increase intracellular Ca2+ concentrations in pancreatic β-cells and cause a loss of cell identity and function. Here, we describe studies of the temporal transcriptomic dynamics induced by sulfonylurea-induced membrane depolarization. Gene expression in isolated islets is highly dynamic, with changes occurring within 30 min of membrane depolarization. Initially, the changes are adaptive and driven mainly by signaling through CREB and several other CREB-dependent transcription factors. However, within several hours, there is a progressive decline in islet function that correlates with the diminished expression of islet identity genes and the expression of dedifferentiation markers, consistent with the responses having become maladaptive. The gene expression adaptations cluster into 19 distinct response patterns driven by multiple transcription factors. We also identify a set of high-concentration glucose/Ca2+-regulated genes and modules of coexpressed genes that are enriched for type 2 diabetes risk genes. Together, these findings establish a close temporal link between membrane depolarization, changes in intracellular Ca2+ concentrations, alterations in the islet transcriptome, and impairments of β-cell identity and function. ARTICLE HIGHLIGHTS: This study was undertaken to establish a temporal link between an increase in intracellular Ca2+ concentration and the loss of pancreatic β-cell identity. We profiled the alterations in Ca2+ dynamics and gene transcription that occur in freshly isolated islets following membrane depolarization. We show that initially adaptive Ca2+-dependent transcription changes, mediated largely by CREB and CREB-dependent transcription factors, rapidly become maladaptive, causing the loss of β-cell identity and function. We also show that many effector genes linked to nearby human type 2 diabetes susceptibility loci are regulated by Ca2+-dependent mechanisms.

Inflammation in the pancreas drives acinar-to-ductal metaplasia (ADM), a progenitor-like state that can be hijacked by mutant Kras in the formation of pancreatic cancer (PDAC). How these cell fate decisions vary according to KRAS mutation remains poorly understood. To define mutation-specific lineage reversion and tumor initiation, we implement novel Ptf1a-TdTomato mice and multiple KRAS mutants across an array of genetic, pharmacologic, and inflammatory perturbations in vivo . Whereas KRAS ^G12D co-opts injury to enable lineage reversion, enhancer reprogramming, and tumor initiation, KRAS ^G12R/V can initiate but not sustain dedifferentiated and neoplastic transcriptional and epigenetic programs. We find the KRAS ^G12R/V defects consist of a failure to invoke robust EGFR signaling and activate Rac1/Vav1, with constitutive Akt activation in vivo sufficient to rescue the tumorigenic potential of KRAS ^G12R . As the marked heterogeneity among KRAS variants begins early in tumorigenesis, these data are crucial to understanding mutation-specific oncogenic trajectories and directing the implementation of KRAS -directed therapeutics. SIGNIFICANCE: Defining how KRAS mutants drive distinct outcomes in human pancreatic cancer is critical for developing allele-specific therapeutic approaches. This study unveils a hierarchy among KRAS ^G12D , KRAS ^G12V , and KRAS ^G12R to drive tumor initiation, owing to heterogeneous activation of EGFR, PI3K/AKT, and RAC1 signaling, thus revealing mutation-specific evolutionary paths in pancreatic tumorigenesis.

Pancreatic β-cells require the coordinated expression of transcription factors such as MAFA to dynamically secrete insulin to maintain euglycemia. A naturally occurring mutation in MAFA (MAFA ^S64F ) produces a long-lived variant protein which predisposes carriers to dichotomous conditions of either maturity (adult)-onset diabetes of the young (MODY) or hypoglycemia in a sex-dependent manner. Here we show that genetic background modulates disease penetrance in MafA ^S64F/+ male mice. Specifically, MafA ^S64F/+ males backcrossed to a C57/Bl6J (“C57”) background prevented dysglycemia, while C57 MafA ^S64F/+ males bred one generation to an SJL/J background (“mixed”) manifested overt diabetes, impaired insulin secretion, and accelerated β-cell senescence. MafA protein levels, phosphorylation status, and target gene expression in C57 MafA ^S64F/+ male islets were more comparable to wildtype males. RNA sequencing of C57 MafA ^S64F/+ male islets revealed fewer differentially expressed genes than mixed background male islets, including transcriptional signatures of β-cell senescence. In addition, retinoic acid signaling, another signature of cellular aging, was uniquely downregulated in C57 MafA ^S64F/+ male islets. Indeed, core retinoic acid signaling receptor RARα and known target genes were downregulated in C57 MafA ^S64F/+ male islets. CUT&RUN mapping revealed that MafA directly impacted retinoic acid receptor alpha Rara expression. In sum, these data show that genetic factors can impact unique pathways to modulate disease penetrance in mice modeling MAFA-MODY. Article Highlights: Genetic background in mice or ancestry in humans can profoundly influence diabetes susceptibility. We asked whether diabetes penetrance in our robust mouse model of MAFA-MODY on a mixed genetic background was impacted when backcrossed to C57/Bl6J (“C57”). Here we show that C57 MafA ^S64F/+ males have wildtype glucose-sensing properties, compared to overt diabetes which could be reproducibly re-created on a mixed background. Further analysis disclosed more muted gene expression changes in C57 MafA ^S64F/+ male islets including unique downregulation of senescence and retinoic acid signaling. These results illustrate the influence of genetic landscape on β-cell function in response to a pathogenic MODY variant.

Expression of SRY-box transcription factor 17 (Sox17) in the endodermal region caudal to the hepatic diverticulum during late gastrulation is necessary for hepato-pancreato-biliary system formation. Analysis of an allelic series of promoter-proximal mutations near the transcription start site (TSS) 2 of Sox17 in mouse has revealed that gallbladder (GB) and extrahepatic bile duct (EHBD) development is exquisitely sensitive to Sox17 expression levels. Deletion of a SOX17-binding cis-regulatory element in the TSS2 promoter impairs GB and EHBD development by reducing outgrowth of the nascent biliary bud. These findings reveal the existence of a SOX17-dependent autoregulatory loop that drives Sox17 expression above a critical threshold concentration necessary for GB and EHBD development to occur, and that minor impairments in Sox17 gene expression are sufficient to impair the expression of SOX17-regulated genes in the nascent GB and EHBD system, impairing or preventing development.

Loss-of-function mutations in ATP-sensitive potassium (KATP) channels cause hyperexcitability and insulin hypersecretion, resulting in congenital hyperinsulinism (CHI). Paradoxically, despite the initial insulin hypersecretion, many CHI cases, as well as KATP knockout (KO) animals, eventually “crossover” to undersecretion and even diabetes. Here, we confirm that Sur1 KO islets exhibit higher intracellular concentration of calcium ion ([Ca2+]i) at all concentrations of glucose but show decreased glucose-stimulated insulin secretion. However, when [Ca2+]i is artificially elevated by increasing extracellular [Ca2+], insulin secretion from Sur1 KO islets increases to the same levels as in wild-type (WT) islets. This indicates that a right-shift in [Ca2+]i dependence of insulin secretion, rather than loss of insulin content or intrinsic secretability, is the primary cause for the crossover. Chronic pharmacological inhibition of KATP channel activity by slow release of glibenclamide in pellet-implanted mice causes a very similar crossover to glucose intolerance and impaired insulin secretion seen in Sur1 KO animals. Whole-islet and single-cell transcriptomic analysis reveal markedly reduced Trpm5 in both conditions. Glibenclamide pellet–implanted Trpm5 KO mice also exhibited significant glucose intolerance. However, this was not as severe as in WT animals, which suggests decreased expression of Trpm5 may play a small role in the disruption of insulin secretion with KATP loss. Article Highlights: Congenital hyperinsulinism caused by loss of ATP-sensitive potassium (KATP) channels crosses over to unexplained undersecretion. Why does loss of β-cell KATP channel activity result in undersecretion of insulin and glucose tolerance, despite elevated intracellular concentration of calcium ion ([Ca2+]i) levels? Superelevation of [Ca2+]i in supraphysiological extracellular [Ca2+] boosted secretion from Sur1 knockout (KO) islets to the same levels as WT, indicating a right-shift in [Ca2+]i dependence of secretion. Transcriptomic analysis revealed markedly reduced β-cell Trpm5 in the absence of KATP. KATP inhibition in Trpm5 KO mice still caused significant glucose intolerance, but slightly less severe than in WT animals. Right-shifted [Ca2+]i dependence of secretion explains crossover. Downregulation of Trpm5 may be involved.