Optogenetic ß cell interrogation in vivo reveals a functional hierarchy directing the Ca2+ response to glucose supported by vitamin B6.

Coordination of cellular activity through Ca2+ enables ß cells to secrete precise quantities of insulin. To explore how the Ca2+ response is orchestrated in space and time, we implement optogenetic systems to probe the role of individual ß cells in the glucose response. By targeted ß cell activation/inactivation in zebrafish, we reveal a hierarchy of cells, each with a different level of influence over islet-wide Ca2+ dynamics. First-responder ß cells lie at the top of the hierarchy, essential for initiating the first-phase Ca2+ response. Silencing first responders impairs the Ca2+ response to glucose. Conversely, selective activation of first responders demonstrates their increased capability to raise pan-islet Ca2+ levels compared to followers. By photolabeling and transcriptionally profiling ß cells that differ in their thresholds to a glucose-stimulated Ca2+ 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 Ca2+ response to glucose in mammalian systems.

Wnt4 is heterogeneously activated in maturing ß-cells to control calcium signaling, metabolism and function.

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic ß-cells. ß-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between ß-cells via gap junctions. Here, we identify the importance of signaling between ß-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 ß-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 ß-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 ß-cells.

A missense mutation in the proprotein convertase gene furinb causes hepatic cystogenesis during liver development in zebrafish.

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.

A single-cell atlas of de novo ß-cell regeneration reveals the contribution of hybrid ß/δ-cells to diabetes recovery in zebrafish.

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 ß-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 ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/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 ß-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 ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo ß-Cell Function.

The pancreatic ß-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ß-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, ß-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying ß-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 ß-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 ß-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate ß-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in ß-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 ß-cell function.

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

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 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.

Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland.

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.

Multicolor Labeling and Tracing of Pancreatic Beta-Cell Proliferation 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.

Nano-Sampling and Reporter Tools to Study Metabolic Regulation in Zebrafish.

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.

Modelling pancreatic ß-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection.

Islet inflammation and cytokine production are implicated in pancreatic ß-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing ß-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-1ß (Il-1ß)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of ß-cells and in vivo drug discovery. Live imaging of immune cells and ß-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 ß-cells. We find that Il-1ß-mediated inflammation does not cause ß-cell destruction but, rather, it impairs ß-cell function and identity. In vivo, ß-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 ß-cells in vivo, while also allowing the identification of therapeutic agents for protecting ß-cells from inflammation.

Leader ß-cells coordinate Ca2+ dynamics across pancreatic islets in vivo.

Pancreatic ß-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 ß-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 ß-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.

Machine learning based classification of cells into chronological stages using single-cell transcriptomics.

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.

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets.

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.

The triumvirate of beta-cell regeneration: solutions and bottlenecks to curing diabetes.

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.

Simultaneous lineage tracing and cell-type identification using CRISPR-Cas9-induced genetic scars.

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.

Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish.

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 signalinghi 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.

Wnt/ß-catenin signaling controls intrahepatic biliary network formation in zebrafish by regulating notch activity.

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/ß-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/ß-catenin signaling, we show that in zebrafish both suppression and overactivation of Wnt/ß-catenin signaling impaired intrahepatic biliary morphogenesis. Hepatocytes, but not BECs, exhibited Wnt/ß-catenin activity; and the global suppression of Wnt/ß-catenin signaling reduced Notch activity in BECs. Hepatocyte-specific suppression of Wnt/ß-catenin signaling also reduced Notch activity in BECs, indicating a cell nonautonomous role for Wnt/ß-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/ß-catenin signaling. Importantly, restoring Notch activity rescued the biliary defects observed in Wnt-suppressed livers. CONCLUSION: Wnt/ß-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).

Catching new targets in metabolic disease with a zebrafish.

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.

Different developmental histories of beta-cells generate functional and proliferative heterogeneity during islet growth.

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 ß-cells in zebrafish switch from proliferative to functional states with increasing time since ß-cell birth, leading to functional and proliferative heterogeneity.