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Aims/hypothesis: The aim of this work was to understand the role of non-beta cells in pancreatic islets at early stages of type 2 diabetes pathogenesis., Methods: Specific clustering was employed to single-cell transcriptome data from islet cells of obese mouse strains differing in their diabetes susceptibility (diabetes-resistant B6.V.Lep ob/ob [OB] and diabetes-susceptible New Zealand Obese [NZO] mice) on a diabetogenic diet., Results: Refined clustering analysis revealed several heterogeneous subpopulations for alpha cells, delta cells and macrophages, of which 133 mapped to human diabetes genes identified by genome-wide association studies. Importantly, a similar non-beta cell heterogeneity was found in a dataset of human islets from donors at different stages of type 2 diabetes. The predominant alpha cell cluster in NZO mice displayed signs of cellular stress and lower mitochondrial capacity (97 differentially expressed genes [DEGs]), whereas delta cells from these mice exhibited higher expression levels of maturation marker genes (Hhex and Sst) but lower somatostatin secretion than OB mice (184 DEGs). Furthermore, a cluster of macrophages was almost twice as abundant in islets of OB mice, and displayed extensive cell-cell communication with beta cells of OB mice. Treatment of beta cells with IL-15, predicted to be released by macrophages, activated signal transducer and activator of transcription (STAT3), which may mediate anti-apoptotic effects. Similar to mice, humans without diabetes possess a greater number of macrophages than those with prediabetes (39 mmol/mol [5.7%] < HbA 1c < 46 mmol/mol [6.4%]) and diabetes., Conclusions/interpretation: Our study indicates that the transcriptional heterogeneity of non-beta cells has an impact on intra-islet crosstalk and participates in beta cell (dys)function., Data Availability: scRNA-seq data from the previous study are available in gene expression omnibus under gene accession number GSE159211 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159211 )., Competing Interests: Acknowledgements: The skilful technical assistance of E. Arlt and A. Helms (Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke [DIfE], Nuthetal, Germany) is gratefully acknowledged. In addition, the assistance of A. Teichmann and C. Gumz (DIfE) for islet picking is gratefully acknowledged. Servier Medical Art was used to create the graphical abstract, which is licensed under CC BY 4.0 ( https://creativecommons.org/licenses/by/4.0/ ). Some of the data were presented as a poster at the DDG conference in 2024 with the title ‘The role of non-beta cell heterogeneity in the pathogenesis of type 2 diabetes’. In addition, the research is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; 491394008). Data availability: scRNA-seq data from the previous study are available in gene expression omnibus under gene accession number GSE159211 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159211 ). Funding: Open Access funding enabled and organized by Projekt DEAL. The study was supported by the German Ministry of Education and Research (BMBF: DZD grant 82DZD03D03) and the Brandenburg State; as well as the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project numbers 407395267, 525802829 and 491394008. Authors’ relationships and activities: The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work. Contribution statement: PG performed data analysis, designed figures and wrote the manuscript, TS performed data analysis, did experiments and wrote the manuscript. MS and JS did experiments. PC and NN performed data analysis and critically edited and revised the manuscript. HL performed study conception and critically edited and revised the manuscript, AS performed study conception and design, and critically edited and revised the manuscript. All authors subsequently edited the manuscript, and approved its final version for publication. AS is the guarantor of this work., (© 2024. The Author(s).)

Coordination of cellular activity through Ca 2+ enables β cells to secrete precise quantities of insulin. To explore how the Ca 2+ response is orchestrated in space and time, we implement optogenetic systems to probe the role of individual β cells in the glucose response. By targeted β cell activation/inactivation in zebrafish, we reveal a hierarchy of cells, each with a different level of influence over islet-wide Ca 2+ dynamics. First-responder β cells lie at the top of the hierarchy, essential for initiating the first-phase Ca 2+ response. Silencing first responders impairs the Ca 2+ response to glucose. Conversely, selective activation of first responders demonstrates their increased capability to raise pan-islet Ca 2+ levels compared to followers. By photolabeling and transcriptionally profiling β cells that differ in their thresholds to a glucose-stimulated Ca 2+ response, we highlight vitamin B6 production as a signature pathway of first responders. We further define an evolutionarily conserved requirement for vitamin B6 in enabling the Ca 2+ response to glucose in mammalian systems.

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic β-cells. β-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between β-cells via gap junctions. Here, we identify the importance of signaling between β-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult β-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between β-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating β-cells., (© 2022. The Author(s).)

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo β-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both β- and δ1-cells. The transcriptomic analysis of β-cell regeneration reveals that β/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of β-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of β-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The pancreatic β-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in β-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, β-cells experience an influx of Ca 2+ . Thus, imaging the glucose-stimulated Ca 2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying β-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca 2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how β-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate β-cell activity in vivo. Here, a detailed protocol for Ca 2+ imaging and glucose stimulation to investigate β-cell function in vivo is presented. This technique allows to monitor the coordinated Ca 2+ dynamics in β-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect β-cell function.

Aims/hypothesis: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B., Methods: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry., Results: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca 2+ . Binding partners for both included secretory-granule-localised PTPRN2/phogrin., Conclusions/interpretation: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans., Data Availability: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.

Currently, we are experiencing a true pandemic of a communicable disease by the virus SARS-CoV-2 holding the whole world firmly in its grasp. Amazingly and unfortunately, this virus uses a metabolic and endocrine pathway via ACE2 to enter our cells causing damage and disease. Our international research training programme funded by the German Research Foundation has a clear mission to train the best students wherever they may come from to learn to tackle the enormous challenges of diabetes and its complications for our society. A modern training programme in diabetes and metabolism does not only involve a thorough understanding of classical physiology, biology and clinical diabetology but has to bring together an interdisciplinary team. With the arrival of the coronavirus pandemic, this prestigious and unique metabolic training programme is facing new challenges but also new opportunities. The consortium of the training programme has recognized early on the need for a guidance and for practical recommendations to cope with the COVID-19 pandemic for the community of patients with metabolic disease, obesity and diabetes. This involves the optimal management from surgical obesity programmes to medications and insulin replacement. We also established a global registry analyzing the dimension and role of metabolic disease including new onset diabetes potentially triggered by the virus. We have involved experts of infectious disease and virology to our faculty with this metabolic training programme to offer the full breadth and scope of expertise needed to meet these scientific challenges. We have all learned that this pandemic does not respect or heed any national borders and that we have to work together as a global community. We believe that this transCampus metabolic training programme provides a prime example how an international team of established experts in the field of metabolism can work together with students from all over the world to address a new pandemic., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line that monitors pax2a expression in the thyrocytes. A population of pax2a-low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a-high and pax2a-low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population., (© 2020 The Authors.)

During embryogenesis, beta-cells arise from the dorsal and ventral bud originating in the endoderm germ layer. As the animal develops to adulthood, the beta-cell mass dramatically increases. The expansion of the beta-cell population is driven by cell division among the embryonic beta-cells and supplanted by neogenesis from post-embryonic progenitors. Here, we describe a protocol for multicolor clonal analysis in zebrafish to define the contribution of individual embryonic beta-cells to the increase in cell numbers. This technique provides insights into the proliferative history of individual beta-cells in an islet. This insight helps in defining the replicative heterogeneity among individual beta-cells during development. Additionally, the ability to discriminate individual cells based on unique color signatures helps quantify the volume occupied by beta-cells and define the contribution of cellular size to the beta-cell mass.

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca 2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca 2+ waves emanated from temporally defined leader β-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory β-cells within coordinated islet networks in vivo. This population may drive Ca 2+ dynamics and pulsatile insulin secretion.

In the past years, evidence has emerged that hallmarks of human metabolic disorders can be recapitulated in zebrafish using genetic, pharmacological or dietary interventions. An advantage of modeling metabolic diseases in zebrafish compared to other "lower organisms" is the presence of a vertebrate body plan providing the possibility to study the tissue-intrinsic processes preceding the loss of metabolic homeostasis. While the small size of zebrafish is advantageous in many aspects, it also has shortcomings such as the difficulty to obtain sufficient amounts for biochemical analyses in response to metabolic challenges. A workshop at the European Zebrafish Principal Investigator meeting in Trento, Italy, was dedicated to discuss the advantages and disadvantages of zebrafish to study metabolic disorders. This perspective article by the participants highlights strategies to achieve improved tissue-resolution for read-outs using "nano-sampling" approaches for metabolomics as well as live imaging of zebrafish expressing fluorescent reporter tools that inform on cellular or subcellular metabolic processes. We provide several examples, including the use of reporter tools to study the heterogeneity of pancreatic beta-cells within their tissue environment. While limitations exist, we believe that with the advent of new technologies and more labs developing methods that can be applied to minimal amounts of tissue or single cells, zebrafish will further increase their utility to study energy metabolism.

Islet inflammation and cytokine production are implicated in pancreatic β-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing β-cells from inflammatory damage. Closing this clinical gap requires the establishment of new disease models of islet inflammation to facilitate screening efforts aimed at identifying new protective agents. Here, we have developed a genetic model of Interleukin-1β (Il-1β)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of β-cells and in vivo drug discovery. Live imaging of immune cells and β-cells in our model revealed dynamic migration, increased visitation and prolonged macrophage retention in the islet, together with robust activation of NF-κB signalling in β-cells. We find that Il-1β-mediated inflammation does not cause β-cell destruction but, rather, it impairs β-cell function and identity. In vivo , β-cells exhibit impaired glucose-stimulated calcium influx and reduced expression of genes involved in function and maturity. These defects are accompanied by α-cell expansion, glucose intolerance and hyperglycemia following a glucose challenge. Notably, we show that a medicinal plant derivative (wedelolactone) is capable of reducing the immune-cell infiltration while also ameliorating the hyperglycemic phenotype of our model. Importantly, these anti-diabetic properties in zebrafish are predictive of wedelolactone's efficacy in protecting rodent and human islets from cytokine-induced apoptosis. In summary, this new zebrafish model of diabetes opens a window to study the interactions between immune and β-cells in vivo , while also allowing the identification of therapeutic agents for protecting β-cells from inflammation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Age-associated deterioration of cellular physiology leads to pathological conditions. The ability to detect premature aging could provide a window for preventive therapies against age-related diseases. However, the techniques for determining cellular age are limited, as they rely on a limited set of histological markers and lack predictive power. Here, we implement GERAS (GEnetic Reference for Age of Single-cell), a machine learning based framework capable of assigning individual cells to chronological stages based on their transcriptomes. GERAS displays greater than 90% accuracy in classifying the chronological stage of zebrafish and human pancreatic cells. The framework demonstrates robustness against biological and technical noise, as evaluated by its performance on independent samplings of single-cells. Additionally, GERAS determines the impact of differences in calorie intake and BMI on the aging of zebrafish and human pancreatic cells, respectively. We further harness the classification ability of GERAS to identify molecular factors that are potentially associated with the aging of beta-cells. We show that one of these factors, junba, is necessary to maintain the proliferative state of juvenile beta-cells. Our results showcase the applicability of a machine learning framework to classify the chronological stage of heterogeneous cell populations, while enabling detection of candidate genes associated with aging.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Malformations of the intrahepatic biliary structure cause cholestasis, a liver pathology that corresponds to poor bile flow, which leads to inflammation, fibrosis, and cirrhosis. Although the specification of biliary epithelial cells (BECs) that line the bile ducts is fairly well understood, the molecular mechanisms underlying intrahepatic biliary morphogenesis remain largely unknown. Wnt/β-catenin signaling plays multiple roles in liver biology; however, its role in intrahepatic biliary morphogenesis remains unclear. Using pharmacological and genetic tools that allow one to manipulate Wnt/β-catenin signaling, we show that in zebrafish both suppression and overactivation of Wnt/β-catenin signaling impaired intrahepatic biliary morphogenesis. Hepatocytes, but not BECs, exhibited Wnt/β-catenin activity; and the global suppression of Wnt/β-catenin signaling reduced Notch activity in BECs. Hepatocyte-specific suppression of Wnt/β-catenin signaling also reduced Notch activity in BECs, indicating a cell nonautonomous role for Wnt/β-catenin signaling in regulating hepatic Notch activity. Reducing Notch activity to the same level as that observed in Wnt-suppressed livers also impaired biliary morphogenesis. Intriguingly, expression of the Notch ligand genes jag1b and jag2b in hepatocytes was reduced in Wnt-suppressed livers and enhanced in Wnt-overactivated livers, revealing their regulation by Wnt/β-catenin signaling. Importantly, restoring Notch activity rescued the biliary defects observed in Wnt-suppressed livers., Conclusion: Wnt/β-catenin signaling cell nonautonomously controls Notch activity in BECs by regulating the expression of Notch ligand genes in hepatocytes, thereby regulating biliary morphogenesis. (Hepatology 2018;67:2352-2366)., (© 2017 by the American Association for the Study of Liver Diseases.)

A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the developmental origin of cells remains challenging. Here we present LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)-a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα -expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signaling hi cells also exhibit premature upregulation of socs2 , an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age., Competing Interests: SJ, SS, AT, SM, PM, JK, SB, DH, GR, GB, NN No competing interests declared, (© 2018, Janjuha et al.)

On 11 January 1922 insulin injection was used for the first time in the treatment of diabetes. Even today, daily insulin injections are the life-saving treatment for patients with Type 1 diabetes and advanced Type 2 diabetes. However, insulin injections often fail to achieve full glucose control, which in the long-term leads to multiple complications and mortality. Beta-cells, the natural producers and secretors of insulin, remain the gold-standard in regulating blood glucose levels. In this review, we focus on three strategies aiming at counteracting beta-cell loss in order to gain insulin independence: replacement, replication and protection. The three approaches, together termed as the triumvirate of beta-cell regeneration, may constitute the basis for a future cure for diabetes.

Traditionally, the development of novel therapeutics for metabolic diseases has relied mainly on high-throughput screening using biochemical or cell-based assays. While this approach represents a driving force in drug discovery, there is also a need to perform large-scale screens without disrupting inter-organ communication and tissue architecture, essential components for understanding the complexity of metabolic regulation and the identification of small molecules with appropriate biological activities in vivo. Hence, the zebrafish Danio rerio is gaining popularity in metabolic research and drug discovery, as this animal model allows screening of small molecules in the context of the whole-organism. Moreover, the zebrafish exhibits conserved function of the pancreas, liver and adipose tissue, which can be leveraged to identify novel targets in metabolic regulation, as well as to study the role of conserved genes associated with the risk of metabolic diseases in humans. Here we highlight recent advances in the identification of targets in metabolic regulation using the zebrafish as a model., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Upon mild liver injury, new hepatocytes originate from preexisting hepatocytes. However, if hepatocyte proliferation is impaired, a manifestation of severe liver injury, biliary epithelial cells (BECs) contribute to new hepatocytes through BEC dedifferentiation into liver progenitor cells (LPCs), also termed oval cells or hepatoblast-like cells (HB-LCs), and subsequent differentiation into hepatocytes. Despite the identification of several factors regulating BEC dedifferentiation and activation, little is known about factors involved in the regulation of LPC differentiation into hepatocytes during liver regeneration. Using a zebrafish model of near-complete hepatocyte ablation, we show that bone morphogenetic protein (Bmp) signaling is required for BEC conversion to hepatocytes, particularly for LPC differentiation into hepatocytes. We found that severe liver injury led to the up-regulation of genes involved in Bmp signaling, including smad5, tbx2b, and id2a, in the liver. Bmp suppression did not block BEC dedifferentiation into HB-LCs; however, the differentiation of HB-LCs into hepatocytes was impaired due to the maintenance of HB-LCs in an undifferentiated state. Later Bmp suppression did not affect HB-LC differentiation but increased BEC number through proliferation. Notably, smad5, tbx2b, and id2a mutants exhibited similar liver regeneration defects as those observed in Bmp-suppressed livers. Moreover, BMP2 addition promoted the differentiation of a murine LPC line into hepatocytes in vitro., Conclusions: Bmp signaling regulates BEC-driven liver regeneration through smad5, tbx2b, and id2a: it regulates HB-LC differentiation into hepatocytes through tbx2b and BEC proliferation through id2a; our findings provide insights into promoting innate liver regeneration as a novel therapy. (Hepatology 2017;66:1616-1630)., (© 2017 by the American Association for the Study of Liver Diseases.)

The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.Βeta-cells have recently been shown to be heterogeneous with regard to morphology and function. Here, the authors show that β-cells in zebrafish switch from proliferative to functional states with increasing time since β-cell birth, leading to functional and proliferative heterogeneity.

The pancreatic beta-cells control glucose homeostasis by secreting insulin in response to nutrient intake. The number of beta-cells is under tight metabolic control, as this number increases with higher nutrient intake. However, the signaling pathways matching nutrition with beta-cell mass plasticity remain poorly defined. By applying pharmacological and genetic manipulations, we show that reactive oxygen species (ROS) regulate dose-dependently beta-cell proliferation in vivo and in vitro. In particular, reducing ROS levels in beta-cells blocks their proliferation in response to nutrients. Using a non-invasive genetic sensor of intracellular hydrogen peroxide (H 2 O 2 ), we reveal that glucose can directly increase the levels of H 2 O 2 . Furthermore, a moderate increase in H 2 O 2 levels can stimulate beta-cell proliferation. Interestingly, while high H 2 O 2 levels are inhibitory to beta-cell proliferation, they expand beta-cell mass in vivo by inducing rapid beta-cell neogenesis. Our study thus reveals a ROS-level-dependent mechanism linking nutrients with beta-cell mass plasticity. Hence, given the requirement of ROS for beta-cell mass expansion, antioxidant therapies should be applied with caution in diabetes.

The fifth EMBO conference on 'The Molecular and Cellular Basis of Regeneration and Repair' took place in the peaceful coastal town of Sant Feliu de Guixols (Spain) on September 2014. The meeting was organised by Emili Saló (U. Barcelona, Spain), Kimberly Mace (U. Manchester, UK), Patrizia Ferretti (University College London, UK) and Michael Brand (Centre for Regenerative Therapies Dresden, Germany) and received the generous support of Society for Developmental Biology, The Company of Biologists, Centre for Regenerative Therapies Dresden, Garland Science and the journals Regeneration and Cell Signalling. The natural surroundings provided an inspiring setting for 185 researchers from all over the world to share their latest findings and views on the field. The conference showcased the great diversity of model organisms used for studying regeneration and tissue repair, including invertebrate and vertebrate species (Fig. 1). Importantly, this diversity in animal models allowed for a global overview of the mechanisms that promote regeneration. In addition, it highlighted some of the unique aspects that confer differences in regenerative capacities among different species. These differences might lie in each of the different steps involved in performing regeneration, including triggering the regenerative response, controlling cellular plasticity, re-stablishing the correct tissue patterns, as well as determining the roles of extrinsic factors, such as the role of inflammation in regeneration. A deeper understanding of these processes in the naturally regenerating species is a prerequisite for advancing the field of regenerative medicine and tissue repair in humans.

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

Background & Aims: Biliary epithelial cells (BECs) are considered to be a source of regenerating hepatocytes when hepatocyte proliferation is compromised. However, there is still controversy about the extent to which BECs can contribute to the regenerating hepatocyte population, and thereby to liver recovery. To investigate this issue, we established a zebrafish model of liver regeneration in which the extent of hepatocyte ablation can be controlled., Methods: Hepatocytes were depleted by administration of metronidazole to Tg(fabp10a:CFP-NTR) animals. We traced the origin of regenerating hepatocytes using short-term lineage-tracing experiments, as well as the inducible Cre/loxP system; specifically, we utilized both a BEC tracer line Tg(Tp1:CreER(T2)) and a hepatocyte tracer line Tg(fabp10a:CreER(T2)). We also examined BEC and hepatocyte proliferation and liver marker gene expression during liver regeneration., Results: BECs gave rise to most of the regenerating hepatocytes in larval and adult zebrafish after severe hepatocyte depletion. After hepatocyte loss, BECs proliferated as they dedifferentiated into hepatoblast-like cells; they subsequently differentiated into highly proliferative hepatocytes that restored the liver mass. This process was impaired in zebrafish wnt2bb mutants; in these animals, hepatocytes regenerated but their proliferation was greatly reduced., Conclusions: BECs contribute to regenerating hepatocytes after substantial hepatocyte depletion in zebrafish, thereby leading to recovery from severe liver damage., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Obese individuals exhibit an increase in pancreatic β cell mass; conversely, scarce nutrition during pregnancy has been linked to β cell insufficiency in the offspring [reviewed in 1, 2]. These phenomena are thought to be mediated mainly through effects on β cell proliferation, given that a nutrient-sensitive β cell progenitor population in the pancreas has not been identified. Here, we employed the fluorescent ubiquitination-based cell-cycle indicator system to investigate β cell replication in real time and found that high nutrient concentrations induce rapid β cell proliferation. Importantly, we found that high nutrient concentrations also stimulate β cell differentiation from progenitors in the intrapancreatic duct (IPD). Furthermore, using a new zebrafish line where β cells are constitutively ablated, we show that β cell loss and high nutrient intake synergistically activate these progenitors. At the cellular level, this activation process causes ductal cell reorganization as it stimulates their proliferation and differentiation. Notably, we link the nutrient-dependent activation of these progenitors to a downregulation of Notch signaling specifically within the IPD. Furthermore, we show that the nutrient sensor mechanistic target of rapamycin (mTOR) is required for endocrine differentiation from the IPD under physiological conditions as well as in the diabetic state. Thus, this study reveals critical insights into how cells modulate their plasticity in response to metabolic cues and identifies nutrient-sensitive progenitors in the mature pancreas., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Genetic studies have implicated Notch signaling in the maintenance of pancreatic progenitors. However, how Notch signaling regulates the quiescent, proliferative or differentiation behaviors of pancreatic progenitors at the single-cell level remains unclear. Here, using single-cell genetic analyses and a new transgenic system that allows dynamic assessment of Notch signaling, we address how discrete levels of Notch signaling regulate the behavior of endocrine progenitors in the zebrafish intrapancreatic duct. We find that these progenitors experience different levels of Notch signaling, which in turn regulate distinct cellular outcomes. High levels of Notch signaling induce quiescence, whereas lower levels promote progenitor amplification. The sustained downregulation of Notch signaling triggers a multistep process that includes cell cycle entry and progenitor amplification prior to endocrine differentiation. Importantly, progenitor amplification and differentiation can be uncoupled by modulating the duration and/or extent of Notch signaling downregulation, indicating that these processes are triggered by distinct levels of Notch signaling. These data show that different levels of Notch signaling drive distinct behaviors in a progenitor population.

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies., Competing Interests: The authors have declared that no competing interests exist.

Tissue remodeling in development and disease involves the coordinated invasion of neighboring territories and/or the replacement of entire cell populations. Cell guidance, cell matching, transitions from passive to migratory epithelia, cell growth and death, and extracellular matrix remodeling all impinge on epithelial spreading. Significantly, the extracellular signals that direct these activities and the specific cellular elements and mechanisms regulated by these signals remain in most cases to be identified. To address these issues, we performed an analysis of histoblasts (Drosophila abdominal epithelial founder cells) on their transition from a dormant state to active migration replacing obsolete larval epidermal cells (LECs). We found that during expansion, Decapentaplegic (Dpp) secreted from surrounding LECs leads to graded pathway activation in cells at the periphery of histoblast nests. Across nests, Dpp activity confers differential cellular behavior and motility by modulating cell-cell contacts, the organization and activity of the cytoskeleton, and histoblast attachment to the substrate. Furthermore, Dpp also prevents the premature death of LECs, allowing the coordination of histoblast expansion to LEC delamination. Dpp signaling activity directing histoblast spreading and invasiveness mimics transforming growth factor-beta and bone morphogenetic proteins' role in enhancing the motility and invasiveness of cancer cells, resulting in the promotion of metastasis., (2010 Elsevier Ltd. All rights reserved.)

Although the molecular elements controlling cell cycle progression are well established, the mechanisms regulating how cell proliferation is triggered in response to extrinsic stimuli and how cell divisions change speed, particularly in stem or tumor cells or regenerative tissues, are poorly understood. One exceptional model system in which these events are precisely defined is Drosophila abdominal morphogenesis, in which stem-like histoblasts build the adult epidermis at metamorphosis by undergoing a series of sequential transitions from a non-proliferative to a growing, and finally to an invasive epithelium. We have recently uncovered in histoblasts an internal logic modulating cell cycle transitions that should constitute a reference paradigm for the study of other equivalent processes in stem cell, cancer or developmental biology.

Regulation of cell proliferation has been extensively studied in cultured cell systems that are characterized by coordinated growth and cell-cycle progression and relatively uniform cell size distribution. During the development of multicellular organisms, however, growth and division can be temporally uncoupled, and the signaling pathways that regulate these growth programs are poorly understood. A good model for analyzing proliferation control in such systems is the morphogenesis of the Drosophila adult abdominal epidermis by histoblasts. These cells undergo a series of temporally regulated transitions during which neither cell size nor division rate is constant. The proliferation of histoblasts during metamorphosis is uniquely amenable to clonal analysis in combination with live imaging. Thereby, we show that abdominal histoblasts, which grow while in G2 arrest during larval stages, enter a proliferative stage in the pupal period that is initiated by ecdysone-dependent string/Cdc25 phosphatase transcription. The proliferating histoblasts have preaccumulated stores of Cyclin E, which trigger an immediate S phase onset after mitosis. These rapid cell cycles lack a G1 phase and result in a progressive reduction of cell size. Eventually, the histoblasts proceed to a stage of slower proliferation that, in contrast to the preceding, depends on epidermal growth factor receptor (EGFR) signaling for progression through the G2/M transition and on insulin receptor/PI3K-mediated signaling for growth. These results uncover the developmentally programmed changes coupling the growth and proliferation of the histoblasts that form the abdominal epidermis of Drosophila. Histoblasts proceed through three distinct stages: growth without division, division without growth, and growth-coupled proliferation. Our identification of the signaling pathways and cell-cycle regulators that control these programs illustrates the power of in vivo time-lapse analyses after clone induction. It sets the stage for the comprehensive understanding of the coordination of cell growth and cell-cycle progression in complex multicellular eukaryotes., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The fusion of epithelial sheets is an essential morphogenetic event. Here, we study the development of the abdomen of Drosophila as a model of bounded epithelia expansion and uncover a complex multistep process for the generation of the adult epidermis from histoblasts, founder cells that replace the larval cells during metamorphosis. We find that histoblasts experience a biphasic cell cycle and emit apical projections that direct their invasive planar intercalation in between larval cells. Coordinately, the larval cells extrude from the epithelia by apical constriction of an actomyosin ring and as a consequence die by apoptosis and are removed by circulating haemocytes. We demonstrate that the proliferation of histoblasts and the death of larval cells are triggered by two independent extrinsic Ecdysone hormonal pulses. Finally, we show that histoblast spreading and the death of larval cells depend on a mutual exchange of signals and are non-autonomous processes.

During larval stages of Drosophila development, the abdominal epidermis is composed of histoblasts (adult precursors) and larval epidermal cells (LECs). During metamorphosis, histoblasts proliferate and colonize the territories occupied by the LECs, which die and become engulfed by macrophages. This morphogenetic process is an excellent model for in vivo analysis of epithelial migration, cell division, cell death, patterning and differentiation. Here, we describe a protocol for time-lapse recording of the developing epidermis during metamorphosis. The protocol describes the removal of the pupal case (which acts as an opaque barrier to effective imaging) and mounting and imaging of specimens of different stages so that normal developmental processes are preserved. This method enables high-resolution studies over long time periods using fluorescent markers and confocal microscopy. The protocol requires 1 h for pupal dissection and mounting and, depending on the stages and genotypes to be analyzed, several more hours for preprocessing and aging and developmental staging of flies and pupae.

Hydrolysate was obtained through enzyme hydrolysis from waste residual fibrin in the production of hyperimmune swine blood. Fibrin hydrolysate and gelatose in 2.5 : 1.5 ratio were tested as protective medium for the freeze-drying of VR2 vaccine against swine erysipelas. It was found that the freeze-drying of the vaccine with the addition of the hydrolysate-gelatose medium at the rate of 4-5 per cent of the vaccine volume met the requirements of the standard for sterility, innocuity, vaccinal titer, and residual moisture.

A method has been developed for discovering of antibody covered bacteria in the urine by means of a coagglutination test with a protein A containing strain--St. aureus Cowan's I. The test is based on the ability of staphylococcal protein A to bind Fc-fragment of IgG. As a specificity control St. aureus Wood 46 was used. 38 patients with urinary infections were examined. As pyelonephritic criteria the following were considered: febrile episodes, lumber pains, polyuria, pollakiuria, leukocyturia, proteinuria, raised arterial pressure, anemia, diminished renal function or chronic renal failure, x-ray, ultrasound and isotopic-nephrographic changes. In the presence of antibody covered bacteria the test is positive--there is coagglutination only with st. aureus Cowan's I. If the bacteria are not antibody coated no coagglutination takes place. If there is coagglutination with both strains the reaction is considered non-specific. II samples gave non-specific reaction. In 75% of the pyelonephritic patients the test was positive. In 70% of the patients with urinary infections of the lower urinary tract, i.e. with bacteria without immunoglobulins, the test was negative. In 74.1% of the cases there is a correlation between the coagglutination test and the localization of the urinary infection by means of other clinical and paraclinical methods. It is suggested that the coagglutination test should be included in the examination of patients with urinary infections, the positive test indicates renal localization of the infection.

The level of the IgM and IgG agglutinating antibody specific hyperimmune serum against coli-bacteriosis and its fractions were studied. The serum was obtained at various stages of the preparation, hyperimmunization and exploitation of serum producing swine by using the 2-mercapto-ethanol test. It was established that serum of the total globulin, alpha-beta-globulin and gamma-globulin fractions contains IgM and IgG agglutinating antibodies against E. coli. These immunoglobulin classes are synthetized at all stages of serum producing swine preparation, hyperimmunization and exploitation. The quantitative IgM:IgG ratios at the individual stages vary. At the initial stages and until the 10th cycle of exsanguination (phlebotomy) IgM antibodies predominate. After that, at the following stages IgG antibodies begin gradually to predominate. Up to the 20th cycle of exsanguination both globulins are synthetized, but the 2-mercapto-ethanol non-sensitive IgG antibodies are in greater quantity.

Comparative testing of the intensity and the continuity of immunity against swine fever was carried out with pigs producers of serum against edema disease and swine erysipelas, vaccinated with an untreated and a freeze-dried K vaccine. Control infection studies revealed that serum-producing pigs with regard to edema disease, immunized with a freeze-dried and an untreated lapinized K vaccine, could acquire sufficiently stable immunity from the 6th to the 9th month following immunization both in the beginning and at the end of the exploitation period. So far as the intensity and the continuity of immunity of pigs producing serum against erysipelas are concerned the animals were shown to acquire sufficiently stable immunity against swine fever when vaccination was carried out with a freeze-dried K vaccine, while not all of the pigs treated with an untreated K vaccine were protected from swine fever upon control challenging. Therefore, instead of immunization with an untreated K vaccine as many as 400 immunization doses of the freeze-dried K vaccine could be used.

An easy and rapid staphylococcal coagulation test is described for detection of bacteria, covered with antibodies in urine of patients with chronic pyelonephritis as an additional index of the etiological role of the isolated microorganisms and localization of the pathological process. Inactivated and stabilized protein A containing strain S. aureus Cowan's (SSM 2352) was used. The coagulation test is negative in infectious processes of the lower segments of urinary system and bacterial contamination of urine.

A live vaccine against coli enteritis in pigs, CA-80, with a protection medium of 5 per cent hemodex, was successfully freeze-dried in ampoules or vials at the rate of 5 to 10 vaccinal doses each, the titer ranging from 10(9) to 10(10) live bacteria per cu. cm, of a 12-month shelf-life period. The vaccine was found to be innocuous for pigs, and was intended for active immunoprophylaxis on both infected and menaced farms and complexes, applying it to sows in the last third of the gestation period.