The Zinc Transporter SLC39A10 Plays an Essential Role in Embryonic Hematopoiesis.

The role of zinc in hematopoiesis is currently unclear. Here, SLC39A10 (ZIP10) is identified as a key zinc transporter in hematopoiesis. The results show that in zebrafish, Slc39a10 is a key regulator of the response to zinc deficiency. Surprisingly, both slc39a10 mutant zebrafish and hematopoietic Slc39a10-deficient mice develop a more severe form of impaired hematopoiesis than animals lacking transferrin receptor 1, a well-characterized iron gatekeeper, indicating that zinc plays a larger role than iron in hematopoiesis, at least in early hematopoietic stem cells (HSCs). Furthermore, it is shown that loss of Slc39a10 causes zinc deficiency in fetal HSCs, which in turn leads to DNA damage, apoptosis, and G1 cell cycle arrest. Notably, zinc supplementation largely restores colony formation in HSCs derived from hematopoietic Slc39a10-deficient mice. In addition, inhibiting necroptosis partially restores hematopoiesis in mouse HSCs, providing mechanistic insights into the requirement for zinc in mediating hematopoiesis. Together, these findings indicate that SLC39A10 safeguards hematopoiesis by protecting against zinc deficiency-induced necroptosis, thus providing compelling evidence that SLC39A10 and zinc homeostasis promote the development of fetal HSCs. Moreover, these results suggest that SLC39A10 may serve as a novel therapeutic target for treating anemia and zinc deficiency-related disorders.

Dietary Intake Levels of Iron, Copper, Zinc, and Manganese in Relation to Cognitive Function: A Cross-Sectional Study.

Background: Previous studies have related circulating levels of trace metal elements, of which dietary intake is the major source, to cognitive outcomes. However, there are still relatively few studies evaluating the associations of dietary intake levels of iron, copper, zinc, and manganese with cognitive function (CF). Methods: We leveraged the data of 6863 participants (mean [standard deviation] age = 66.7 [10.5] years) in the Health and Retirement Study (2013/2014). Dietary intake levels of iron, copper, zinc, and manganese were calculated from a semi-quantitative food frequency questionnaire. CF was assessed using the 27-point modified Telephone Interview for Cognitive Status (TICS). We used linear regression models to calculate the mean differences in global CF scores by quintiles of dietary intake levels of trace metal elements. Results: Among the study participants, the mean (SD) values of daily dietary intake were 13.3 (6.3) mg for iron, 1.4 (0.7) mg for copper, 10.7 (4.6) mg for zinc, and 3.3 (1.6) mg for manganese. Compared with the lowest quintile of dietary iron intake (<8.1 mg), the highest quintile (≥17.7 mg) was associated with a lower cognitive score (-0.50, -0.94 to -0.06, P-trend = 0.007). Higher dietary copper was significantly associated with poorer CF (P-trend = 0.002), and the mean difference in cognitive score between extreme quintiles (≥1.8 vs. <0.8 mg) was -0.52 (95% confidence interval: -0.94 to -0.10) points. We did not observe significant associations for dietary intake of zinc (P-trend = 0.785) and manganese (P-trend = 0.368). Conclusion: In this cross-sectional study, higher dietary intake of iron and copper was related to worse CF, but zinc and manganese intake levels were not significantly associated with CF.

Zinc transporter Slc30a1 regulates melanocyte development by interacting with mt2 in zebrafish.

The essential trace element zinc is involved in multiple biological processes including development and metabolism, while its role in melanocyte formation is still unclear. Slc30a1a and Slc30a1b are zinc exporters in zebrafish. Here, we found that melanocytes were increased in slc30a1a and slc30a1b double mutant zebrafish. SMART-seq data revealed that genes involved in the melanoma pathway and the gene mt2, which encodes zinc-binding protein, were significantly upregulated in the mutants. In addition, the expression of mt2 was specifically increased in mutant melanocytes, as detected by in situ hybridization, suggesting an essential role of this gene in the tissue. Mechanistically, we demonstrated that elevated zinc levels resulting from Slc30a1 deficiency promoted melanocyte proliferation and that mt2 played a protective role in the process of Slc30a1/zinc-mediated melanocyte hyperplasia. This study uncovered the critical function of Slc30a1-mediated zinc homeostasis in melanocyte development and suggests that accumulated zinc in melanocytes would be a risk for inducing melanoma and that mt2 is a potential target for controlling diseases related to abnormal melanocyte development.

Manganese homeostasis at the host-pathogen interface and in the host immune system.

Manganese serves as an indispensable catalytic center and the structural core of various enzymes that participate in a plethora of biological processes, including oxidative phosphorylation, glycosylation, and signal transduction. In pathogenic microorganisms, manganese is required for survival by maintaining basic biochemical activity and virulence; in contrast, the host utilizes a process known as nutritional immunity to sequester manganese from invading pathogens. Recent epidemiological and animal studies have shown that manganese increases the immune response in a wide range of vertebrates, including humans, rodents, birds, and fish. On the other hand, excess manganese can cause neurotoxicity and other detrimental effects. Here, we review recent data illustrating the essential role of manganese homeostasis at the host-pathogen interface and in the host immune system. We also discuss the accumulating body of evidence that manganese modulates various signaling pathways in immune processes. Finally, we discuss the key molecular players involved in manganese's immune regulatory function, as well as the clinical implications with respect to cancer immunotherapy.

Metal transporter Slc30a1 controls pharyngeal neural crest differentiation via the zinc-Snai2-Jag1 cascade.

The pharyngeal arch (PA) is a neural crest (NC)-derived organ that is transiently developed during embryogenesis and is required for the subsequent development of various tissues. However, the role of zinc during PA differentiation from NC progenitor cells is unknown. Here, we found that the metal transporters Slc30a1a and Slc30a1b mediate zinc homeostasis during PA differentiation. Slc30a1-deficient zebrafish develop zinc accumulation in NC cells, with increased expression of stemness markers and PA dorsal genes, and SMART-seq analyses revealed that the genes snai2 and jag1b may serve as downstream targets. Furthermore, functional studies showed that knocking down either snai2 or jag1b rescues PA development in Slc30a1-deficient zebrafish. Notably, we identified the double zinc-finger domain in the transcription factor Snai2 as a zinc-responsive element that regulates jag1b expression. Our findings indicate that the Slc30a1/zinc-snai2-jag1b axis is an essential regulatory network controlling PA differentiation, shedding new light on the function of zinc homeostasis in maintaining NC cell stemness and multipotency in vertebrates.

Interactions between methanotrophs and ammonia oxidizers modulate the response of in situ methane emissions to simulated climate change and its legacy in an acidic soil.

Methane (CH4) is one of the most important greenhouse gases which can be formed by methanogens and oxidized by methanotrophs, as well as ammonia oxidizers. Agricultural soils can be both a source and sink for atmospheric CH4. However, it is unclear how climate change, will affect CH4 emissions and the underlying functional guilds. In this field study, we determined the impact of simulated climate change (a warmer and drier condition) and its legacy effect on CH4 emissions and the methanogenic and methanotrophic communities, as well as their relationships with ammonia oxidizers in an acidic soil with urea application. The climate change conditions were simulated in a greenhouse, and the legacy effect was simulated by removing the greenhouse after twelve months. Simulated climate change significantly decreased the in situ CH4 emissions in the urea-treated soils while the legacy effect significantly decreased the in situ CH4 emissions in the control plots, but had very little effect in the urea-treated soils. This indicates that the impact of simulated climate change and its legacy on CH4 emissions was significantly modified by nitrogen fertilization. Methanotrophs were more sensitive than methanogens in response to simulated climate change and its legacy effect, especially in the urea treated soil. Significant negative correlations were observed between the abundances of ammonia oxidizers and methanotrophs. Additionally, results of partial least path modeling (PLS-PM) indicated that the interactions of methanogens and methanotrophs with ammonia oxidizing archaea (AOA) had significant positive relationships with in situ CH4 emissions under the simulated climate change condition. Our work highlights the important role of AOA for CH4 emissions under climate change conditions. Further research is needed to better understand this effect in other ecosystems.

Slc39a5-mediated zinc homeostasis plays an essential role in venous angiogenesis in zebrafish.

Angiogenesis is a precise process mediated by a variety of signals and the environmental niche. Although the essential trace element zinc and its homeostasis are essential for maintaining proper cellular functions, whether zinc plays a role in angiogenesis is currently unknown. Using zebrafish embryos as a model system, we found that zinc treatment significantly increased the expression of the slc39a5 gene, which encodes the zinc transporter Slc39a5. Moreover, knocking down slc39a5 expression using either a morpholino or CRISPR/Cas9-mediated gene editing led to cardiac ischaemia and an accumulation of red blood cells in the caudal vein plexus (CVP), as well as delayed venous sprouting and fewer vascular loops in the CVP region during early development. Further analysis revealed significantly reduced proliferation and delayed cell migration in the caudal vein of slc39a5 morphants. At the mechanistic level, we found increased levels of systemic zinc in slc39a5-deficient embryos, and chelating zinc restored CVP development. In addition, we found that zinc overload in wild-type embryos leads to impaired CVP formation. Taken together, these results indicate that Slc39a5 plays a critical role in endothelial sprouting and migration in venous angiogenesis by regulating zinc homeostasis.

Smad7 deficiency decreases iron and haemoglobin through hepcidin up-regulation by multilayer compensatory mechanisms.

To maintain iron homoeostasis, the iron regulatory hormone hepcidin is tightly controlled by BMP-Smad signalling pathway, but the physiological role of Smad7 in hepcidin regulation remains elusive. We generated and characterized hepatocyte-specific Smad7 knockout mice (Smad7Alb/Alb ), which showed decreased serum iron, tissue iron, haemoglobin concentration, up-regulated hepcidin and increased phosphor-Smad1/5/8 levels in both isolated primary hepatocytes and liver tissues. Increased levels of hepcidin lead to reduced expression of intestinal ferroportin and mild iron deficiency anaemia. Interestingly, we found no difference in hepcidin expression or phosphor-Smad1/5/8 levels between iron-challenged Smad7Alb/Alb and Smad7flox/flox , suggesting other factors assume the role of iron-induced hepcidin regulation in Smad7 deletion. We performed RNA-seq to identify differentially expressed genes in the liver. Significantly up-regulated genes were then mapped to pathways, revealing TGF-ß signalling as one of the most relevant pathways, including the up-regulated genes Smad6, Bambi and Fst (Follistatin). We found that Smad6 and Bambi-but not Follistatin-are controlled by the iron-BMP-Smad pathway. Overexpressing Smad6, Bambi or Follistatin in cells significantly reduced hepcidin expression. Smad7 functions as a key regulator of iron homoeostasis by negatively controlling hepcidin expression, and Smad6 and Smad7 have non-redundant roles. Smad6, Bambi and Follistatin serve as additional inhibitors of hepcidin in the liver.

Characterization of acrylamide-induced oxidative stress and cardiovascular toxicity in zebrafish embryos.

Acrylamide (AA) is a high production volume chemical in industrial applications and widely found in baked or fried carbohydrate-rich foods. In this study, we unravelled that AA induced developmental toxicity associated with oxidative stress status and disordered lipid distribution in heart region of developing zebrafish. Treatment with AA caused a deficient cardiovascular system with significant heart malformation and dysfunction. We also found that AA could reduce the number of cardiomyocytes through the reduced capacity of cardiomyocyte proliferation rather than cell apoptosis. The cardiac looping and ballooning appeared abnormal though cardiac chamber-specific identity in the differentiated myocardium was maintained well after AA treatment through MF20/S46 immunofluorescence assay. Furthermore, treatment with AA disturbed the differentiation of atrioventricular canal, which was demonstrated by the disordered expressions of the atrioventricular boundary markers bmp4, tbx2b and notch1b and further confirmed by the ectopic expressions of the cardiac valve precursor markers has2, klf2a and nfatc1 through whole-mount in situ hybridization. Thus, our studies provide the evidence of cardiac developmental toxicity of AA in the cardiovascular system, and also raised health concern about the harm of trans-placental exposure to high level of AA for foetuses and the risk of high exposure to AA for the pregnant women.

Exposure to acrylamide induces cardiac developmental toxicity in zebrafish during cardiogenesis.

Acrylamide (AA), an environmental pollutant, has been linked to neurotoxicity, genotoxicity and carcinogenicity. AA is widely used to synthesize polymers for industrial applications, is widely found in Western-style carbohydrate-rich foods and cigarette smoke, and can also be detected in human umbilical cord blood and breast milk. This is the first study that demonstrated the cardiac developmental toxicity of AA in zebrafish embryos. Post-fertilization exposure to AA caused a clearly deficient cardiovascular system with a shrunken heart and abortive morphogenesis and function. Disordered expression of the cardiac genes, myl7, vmhc, myh6, bmp4, tbx2b and notch1b, as well as reduced number of myocardial cells and endocardial cells, indicated the collapsed development of ventricle and atrium and failed differentiation of atrioventricular canal (AVC). Although cell apoptosis was not affected, the capacity of cardiomyocyte proliferation was significantly reduced by AA exposure after fertilization. Further investigation showed that treatment with AA specifically reduced the expressions of nkx2.5, myl7 and vmhc in the anterior lateral plate mesoderm (ALPM) during the early cardiogenesis. In addition, AA exposure disturbed the restricted expressions of bmp4, tbx2b and notch1b during atrioventricular (AV) valve development and cardiac chambers maturation. Our results showed that AA-induced cardiotoxicity was related to decreased cardiac progenitor genes expression, reduced myocardium growth, abnormal cardiac chambers morphogenesis and disordered AVC differentiation. Our study demonstrates that AA exposure during a time point analogous to the first trimester in humans has a detrimental effect on early heart development in zebrafish. A high ingestion rate of AA-containing products may be an underlying risk factor for cardiogenesis in fetuses.

Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

[Effect of oxidative damage on expression of myocardin and NF-E2-related factor 2 in myocardial cells].

With the oxidative damage model established in rat myocardial cells by hydrogen peroxide (H 2O 2), the expression of myocardin and nuclear factor erythroid 2-related factor 2 (Nrf2) during oxidative damage and effect of myocardin on Nrf2 were preliminarily explored. The expression of the target gene was increased or decreased by transfection of plasmid DNA or shRNA, respectively. Cell proliferation was detected by sulforhodamine B (SRB) assay. The expression of myocardin mRNA and Nrf2 mRNA was detected by Real-time PCR, and their protein levels were detected by Western blot. The results showed that oxidative damage was induced by H 2O 2 with an optimized incubation condition of 200 µmol/L H 2O 2 for 24 hours. H 2O 2 inhibited expression of myocardin in mRNA and protein levels, and increased expression of Nrf2 in mRNA and protein levels. The overexpression of myocardin or the knockdown of Nrf2 significantly decreased cell viability compared with the control group, while the knockdown of myocardin or the overexpression of Nrf2 significantly increased cell viability. The overexpression of myocardin significantly down-regulated the expression of Nrf2 in mRNA and protein levels, while the knockdown of myocardin dramatically up-regulated the expression of Nrf2. Thus, it is deduced that myocardin may inhibit cell proliferation and Nrf2 may promote cell proliferation. Oxidative damage induced by H 2O 2 in rat myocardial cell might activate Nrf2-related signaling pathway through down-regulation of myocardin.

Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism.

Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases.

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish.

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.

ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.