Generation of a Slc39a8 hypomorph mouse: markedly decreased ZIP8 Zn²?/(HCO??)? transporter expression.
ABSTRACT: Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn(2+)/(HCO(3)(-))(2) symporter, moving both ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.
Project description:Slc39a8 encodes ZIP8, a divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cells, and therefore ubiquitous in adult tissues; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice exhibit 10-15% of wild-type ZIP8 mRNA and protein levels, and show pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed RNA-seq analysis of gestational day (GD)13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8(neo/neo) with Slc39a8(+/+) wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues - having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and coagulation cascade - consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, we identified numerous genes encoding zinc-finger and other TFs associated with hematopoietic stem cell functions. We conclude that, in this mouse model, deficient ZIP8-mediated divalent cation transport affects zinc-finger (e.g. GATA proteins) and other TFs interacting with GATA proteins (e.g. TAL1), predominantly in yolk sac. These data strongly support the phenotype of dysmorphogenesis and anemia seen in Slc39a8(neo/neo) mice in utero.
Project description:Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.
Project description:SLC39A8 is an evolutionarily highly conserved gene that encodes the ZIP8 metal cation transporter in all vertebrates. SLC39A8 is ubiquitously expressed, including pluripotent embryonic stem cells; SLC39A8 expression occurs in every cell type examined. Uptake of ZIP8-mediated Mn2+, Zn2+, Fe2+, Se4+, and Co2+ represents endogenous functions-moving these cations into the cell. By way of mouse genetic differences, the phenotype of "subcutaneous cadmium-induced testicular necrosis" was assigned to the Cdm locus in the 1970s. This led to identification of the mouse Slc39a8 gene, its most closely related Slc39a14 gene, and creation of Slc39a8-overexpressing, Slc39a8(neo/neo) knockdown, and cell type-specific conditional knockout mouse lines; the Slc39a8(-/-) global knockout mouse is early-embryolethal. Slc39a8(neo/neo) hypomorphs die between gestational day 16.5 and postnatal day 1-exhibiting severe anemia, dysregulated hematopoiesis, hypoplastic spleen, dysorganogenesis, stunted growth, and hypomorphic limbs. Not surprisingly, genome-wide association studies subsequently revealed human SLC39A8-deficiency variants exhibiting striking pleiotropy-defects correlated with clinical disorders in virtually every organ, tissue, and cell-type: numerous developmental and congenital disorders, the immune system, cardiovascular system, kidney, lung, liver, coagulation system, central nervous system, musculoskeletal system, eye, and gastrointestinal tract. Traits with which SLC39A8-deficiency variants are currently associated include Mn2+-deficient hypoglycosylation; numerous birth defects; Leigh syndrome-like mitochondrial redox deficiency; decreased serum high-density lipoprotein-cholesterol levels; increased body mass index; greater risk of coronary artery disease, hypotension, cardiovascular death, allergy, ischemic stroke, schizophrenia, Parkinson disease, inflammatory bowel disease, Crohn disease, myopia, and adolescent idiopathic scoliosis; systemic lupus erythematosus with primary Sjögren syndrome; decreased height; and inadvertent participation in the inflammatory progression of osteoarthritis.
Project description:Zinc ion (Zn2+) is essential for life; its deficiency in the human body could cause stunted growth, anemia and susceptibility to infection. The Zn transporter ZIP8 (also known as SLC39A8) is an important Zn2+ importer; aberrant Zn2+ influx mediated by ZIP8 can lead to the pathogenesis of osteoarthritis and inflammatory diseases. ZIP8 also mediates the cellular uptake of divalent metal ions including iron, manganese, and the toxic heavy metal cadmium. Individuals with SLC39A8 mutations and transgenic mouse models are starting to reveal the critical role that this gene plays in embryonic development and the metabolism of essential metal ions. Here we summarize our current understanding of ZIP8's function and regulation, at both the molecular and biological levels. We also review the association of ZIP8 with various diseases and its linkage with complex disorders like obesity, hypertension, and schizophrenia as revealed by several large genome-wide association studies.
Project description:Slc39a8 encodes ZIP8, a ubiquitous divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cell; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice globally exhibit 10-15% of wild-type ZIP8 mRNA and protein levels, and show a pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed transcriptomics of GD13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8(neo/neo) with wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues –– having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and the coagulation cascade –– consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, numerous genes encoding zinc-finger TFs and associated with hematopoietic stem cell functions were identified. We conclude that, in this mouse model, deficient ZIP8-mediated Zn2+ transport affects zinc-finger (e.g. GATA proteins) and other transcription-factors (e.g. TAL1) predominantly in yolk sac, strongly supporting the observed dysmorphogenesis and anemia phenotype. Overall design: mRNA samples of Slc39a8(+/+) and Slc39a8(neo/neo) yolk sacs and placentas at GD13.5, fetal liver, kidney, lung, heart and cerebellum at GD16.5 were profiled in triplicate using Illumina HiSeq.
Project description:Abstract Objectives The SLC39A8 gene encodes a divalent metal transporter, ZIP8. ZIP8 polymorphisms are associated with pleiotropic effects including altered risks for schizophrenia. Our objective is to determine the different brain MRI phenotypes associated at or near the SLC39A8 (ZIP8) genetic locus using a phenome-wide association (PheWAS) approach followed by joint and conditional association analysis. Methods Using the summary statistics database containing brain MRI genome-wide association study (GWAS) data, we systematically selected all brain MRI phenotypes which were associated with single-nucleotide polymorphisms (SNPs) within 1 million bp of the SLC39A8 genetic locus, as defined as reaching a P-value significance cutoff of P < 5.0 × 10–8. For all brain MRI phenotypes reaching significance, we used GCTA-COJO to determine the number of independent association signals using settings of P < 1.0 × 10–5 and a window of 10 million base pairs. Using SNPclip and the European 1000 Genomes linkage panel with a linkage disequilibrium cutoff of r2 > 0.8, we identified SNP candidates at each index SNP. Linkage equilibrium for brain phenotypes with multiple independent signals was confirmed by LDpair. Results We identified 25 brain MRI phenotypes that vary due to MRI type and brain region that all contain a SNP associated with the SLC39A8 locus. All of these datasets have at least 1 index SNP directly labeling or in high linkage disequilibrium with rs13107325, which encodes a missense mutation in the SLC39A8 (ZIP8). Among the 25 datasets, an additional 4 association signals were identified by GCTA-COJO and confirmed to be in linkage equilibrium with rs13107325 using LDpair. For these additional association signals, probable causative SNPs were identified from the index SNP using SNPclip. Conclusions From the 25 brain MRI phenotypes, we identified new probable causative SNPs in addition to a previously reported missense SNP (rs13107325) associated with schizophrenia. This study provides leads into how SNPs in genes involved in trace metal transport influence brain structures and affect risks for schizophrenia. Funding Sources This work was funded by grants from the Oklahoma Center for the Advancement of Science and Technology and the Oklahoma Agricultural Experiment Station.
Project description:Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.
Project description:Gene expression profiling of primary mouse articular chondrocyte infected with recombinant adenovirus expressing the zinc transporter ZIP8 (SLC39A8) protein. In this study, we have attempted to explore the effects of ZIP8 overexpression on mouse transcriptome and have identified numerous genes which are involved in osteoarthritis pathogenesis. Overall design: Primary mouse articular chondrocytes were isolated from mouse femoral condyles and tibial plateaus cartilage tissue (WT ICR 5-day-old mice) and were infected with null(control) or recombinant adenovirus expressing zinc transporter ZIP8 for 24 hours. At 24 hours post-infection, total RNA was extracted from infected with null(control) adenovirus (4 biological replicates) or infected with recombinant adenovirus expressing the zinc transporter ZIP8 (4 biological replicates) on primary mouse articular chondrocytes and subjected to microarray analysis.
Project description:Testicular necrosis is a sensitive endpoint for cadmium (Cd(2+), Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.
Project description:Mouse Slc39a8 and Slc39a14 genes encode ZIP8 and ZIP14, respectively, which are ubiquitous divalent cation/(HCO3-)2 symporters responsible for uptake of Zn2+, Fe2+, and Mn2+ into cells. Cd2+ and other toxic nonessential metals can displace essential cations, thereby entering vertebrate cells. Whereas Slc39a8 encodes a single protein, Slc39a14 has 2 exons 4 which, via alternative splicing, give rise to ZIP14A and ZIP14B; why differences exist in cell type-specific expression of ZIP14A and ZIP14B remains unknown. Inflammatory stimuli have been associated with upregulation of ZIP8 and ZIP14, but a systematic study of many tissues simultaneously in a laboratory animal following inflammatory cytokine exposure has not yet been reported. Herein, we show that C57BL/6J male mice--treated intraperitoneally with lipopolysaccharide or the proinflammatory cytokines tumor necrosis factor (TNF) or interleukin-6 (IL6)--exhibited quantatively very different, highly tissue-specific, and markedly time-dependent up- and downregulation of ZIP8, ZIP14A, and ZIP14B messenger RNA (mRNA) levels in 12 tissues. The magnitude of inflammatory response was confirmed by measuring the proinflammatory cytokine TNF, IL6, and interleukin-1? mRNA levels in the same tissues of these animals. Our data suggest that most if not all tissues use ZIP8, ZIP14A, and/or ZIP14B for Zn2+ uptake, some tissues under basal conditions and others moreso when inflammatory stressors are present; collectively, this might lead to substantial alterations in plasma Zn2+ levels due to Zn2+ redistribution not just in liver but across many vital organs. In the context of cadmium-mediated toxicity, our data suggest that tissues other than liver, kidney, and lung should also be considered.