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      Hrg1 promotes heme-iron recycling during hemolysis in the zebrafish kidney

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          Abstract

          Heme-iron recycling from senescent red blood cells (erythrophagocytosis) accounts for the majority of total body iron in humans. Studies in cultured cells have ascribed a role for HRG1/SLC48A1 in heme-iron transport but the in vivo function of this heme transporter is unclear. Here we present genetic evidence in a zebrafish model that Hrg1 is essential for macrophage-mediated heme-iron recycling during erythrophagocytosis in the kidney. Furthermore, we show that zebrafish Hrg1a and its paralog Hrg1b are functional heme transporters, and genetic ablation of both transporters in double knockout ( DKO) animals shows lower iron accumulation concomitant with higher amounts of heme sequestered in kidney macrophages. RNA-seq analyses of DKO kidney revealed large-scale perturbation in genes related to heme, iron metabolism and immune functions. Taken together, our results establish the kidney as the major organ for erythrophagocytosis and identify Hrg1 as an important regulator of heme-iron recycling by macrophages in the adult zebrafish.

          Author summary

          Total body iron stores in mammals is a composite of iron absorption from diet and iron recycled by macrophages from dying red blood cells (RBCs). Upon erythrophagocytosis of RBCs, the hemoglobin is degraded and heme is imported from the phagosomal compartment into the cytoplasm so that the iron can be released from heme. Defects in these pathways can lead to aberrant iron homeostasis. The Heme Responsive Gene-1 (HRG1, SLC48A1) was identified previously as a heme importer in the intestine of the roundworm, Caenorhabditis elegans. In cell culture studies, HRG1 was demonstrated to mobilize heme from the erythrophagosome of mouse macrophages into the cytosol. However, the in vivo function of HRG1 remains to be elucidated. The zebrafish is a powerful genetic animal model for studying vertebrate development and ontogeny of hematopoiesis. In zebrafish, the kidney marrow is the adult hematopoietic organ that is functionally analogous to the mammalian bone marrow. In this study, we show that Hrg1 plays an essential in vivo role in recycling of damaged RBCs, and that the kidney macrophages are primarily responsible for recycling heme-iron in the adult zebrafish.

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          Most cited references35

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          Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

          A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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            Transformation of intact yeast cells treated with alkali cations.

            Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.
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              Zebrafish hox clusters and vertebrate genome evolution.

              HOX genes specify cell fate in the anterior-posterior axis of animal embryos. Invertebrate chordates have one HOX cluster, but mammals have four, suggesting that cluster duplication facilitated the evolution of vertebrate body plans. This report shows that zebrafish have seven hox clusters. Phylogenetic analysis and genetic mapping suggest a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-finned and lobe-finned fishes but before the teleost radiation. Thus, teleosts, the most species-rich group of vertebrates, appear to have more copies of these developmental regulatory genes than do mammals, despite less complexity in the anterior-posterior axis.
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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Software
                Role: Formal analysisRole: MethodologyRole: Resources
                Role: Formal analysisRole: Funding acquisitionRole: ResourcesRole: SoftwareRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                24 September 2018
                September 2018
                : 14
                : 9
                : e1007665
                Affiliations
                [1 ] Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, Maryland, United States of America
                [2 ] Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney, Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
                [3 ] Division of Hematology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America
                Marine Biological Laboratory, UNITED STATES
                Author notes

                IH is the President and Founder of Rakta Therapeutics Inc. (College Park, MD), a company involved in the development of heme transporter-related diagnostics. He declares no other competing financial interests.

                Author information
                http://orcid.org/0000-0003-2928-2227
                http://orcid.org/0000-0003-1948-3690
                http://orcid.org/0000-0003-1567-1678
                http://orcid.org/0000-0001-6127-3940
                http://orcid.org/0000-0003-0045-0610
                Article
                PGENETICS-D-18-00461
                10.1371/journal.pgen.1007665
                6171960
                30248094
                4583db98-3614-4e33-bc27-09081d37eaf5

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 9 March 2018
                : 28 August 2018
                Page count
                Figures: 5, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases;
                Award ID: DK85035
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases;
                Award ID: Intramural grant
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000062, National Institute of Diabetes and Digestive and Kidney Diseases;
                Award ID: DK090257
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000066, National Institute of Environmental Health Sciences;
                Award ID: ES025661
                Award Recipient :
                This work was supported by funding from the National Institutes of Health DK85035 (IH); support was also provided by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases (MK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Model Organisms
                Zebrafish
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Animal Models
                Zebrafish
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Fish
                Osteichthyes
                Zebrafish
                Biology and Life Sciences
                Anatomy
                Renal System
                Kidneys
                Medicine and Health Sciences
                Anatomy
                Renal System
                Kidneys
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Heme
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Embryos
                Biology and Life Sciences
                Physiology
                Immune Physiology
                Spleen
                Medicine and Health Sciences
                Physiology
                Immune Physiology
                Spleen
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and Life Sciences
                Genetics
                Gene Expression
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-10-04
                All the sequencing data including read counts per gene were deposited to GEO with the accession number of GSE109978. All other relevant data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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