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.

Circulating factors cause proteinuria in parabiotic zebrafish.

Proteinuria can be induced by impairment of any component of the glomerular filtration barrier (GFB). To determine the role of circulating permeability factors on glomerular damage, we developed a parabiosis-based zebrafish model to generate a common circulation between zebrafish larvae. A morpholino-mediated knockdown of a podocyte specific gene (nephronectin) was induced in one zebrafish larva which was then fused to an un-manipulated fish. Notably, proteinuria and glomerular damage were present in the manipulated fish and in the parabiotically-fused partner. Thus, circulating permeability factors may be induced by proteinuria even when an induced podocyte gene dysregulation is the initiating cause.

Cadmium exposure exacerbates severe hyperlipidemia and fatty liver changes in zebrafish via impairment of high-density lipoproteins functionality.

Cadmium (Cd) is a heavy metal with several toxicities that have destructive effect on most organ systems. However, its toxic effects on human lipoproteins are largely remained unknown especially in hyperlipidemic zebrafish model. Treatment of human high-density lipoprotein (HDL) with cadmium chloride (CdCl2, final 12 and 24µM) caused spontaneous formation of multimeric apoA-I as well as increased production of glycated extent products. Cd-HDL3 accelerated uptake of oxidized LDL (oxLDL) into macrophages and induced severe senescence in human dermal fibroblast (HDF) cells. Microinjection of Cd-HDL3 into zebrafish embryos resulted in acute embryonic toxicity with high mortality. Exposure of zebrafish embryos to water containing CdCl2 (final 12 and 24µM) caused early embryonic death along with increased production of oxidized products and impairment of skeletal development. Consumption of CdCl2 (12 and 24µM) by zebrafish for 4weeks resulted in severe elevation of plasma total cholesterol (TC) and triglyceride (TG) levels as well as cholesteryl ester (CE) transfer activity. Furthermore, consumption of CdCl2 resulted in acceleration of fatty liver changes and increased production of reactive oxygen species (ROS). In conclusion, CdCl2 caused structural modification of HDL3 and impaired the beneficial functions of HDL3, including anti-oxidation, anti-atherosclerosis, and anti-senescence effects. Consumption of CdCl2 also resulted in exacerbated hyperlipidemia and fatty liver changes in zebrafish via enhancement of cholesteryl ester transfer protein (CETP) activity.

Autotaxin-Lysophosphatidic Acid Axis Acts Downstream of Apoprotein B Lipoproteins in Endothelial Cells.

OBJECTIVE: As they travel through the blood stream, plasma lipoproteins interact continuously with endothelial cells (ECs). Although the focus of research has mostly been guided by the importance of lipoproteins as risk factors for atherosclerosis, thrombosis, and other cardiovascular diseases, little is known about the mechanisms linking lipoproteins and angiogenesis under physiological conditions, and particularly, during embryonic development. In this work, we performed global mRNA expression profiling of endothelial cells from hypo-, and hyperlipidemic zebrafish embryos with the goal of uncovering novel mediators of lipoprotein signaling in the endothelium. APPROACH AND RESULTS: Microarray analysis was conducted on fluorescence-activated cell sorting-isolated fli1:EGFP(+) ECs from normal, hypo-, and hyperlipidemic zebrafish embryos. We found that opposed levels of apoprotein B lipoproteins result in differential expression of the secreted enzyme autotaxin in ECs, which in turn affects EC sprouting and angiogenesis. We further demonstrate that the effects of autotaxin in vivo are mediated by lysophosphatidic acid (LPA)-a well-known autotaxin activity product-and that LPA and LPA receptors participate as well in the response of ECs to lipoprotein levels. CONCLUSIONS: Our findings provide the first in vivo gene expression profiling of ECs facing different levels of plasma apoprotein B lipoproteins and uncover a novel lipoprotein-autotaxin-LPA axis as regulator of EC behavior. These results highlight new roles for lipoproteins as signaling molecules, which are independent of their canonical function as cholesterol transporters.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1.

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

Adequate Th2-type response associates with restricted bacterial growth in latent mycobacterial infection of zebrafish.

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.

Novel blood collection method allows plasma proteome analysis from single zebrafish.

Zebrafish is an important model organism in biological research. One of the least explored tissues of zebrafish is blood, because the existing methods for isolating blood from this organism are tedious and irreproducible. The small volume of blood collected by these methods also prohibits many biochemical and cytological analyses. This technical obstacle limits the utilization of zebrafish in many applications, particularly in hematological research and plasma biomarker discovery. To overcome this limitation, we have established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a wound. This method consistently recovers more blood than traditional methods. Gel electrophoresis and flow cytometry showed that composition of blood harvested by this method is indistinguishable from traditional methods. The increase in yield enables us to perform biochemical experiments on zebrafish blood. In particular, we have demonstrated that quantitative proteomics can be performed on plasma collected from single zebrafish. Here, we have compared, by using shotgun proteomic analysis, the plasma proteomes of adult male and female zebrafish. Twenty-seven gender-dependent plasma proteins are identified and their biochemical importance discussed. Taken together, this novel technique enables analyses that were previously difficult to perform on zebrafish blood.

Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation.

BACKGROUND: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. METHODS AND RESULTS: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. CONCLUSION: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Deficiency of ribosomal protein S19 during early embryogenesis leads to reduction of erythrocytes in a zebrafish model of Diamond-Blackfan anemia.

Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79 ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in blood cells as well as deformities in the head and tail regions at early developmental stages. These phenotypes were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hematopoietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valuable tool for investigating the molecular mechanisms of DBA development in humans.

A global role for zebrafish klf4 in embryonic erythropoiesis.

There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions.

Evaluation of the estrogenic potential of river and treated waters in the Paris area (France) using in vivo and in vitro assays.

For many years, surface waters have been shown to be contaminated by endocrine-disrupting compounds (EDCs), which can cause adverse effects on human and wildlife growth, development, and reproduction. It is therefore of primary importance to determine if drinking water could be contaminated by EDCs when produced from polluted surface waters. It is also essential to determine if disinfection by-products can account for estrogenic activity in treated waters. The estrogenic potential of river and treated waters was investigated using an in vivo assay. Adult male zebrafish were placed in three drinking water treatment plants (DWTPs) in the Paris area and exposed for 1 month to the two types of waters. After exposure, vitellogenin (VTG) was measured in the plasma of fish using a competitive ELISA. In addition, an in vitro assay (MELN cells) was used to assess the estrogenic potential of 10 major chlorination by-products. No significant induction of VTG was observed in fish exposed to river or treated waters. Among the 10 chlorination by-products tested, only 2-chlorophenol was found to be weakly estrogenic at concentrations up to 1mg/L. Therefore, the risk for the three DWTPs studied to produce drinking water with significant level of estrogenic substances appears to be low.

Xenoestrogenic effects of ethinylestradiol in zebrafish (Danio rerio).

To assess the estrogenic effects of ethinylestradiol on zebrafish, zebrafish at different developmental stages (embryos, juveniles, and adults) were exposed to the synthetic hormone ethinylestradiol (EE2) in concentrations of 1, 10, and 100 ng/L for up to 33 days. Survival, hatching, length, weight, growth, condition, hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) production were examined. Exposure of zebrafish juveniles and embryos to 100 ng EE2/L for up to 33 days had significant effects on survival, growth, and hatching. Two VTG fragments with molecular weights of approximately 140 and 170 kDa were detected with protein electrophoresis and Western blotting in the blood of exposed males and exposed and unexposed females, as well as in whole-body homogenates of exposed and unexposed juveniles. Significantly higher VTG concentrations (compared to controls) were measured in adults exposed to 10 and 100 ng EE2/L for 14 days, but not in fish exposed to 1 ng EE2/L. This study demonstrated that (1) zebrafish juveniles, larvae, and embryos are sensitive to the toxic effects of the endocrine disrupter EE2; (2) the effects on VTG production in adults are detected after exposure to environmentally relevant concentrations of EE2; (3) unexposed juvenile zebrafish produce measurable concentrations of VTG.