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

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

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

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

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

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

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

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

Inhibition of Cdk5 Promotes ß-Cell Differentiation From Ductal Progenitors.

Inhibition of notch signaling is known to induce differentiation of endocrine cells in zebrafish and mouse. After performing an unbiased in vivo screen of ∼2,200 small molecules in zebrafish, we identified an inhibitor of Cdk5 (roscovitine), which potentiated the formation of ß-cells along the intrapancreatic duct during concurrent inhibition of notch signaling. We confirmed and characterized the effect with a more selective Cdk5 inhibitor, (R)-DRF053, which specifically increased the number of duct-derived ß-cells without affecting their proliferation. By duct-specific overexpression of the endogenous Cdk5 inhibitors Cdk5rap1 or Cdkal1 (which previously have been linked to diabetes in genome-wide association studies), as well as deleting cdk5, we validated the role of chemical Cdk5 inhibition in ß-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of ß-cells independently of inhibition of notch signaling, in both the basal state and during ß-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote ß-cell formation was conserved in mouse embryonic pancreatic explants, adult mice with pancreatic ductal ligation injury, and human induced pluripotent stem (iPS) cells. Thus, we have revealed a previously unknown role of Cdk5 as an endogenous suppressor of ß-cell differentiation and thereby further highlighted its importance in diabetes.

Spatiotemporal expression analysis of nuclear estrogen receptors in the zebrafish ovary and their regulation in vitro by endocrine hormones and paracrine factors.

Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression via nuclear estrogen receptors (nERs) in the zebrafish ovary. We have demonstrated that endocrine hormones such as gonadotropin (hCG) and paracrine factors such as epidermal growth factor (EGF) and pituitary adenylate cyclase-activating peptide (PACAP) can modulate E2-induced lhcgr expression in vitro. These observations raised a question on whether these hormones and factors exert their effects via regulating the expression of nERs. In this study, we first characterized the spatiotemporal expression profiles of three nER subtypes in the zebrafish ovary, including esr1 (ERα), esr2a (ERß2) and esr2b (ERß1). All three nERs increased their expression at the pre-vitellogenic stage and peaked at mid- (esr1 and esr2a) or late vitellogenic (esr2b) stage, followed by a significant decline at the full-grown stage. RT-PCR analysis showed that esr1 and esr2b were exclusively expressed in the follicle layer while esr2a was expressed in both compartments. We then examined how E2, hCG, PACAP and EGF regulated the expression of nERs in cultured zebrafish follicle cells. E2 quickly increased esr1 but reduced esr2a and esr2b expression from 1.5 to 12h of treatment. Similarly, EGF down-regulated esr2a significantly at 1.5h and this effect was further intensified at 24h. hCG decreased the expression of all three nER subtypes with similar potency throughout the 24-h time-course. Interestingly, PACAP exerted a biphasic regulation on esr2a. Our present study suggests that nERs, especially esr2a, provide potential target points for other hormones and factors to modulate E2 activity during folliculogenesis in the zebrafish.

Critical role for adenosine receptor A2a in ß-cell proliferation.

OBJECTIVE: Pharmacological activation of adenosine signaling has been shown to increase ß-cell proliferation and thereby ß-cell regeneration in zebrafish and rodent models of diabetes. However, whether adenosine has an endogenous role in regulating ß-cell proliferation is unknown. The objective of this study was to determine whether endogenous adenosine regulates ß-cell proliferation-either in the basal state or states of increased demand for insulin-and to delineate the mechanisms involved. METHODS: We analyzed the effect of pharmacological adenosine agonists on ß-cell proliferation in in vitro cultures of mouse islets and in zebrafish models with ß- or δ-cell ablation. In addition, we performed physiological and histological characterization of wild-type mice and mutant mice with pancreas- or ß-cell-specific deficiency in Adora2a (the gene encoding adenosine receptor A2a). The mutant mice were used for in vivo studies on the role of adenosine in the basal state and during pregnancy (a state of increased demand for insulin), as well as for in vitro studies of cultured islets. RESULTS: Pharmacological adenosine signaling in zebrafish had a stronger effect on ß-cell proliferation during ß-cell regeneration than in the basal state, an effect that was independent of the apoptotic microenvironment of the regeneration model. In mice, deficiency in Adora2a impaired glucose control and diminished compensatory ß-cell proliferation during pregnancy but did not have any overt phenotype in the basal state. Islets isolated from Adora2a-deficient mice had a reduced baseline level of ß-cell proliferation in vitro, consistent with our finding that UK432097, an A2a-specific agonist, promotes the proliferation of mouse ß-cells in vitro. CONCLUSIONS: This is the first study linking endogenously produced adenosine to ß-cell proliferation. Moreover, we show that adenosine signaling via the A2a receptor has an important role in compensatory ß-cell proliferation, a feature that could be harnessed pharmacologically for ß-cell expansion and future therapeutic development for diabetes.

IGFBP1 increases ß-cell regeneration by promoting α- to ß-cell transdifferentiation.

There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of ß cells in people with diabetes. By performing whole-genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during ß-cell regeneration. We then tested the proteins' ability to potentiate ß-cell regeneration in zebrafish at supraphysiological levels. One protein, insulin-like growth factor (Igf) binding-protein 1 (Igfbp1), potently promoted ß-cell regeneration by potentiating α- to ß-cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1's effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co-expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type-2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of ß-cell regeneration and highlight its clinical importance in diabetes.

Luteinizing hormone receptor (lhcgr) as a marker gene for characterizing estrogenic endocrine-disrupting chemicals in zebrafish ovarian follicle cells.

The adverse effects of endocrine-disrupting chemicals (EDCs) have been well documented; however, the action mechanisms of many EDCs remain elusive and controversial. Furthermore, the highly diversified chemical structures and low environmental concentrations of EDCs present a major challenge to their chemical detection. Clearly, there is an urgent need for simple and reliable bioassays to detect EDCs in the environment and unravel their action mechanisms. We have recently identified luteinizing hormone receptor (lhcgr) as a robust estradiol (E2)-responsive gene in cultured zebrafish ovarian follicle cells. The expression of lhcgr exhibited a distinct biphasic response to E2 over a 24-h time-course treatment, making this a unique system for characterizing estrogenic EDCs. This study was undertaken to validate this platform by testing a wide range of EDCs, including 17α-ethinylestradiol (EE2), diethylstilbestrol (DES), bisphenol A (BPA), genistein (GEN), 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p'-DDT), vinclozolin (VIN), bis(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Diethylstilbestrol (DES), EE2 and o,p'-DDT mimicked E2 and induced a biphasic expression of lhcgr while BPA and GEN stimulated a monophasic expression in the 24-h time-course. In contrast, BDE-47, DEHP and VIN had no effect, whereas TCDD decreased lhcgr expression. Dose-response experiment showed that E2, EE2 and DES had the highest potency, which was followed by GEN, BPA and o,p'-DDT. The effects of estrogenic EDCs were further confirmed by their potentiation of hCG-induced activin ßA2 subunit (inhbab) expression. In conclusion, the present study showed that the expression of lhcgr in cultured zebrafish follicle cells and its biphasic response to estrogens provide a unique in vitro platform for screening and categorizing estrogenic substances and deciphering their action mechanisms.

Evidence for gating roles of protein kinase A and protein kinase C in estradiol-induced luteinizing hormone receptor (lhcgr) expression in zebrafish ovarian follicle cells.

Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression in zebrafish follicle cells via nuclear estrogen receptors (nERs) that are likely expressed on the membrane, and lhcgr responds to E2 in a biphasic manner during 24-h treatment. These observations raise an interesting question on the signaling mechanism underlying E2 regulation, in particular the biphasic response of lhcgr expression. In the present study, we demonstrated that E2 regulation of lhcgr was significantly influenced by the activity of cAMP-PKA pathway. Activation of cAMP-PKA pathway by forskolin or db-cAMP suppressed E2-stimulated lhcgr expression in short-term (3 h) but enhanced its effect in long-term (24 h), suggesting differential roles of PKA at these two phases of lhcgr response. PKA inhibitor H89 showed reversed effects. In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h. One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a. Despite the strong influence of PKA and PKC, our data did not suggest direct mediating roles for these two pathways in E2 stimulation of lhcgr expression; yet they likely play critical gating roles in E2 signal transduction. As a follow-up study to our previous report on E2 regulation of gonadotropin receptors in the zebrafish ovary, the present study provides further evidence for the involvement of classical intracellular signal transduction pathways in E2 stimulation of lhcgr expression in the follicle cells.

Differential regulation of gonadotropin receptors (fshr and lhcgr) by epidermal growth factor (EGF) in the zebrafish ovary.

Epidermal growth factor (egf) is expressed in the zebrafish oocyte whereas its receptor EGF receptor (egfr) is expressed in the somatic follicle layer, strongly suggesting a role for Egf in the intrafollicular paracrine communication that mediates an oocyte-to-follicle cell signaling pathway. However, the exact function of Egf in the follicle remains largely unknown. The present study aimed to explore the possible role of Egf in regulating gonadotropin receptors (fshr and lhcgr) in cultured zebrafish follicle cells. EGF down-regulated lhcgr expression dose-dependently in a biphasic manner with significant effect observed at 1.5 and 24 h. The effect was mediated via Egfr on the follicle cells. On the contrary, EGF also tended to decrease fshr expression at 1.5 h but it appeared to up-regulate fshr at 24 h. The EGF suppression of lhcgr expression was functionally relevant as pre-exposure to EGF reduced the follicle cell responsiveness to LH/hCG. We have recently reported that estradiol (E2) strongly stimulated lhcgr expression in the zebrafish ovary. In the current study, we further demonstrated that EGF and other EGF family members, heparin-binding EGF-like growth factor (HBEGF), transforming growth factor α (TGFα) and betacellulin (BTC), all reduced basal and E2-induced lhcgr expression. This study provides evidence for a potential paracrine role of Egf and its related peptides in the zebrafish follicle. The oocyte-derived EGF family ligands may actively control the process of follicle growth and maturation by differentially controlling the expression of fshr and lhcgr in the follicle cells in a paracrine manner.

Differential regulation of gonadotropin receptors (fshr and lhcgr) by estradiol in the zebrafish ovary involves nuclear estrogen receptors that are likely located on the plasma membrane.

FSH and LH are gonadotropins (GTH) that control all major events of gonadal function. FSH and LH signal through their cognate receptors, FSH receptor and LH/choriogonadotropin receptor, respectively, across vertebrates. Compared with the information in mammals, very little is known about these receptors in fish, especially the regulation of their expression. In female zebrafish, fshr and lhcgr exhibit significant temporal difference in expression, with fshr increasing first when the follicles are activated to enter the vitellogenic growth phase and lhcgr lagging behind. This raises an interesting question on the differential regulation of these two GTH receptors (GTHR) during folliculogenesis. Using a primary follicle cell culture, the present study demonstrated that 17ß-estradiol (E2), but not testosterone, was a potent endocrine hormone that differentially regulated the expression of fshr and lhcgr. Although E2 stimulated both receptors, its effect on the steady-state level of lhcgr mRNA was much higher (>8-fold up-regulation) than that of fshr (∼0.5-fold increase). E2 likely acted at the transcription level via its nuclear estrogen receptors (ERα and ERß), because ICI 182,780 could abolish its effects. However, our evidence suggested that these receptors might be localized on the plasma membrane, because ß-estradiol 6-(O-carboxy methyl)oxime:BSA could fully mimic the effects of E2. Demonstrating that E2 is likely one of the differentiating factors for the distinct expression of the two GTHR in the zebrafish ovary, this study sheds important light on the functions of the two GTH and their receptors in fish as well as the conservation and diverse aspects of GTHR regulation across vertebrates.