Leveraging zebrafish to investigate pancreatic development, regeneration, and diabetes.

The zebrafish has become an outstanding model for studying organ development and tissue regeneration, which is prominently leveraged for studies of pancreatic development, insulin-producing ß-cells, and diabetes. Although studied for more than two decades, many aspects remain elusive and it has only recently been possible to investigate these due to technical advances in transcriptomics, chemical-genetics, genome editing, drug screening, and in vivo imaging. Here, we review recent findings on zebrafish pancreas development, ß-cell regeneration, and how zebrafish can be used to provide novel insights into gene functions, disease mechanisms, and therapeutic targets in diabetes, inspiring further use of zebrafish for the development of novel therapies for diabetes.

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling.

The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.

Multivalent insulin receptor activation using insulin-DNA origami nanostructures.

Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin-DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin-DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent-but not monovalent-insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis.

AIMS/HYPOTHESIS: Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. METHODS: In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. RESULTS: Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. CONCLUSIONS/INTERPRETATION: Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. DATA AVAILABILITY: Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398 ).

Decoding pancreatic endocrine cell differentiation and ß cell regeneration in zebrafish.

In contrast to mice, zebrafish have an exceptional yet elusive ability to replenish lost ß cells in adulthood. Understanding this framework would provide mechanistic insights for ß cell regeneration, which may be extrapolated to humans. Here, we characterize a krt4-expressing ductal cell type, which is distinct from the putative Notch-responsive cells, showing neogenic competence and giving rise to the majority of endocrine cells during postembryonic development. Furthermore, we demonstrate a marked ductal remodeling process featuring a Notch-responsive to krt4+ luminal duct transformation during late development, indicating several origins of krt4+ ductal cells displaying similar transcriptional patterns. Single-cell transcriptomics upon a series of time points during ß cell regeneration unveil a previously unrecognized dlb+ transitional endocrine precursor cell, distinct regulons, and a differentiation trajectory involving cellular shuffling through differentiation and dedifferentiation dynamics. These results establish a model of zebrafish pancreatic endocrinogenesis and highlight key values of zebrafish for translational studies of ß cell regeneration.

Excess pancreatic elastase alters acinar-ß cell communication by impairing the mechano-signaling and the PAR2 pathways.

Type 1 (T1D) or type 2 diabetes (T2D) are caused by a deficit of functional insulin-producing ß cells. Thus, the identification of ß cell trophic agents could allow the development of therapeutic strategies to counteract diabetes. The discovery of SerpinB1, an elastase inhibitor that promotes human ß cell growth, prompted us to hypothesize that pancreatic elastase (PE) regulates ß cell viability. Here, we report that PE is up-regulated in acinar cells and in islets from T2D patients, and negatively impacts ß cell viability. Using high-throughput screening assays, we identified telaprevir as a potent PE inhibitor that can increase human and rodent ß cell viability in vitro and in vivo and improve glucose tolerance in insulin-resistant mice. Phospho-antibody microarrays and single-cell RNA sequencing analysis identified PAR2 and mechano-signaling pathways as potential mediators of PE. Taken together, our work highlights PE as a potential regulator of acinar-ß cell crosstalk that acts to limit ß cell viability, leading to T2D.

Efficient knock-in method enabling lineage tracing in zebrafish.

Here, we devised a cloning-free 3' knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5' AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92, nkx6.1, krt4, and id2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested that nkx6.1 + cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas id2a + cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic id2a + duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

In vivo drug discovery for increasing incretin-expressing cells identifies DYRK inhibitors that reinforce the enteroendocrine system.

Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput in vivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4, a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes.

Association between folic acid use during pregnancy and gestational diabetes mellitus: Two population-based Nordic cohort studies.

INTRODUCTION: Inconsistent results have been reported on the association between folic acid use in pregnancy and risk of GDM. The aim of this study was to estimate the association between folic acid use and GDM in two population-based Nordic cohorts. MATERIAL AND METHODS: Two cohort studies were conducted using data from the national population registers in Norway (2005-2018, n = 791,709) and Sweden (2006-2016, n = 1,112,817). Logistic regression was used to estimate the associations between GDM and self-reported folic acid use and prescribed folic acid use, compared to non-users, adjusting for covariates. To quantify how potential unmeasured confounders may affect the estimates, E-values were reported. An exposure misclassification bias analysis was also performed. RESULTS: In Norwegian and Swedish cohorts, adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for maternal self-reported folic acid use were 1.10 (1.06-1.14) and 0.89 (0.85-0.93), with E-values of 1.43 (1.31) and 1.50 (1.36), respectively. For prescribed folic acid use, ORs were 1.33 (1.15-1.53) and 1.56 (1.41-1.74), with E-values of 1.99 (1.57) and 2.49 (2.17), in Norway and Sweden respectively. CONCLUSIONS: The slightly higher or lower odds for GDM in self-reported users of folic acid in Norway and Sweden respectively, are likely not of clinical relevance and recommendations for folic acid use in pregnancy should remain unchanged. The two Nordic cohorts showed different directions of the association between self-reported folic acid use and GDM, but based on bias analysis, exposure misclassification is an unlikely explanation since there may still be differences in prevalence of use and residual confounding. Prescribed folic acid is used by women with specific comorbidities and co-medications, which likely underlies the higher odds for GDM.

MNK2 deficiency potentiates ß-cell regeneration via translational regulation.

Regenerating pancreatic ß-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of ß-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased ß-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and ß-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in ß-cell regeneration, a role that warrants further investigation in diabetes.

CERBERUS in the DARPA Subterranean Challenge.

This article presents the core technologies and deployment strategies of Team CERBERUS that enabled our winning run in the DARPA Subterranean Challenge finals. CERBERUS is a robotic system-of-systems involving walking and flying robots presenting resilient autonomy, as well as mapping and navigation capabilities to explore complex underground environments.

Investigating the association between prenatal exposure to folic acid and risk of neonatal diabetes/hyperglycemia and type 1 diabetes: A Norwegian register-based study.

BACKGROUND: Experimental animal studies suggest a novel role for the folate receptor 1 in ß-cell differentiation in the pancreas, with potential implications for glycemic control. We tested the hypothesis of a protective association between prenatal folic acid use and neonatal diabetes or hyperglycemia and type 1 diabetes in an observational cohort study using data from the national population health registers in Norway. METHODS: All singleton pregnancies resulting in live births from 2005 to 2018 were identified. Prenatal exposure to folic acid was determined based on maternal report at antenatal care in early pregnancy. Diagnoses of neonatal diabetes, hyperglycemia, and type 1 diabetes for the children were identified. Associations were estimated with logistic regression or Cox proportional hazard model and included crude and adjusted estimates. RESULTS: Among 781,567 children, 69% had prenatal exposure to folic acid, 264 were diagnosed with neonatal diabetes or hyperglycemia, and 1390 with type 1 diabetes. Compared to children with no prenatal exposure to folic acid, children with prenatal exposure to folic acid had similar odds of having a neonatal diabetes or hyperglycemia diagnosis (adjusted odds ratio 0.95, 95% confidence interval [CI] 0.72, 1.25) and similar risk of being diagnosed with type 1 diabetes (adjusted hazard ratio 1.05, 95% CI 0.93, 1.18). CONCLUSIONS: No association between prenatal folic acid exposure and neonatal diabetes/hyperglycemia or type 1 diabetes was found. These findings do not rule out a translational effect of the experimental results and future studies with longer follow-up and more precise information on the window of prenatal exposure are needed.

Insulin-producing ß-cells regenerate ectopically from a mesodermal origin under the perturbation of hemato-endothelial specification.

To investigate the role of the vasculature in pancreatic ß-cell regeneration, we crossed a zebrafish ß-cell ablation model into the avascular npas4l mutant (i.e. cloche). Surprisingly, ß-cell regeneration increased markedly in npas4l mutants owing to the ectopic differentiation of ß-cells in the mesenchyme, a phenotype not previously reported in any models. The ectopic ß-cells expressed endocrine markers of pancreatic ß-cells, and also responded to glucose with increased calcium influx. Through lineage tracing, we determined that the vast majority of these ectopic ß-cells has a mesodermal origin. Notably, ectopic ß-cells were found in npas4l mutants as well as following knockdown of the endothelial/myeloid determinant Etsrp. Together, these data indicate that under the perturbation of endothelial/myeloid specification, mesodermal cells possess a remarkable plasticity enabling them to form ß-cells, which are normally endodermal in origin. Understanding the restriction of this differentiation plasticity will help exploit an alternative source for ß-cell regeneration.

Reinforcing one-carbon metabolism via folic acid/Folr1 promotes ß-cell differentiation.

Diabetes can be caused by an insufficiency in ß-cell mass. Here, we performed a genetic screen in a zebrafish model of ß-cell loss to identify pathways promoting ß-cell regeneration. We found that both folate receptor 1 (folr1) overexpression and treatment with folinic acid, stimulated ß-cell differentiation in zebrafish. Treatment with folinic acid also stimulated ß-cell differentiation in cultures of neonatal pig islets, showing that the effect could be translated to a mammalian system. In both zebrafish and neonatal pig islets, the increased ß-cell differentiation originated from ductal cells. Mechanistically, comparative metabolomic analysis of zebrafish with/without ß-cell ablation and with/without folinic acid treatment indicated ß-cell regeneration could be attributed to changes in the pyrimidine, carnitine, and serine pathways. Overall, our results suggest evolutionarily conserved and previously unknown roles for folic acid and one-carbon metabolism in the generation of ß-cells.

In vivo screen identifies a SIK inhibitor that induces ß cell proliferation through a transient UPR.

It is known that ß cell proliferation expands the ß cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for ß cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human ß cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in ß cells blocks the effect of HG-9-91-01 on ß cell proliferation. Single-cell transcriptomic analyses of mouse ß cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive ß cell proliferation.

NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Corrigendum: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

This corrects the article DOI: 10.1038/nature18614.

SOX2 regulates common and specific stem cell features in the CNS and endoderm derived organs.

Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.