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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 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 Ca2+. 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. Graphical abstract

One of the earliest studies on human pancreas regeneration dates from the beginning of the 1900s when the American pathologist Russel Cecil noted signs of attempted islet regeneration in samples from organ donors with diabetes. A century later, the quest is on to enhance the limited regenerative potential of the pancreas in order to develop new therapies for diabetes. While regeneration is incomplete in mammals, zebrafish can naturally recover from extreme β-cell destruction and hyperglycemia by producing new β-cells. Here, we discuss how a unique blend of in vivo imaging, genetics and drug discovery in zebrafish could reveal the secrets of natural β-cell regeneration. Specifically, we explore the essential steps for successful regeneration, from the induction of pancreas plasticity to the integration of cells into networks that establish functional islet communities.

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 develops hepatic cysts during larval stages. Cyst formation in these mutants is 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 an excessive and 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 alters Furinb localization and causes 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 livers also exhibited increased mTOR signaling and treatment with the mTOR inhibitor rapamycin partially blocked cyst formation by reducing ER stress. Our study has established a novel vertebrate model for studying hepatic cystogenesis and illustrated the role of ER stress in the disease pathogenesis.

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 Ca2+. Thus, imaging the glucose-stimulated Ca2+ 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 Ca2+ 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 Ca2+ imaging and glucose stimulation to investigate β-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ 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.

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.

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 use 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 constitute 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

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.

Upon mild liver injury, new hepatocytes originate from pre-existing 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 here show that Bmp signaling is required for BEC conversion to hepatocytes, particularly for LPC differentiation into hepatocytes. We found that severe liver injury led to the upregulation 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 cell line into hepatocytes in vitro. Conclusions: Bmp signaling regulates BEC-driven liver regeneration via smad5, tbx2b and id2a: it regulates HB-LC differentiation into hepatocytes via tbx2b and BEC proliferation via id2a. Our findings provide insights into promoting innate liver regeneration as a novel therapy. This article is protected by copyright. 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 genetic heterogeneity in the thyrocyte population, and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells and fibroblasts. Further, the thyrocytes could be split into two sub-populations with unique transcriptional signature, including differential expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line, which demonstrated specific pax2a expression in the thyrocytes. However, a population of pax2a-low mature thyrocytes interspersed within individual follicles could be distinguished, corroborating heterogeneity within the thyrocyte population. Our results identify and validate transcriptional differences within the nominally homogenous thyrocyte population., info:eu-repo/semantics/published

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.

Satin et al. (1) present a critique of a recent model for “hub/follower” dynamics in intra-islet regulation of insulin secretion presented in two recent articles from our laboratories (2,3). Based on older work and our own demonstrations of heterogeneity between individual β-cells, these studies used a combination of multicellular Ca2+ imaging, network theory, and interventional approaches to demonstrate that a subpopulation of β-cells within the islet displays earlier responses to stimulation with glucose and is required for fully coordinated behavior across sections of the islet. Studies from others using some of these approaches have reached broadly similar conclusions (4,5). The critique of Satin et al. (1) is based in large …

Aim: In-vitro studies reveal a subpopulation of β cells which co-ordinate insulin release from pancreatic islets. This connectivity has not been established in 3 dimensions across the islet or in the living animal. We investigated this in the living zebrafish D. rario or in mouse islets engrafted in the anterior eye chamber (ACE), using the recombinant Ca2+ sensor GCaMP6 under the insulin promoter/C57Bl6, Ins1Cre:GCaM6ff/f. Methods: Whole islet live imaging of D. rario primary islets in situ at an acquisition rate of 0.8Hz, covering 700μm, was achieved using resonant scanner technology with a Leica inverted laser scanning confocal system, using a 25X/N.A. 0.95 water lens. Fully vascularized islets in the murine ACE were imaged under isofluorane anaesthesia on a modified Nikon Ti-E spinning disc confocal microscope (20x 0.75 NA water immersion objective, 3 Hz frame rate). Results: 3-dimensional β cell connectivity (Pearson R statistic) significantly rose to 88% across the zebrafish islet in response to glucose stimulation. Focused ablation of the co-ordinating “hub” (but not follower) β cells significantly attenuated the subsequent response to a glucose challenge. In mouse islets, high circulating glucose levels were also associated with a significant rise in 3D beta cell connectivity from 65 to 86% (n=3; p=0.02). Granger causality analysis on these islets revealed that even on prolonged (>10 minute) glucose stimulation, pan-islet connectivity was sustained and the most highly connected cells were those in the region of the islet from which Ca2+ waves emanated. Binarized signal analysis (n=6) separately confirmed the presence of a highly-connected β cell subpopulation (8.7 ± 3.6% of cells). Conclusions: These studies evidence beta cell “hubs” which are responsible for the initiation and sustained coordination of the Ca2+ response to glucose across fish and mouse islets in vivo. Disclosure V. Salem: None. L.F. Delgadillo Silva: None. K. Suba: None. A. Martin Alonso: None. W. Kim: None. V. Kalogianni: None. N. Akhtar: None. N. Mousavy Gharavy: None. E. Georgiadou: None. I. Leclerc: None. L. Briant: None. D. Hodson: None. W. Distaso: None. N.N. Ninov: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding Diabetes UK; UK Wellcome Trust

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 Ca2+ 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, Ca2+ 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 Ca2+ dynamics and pulsatile insulin secretion., info:eu-repo/semantics/published

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.

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.

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

The negative demographic trend over the last more than two decades in the Republic of Bulgaria raises the question of the reasons for the falling birth rate in the country. Some of these reasons led to the negative natural growth are purely negative. Some couples give up the opportunity for second and following children due to the fact that, growing them is related to newly created additional, often significant family budget expenses. An alternative to overcome the situation concerning the demographic decline in the country is the use of economic incentives to young families for child’s birth and childcare. Payments to which parents are entitled under the social security system (in case they are insured persons) and the social assistance system (in case they are uninsured persons) unfortunately are of small size that does not allow full satisfaction of newly emerging needs. In the process of clarifying this type of payment, it is logical to conclude that it is necessary to develop the potential of additional activities such as insurance.

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 trans criptomes. 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 predictive power of GERAS to identify genome-wide molecular factors that correlate with aging. We show that one of these factors, junb, 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 to detect pro-aging factors and candidate genes associated with aging.

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 Salo (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.

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.

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.

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.

SummaryObese 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.

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).

Report.-- et al., Tissue remodeling in development and disease [[1] and [2]] 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 [[3] and [4]]) 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-β and bone morphogenetic proteins' role in enhancing the motility and invasiveness of cancer cells, resulting in the promotion of metastasis [[5] and [6]]., We thank the Vienna Drosophila RNAi Center, the Bloomington Stock Center, and the Transgenic RNAi Resource Project at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing fly stocks used in this study. N.N. held a Formación de Profesorado Universitario PhD studentship, C.P.-R. held a “La Caixa” master fellowship, and S.M.-C. is supported by a PhD fellowship from the Fundação para a Ciência e a Tecnologia (Portugal). N.N. would like to thank U. Tepass for his assistance and support. Research in E.M.-B.'s laboratory is funded by grants from the Spanish Ministry of Science and the European Union. Research in M.A.'s laboratory is supported by the Swiss National Science Foundation, Kanton Basel-Stadt, Kanton Basel-Landschaft, and the European Union.

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.

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.

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.

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.

12 pages, 7 figures.-- PMID: 19355788 [PubMed].-- Supporting information (Suppl. figures S1-S3, movies S1-S7) available at: http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.1000079#toclink6, 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., [Author summary] A fundamental issue in biology is the question of how the rate of cell division is coupled to cell growth. Coordination of these processes has been studied extensively in cultured cell systems but to a much lesser extent in intact organisms. To study this phenomenon in a physiological setting, we developed a methodology to observe and manipulate cell division and growth in a population of Drosophila abdominal cells called histoblasts. The various developmental stages of histoblast morphogenesis include exit from cell-cycle arrest, initially rapid growth in the absence of cell division, and subsequent coupling of proliferation and growth. We identified several critical developmental signaling pathways (including signaling via ecdysone, the EGF receptor, and PI 3-kinase) that regulate and coordinate cell growth and division cycles during these different types of cell-cycle phenomena. We propose that the internal logic of the Drosophila histoblast system may serve as a basic framework for understanding not only how coordinated cell growth and division operate in a number of other developmental contexts, but also how misregulation of cell growth and division occurs in contexts such as cancer cell populations., NN was supported by a Formación de Profesorado Universitario (FPU) fellowship from the Spanish Ministry of Education and Science (MEC) and CM by the "Juan de la Cierva" MEC program. Work in EM-B's lab is supported by grants of the DGICYT (MEC) and the European Union (EU) (WOUND-STREP Project).

According to the World Bank 1.5% of the Bulgarian population lives below $1.90 per day. For a population of Bulgaria as of 31st December 2014, comprising 7 202 198 people, this share covers approximately 108 032 people. At the same time, in relation to the implementation of social assistance activities, according to the Social Assistance Act (SAA) and the Regulation on the Implementation of the Social Assistance Act (RISAA), from the begging of 2014 until the end of December of the same year under the Regulation on the Implementation of the Social Assistance Act with one-of, targeted and monthly social benefits in the country, a total of 63 932 persons and families were supported. This fact demonstrates an alarming trend in particular that not a small number of people remain out of the coverage of the publicly organized social assistance system – approximately 44 100 people, who are not eligible for social benefits for various reasons. All this gives us reason to look for alternative solutions to improve social protection, especially to those being absolutely poor in our country. One of these alternatives, which is widespread in the world and unfortunately almost unknown in our country, is microinsurance. The latter has the potential to increase the social security of those who are in extreme poverty and to expand the activity of the insurance companies in Bulgaria.