The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88-DLA-12 and DLA-88-DLA-88L, were identified, and the novel haplotype DLA-88-DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

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The major histocompatibility complex (MHC) is a highly polymorphic and duplicated genomic region that encodes transplantation and immune regulatory molecules. Although it is well-known that particular MHC allelic polymorphisms and haplotypes are genetically relate to immune-mediated diseases detailed information of the cat MHC (Feline Leukocyte Antigen; FLA) genetic and haplotypic structure and diversity is limited in comparison to humans and many other species. In this study, to better understand the degree and types of allele and allelic haplotype diversity of FLA-class I (FLA-I) and FLA-DRB loci in domestic cats, we identified six expressible FLA-I loci in peripheral white blood cells by in silico estimation of the coding exons and NGSbased amplicon sequencing using five unrelated cats. We then used a newly developed NGS-based genotyping method to genotype and annotate 32 FLA-I and 16 FLA-DRB sequences in two families of 20 domestic cats. A total of 14 FLA-I and seven FLA-DRB were identified as novel polymorphic sequences. Phylogenetic analyses grouped the sequences into six FLA-I (FLA-E/H/K, FLA-A, FLA-J, FLA-L, FLA-O and a tentatively named FLA-E/H/K_Rec) and four FLA-DRB (FLA-DRB1, FLA-DRB3, FLA-DRB4, and FLA-DRB5) lineages. Pedigree analysis of two cat families revealed eight distinct FLA structural haplotypes (Class I-DRB) with five to eight FLA-I and two to three FLA-DRB transcribed loci per haplotype. It is evident that the eight FLA haplotypes were generated by gene duplications and deletions, and rearrangements by genetic recombination with the accumulation and/or inheritance of novel polymorphisms. These findings are useful for further genetic diversity analysis and disease association studies among cat breeds and in veterinary medicine.

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Hematopoietic stem cells (HScs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. it has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. the double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a + runx1 + ) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. transcriptome analysis revealed that gata2a + runx1 + cells showed typical molecular signatures of HScs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. transplantation assays demonstrated that long-term repopulating HScs were highly enriched within the gata2a + runx1 + fraction. in contrast, colony-forming assays showed that gata2a − runx1 + cells abundantly contain erythroid-and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.Hematopoietic stem cells (HSCs) are self-renewing multipotent cells that can generate all types of blood cells over the lifetime of an individual and can be used therapeutically to treat hematopoietic diseases 1 . In the adult, most HSCs present in bone marrow are quiescent and divide rarely under homeostatic conditions. HSCs produce a heterogeneous pool of hematopoietic progenitor cells (HPCs), which have limited or no self-renewal ability, but rapidly proliferate and differentiate to satisfy the requirements for new mature blood cells 2,3 . Although the frequency of HSCs is extremely rare in bone marrow, HSC potential can be evaluated by transplantation assays, whereby the relative hematopoietic reconstitution activity of co-transplanted donor and competitor cells are compared in a recipient 4 . Purification of HSCs from murine and human bone marrow has been facilitated via transplantation assays using combinations of multiple cell-surface markers [5][6][7][8][9] . Studies in mice revealed that a single CD150 + CD34 − c-kit + Sca-1 + Lineage-marker − cell in the bone marrow showed long-term and multilineage hematopoietic reconstitution following transplantation 10,11 . Prospective isolation of highly purified HSCs thus elucidated many aspects of HSC biology, including self-renewal, differentiation, and HSC niches.The zebrafish is an excellent model for the study of HSCs due to its many unique advantages. Many valuable tools and experimental methods have been established for the study of hematopoietic cells in zebrafish (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.) 12,13 . Moreover, genome-editing technology based on...

Despite 400 million years of evolutionary divergence, hematopoiesis is highly conserved between mammals and teleost fish. All types of mature blood cells including the erythroid, myeloid, and lymphoid lineages show a high degree of similarity to their mammalian counterparts at the morphological and molecular level. Hematopoietic stem cells (HSCs) are cells that are capable of self-renewal and differentiating into all hematopoietic lineages over the lifetime of an organism. The study of HSCs has been facilitated through bone marrow transplantation experiments developed in the mouse model. In the last decade, the zebrafish and clonal ginbuna carp (Carassius auratus langsdorfii) have emerged as new models for the study of HSCs. This review highlights the recent progress and future prospects of studying HSCs in teleost fish.Transplantation assays using these teleost models have demonstrated the presence of HSCs in the kidney, which is the major hematopoietic organ in teleost fish. Moreover, it is possible to purify HSCs from the kidney utilizing fluorescent dyes or transgenic animals. These teleost models will provide novel insights into the universal mechanisms of HSC maintenance, homeostasis, and differentiation among vertebrates.

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Erythropoietin is the principal regulator of erythropoiesis and promotes the survival, proliferation and differentiation of erythroid progenitor cells in mammals. In this study we report on the molecular and functional characterization of erythropoietin from the goldfish. Quantitative expression analysis of goldfish epo revealed the highest mRNA levels in heart, followed by brain, liver, spleen and kidney tissues. There was no marked change of epo expression in goldfish primary kidney macrophage cultures, as progenitor cell to macrophage development progressed, indicating that erythropoietin is not involved in monopoiesis. Recombinant goldfish erythropoietin induced proliferation of progenitor cells in a dose-dependent manner, and up-regulated the expression of erythroid transcription factors gata1 and lmo2 in progenitor cells. Furthermore, recombinant goldfish erythropoietin stimulated erythroid colony formation in a dose-dependent manner and promoted survival of erythroid progenitor cells as colony-forming cells. Our results demonstrate that the function of erythropoietin in the goldfish is similar to that of mammals and suggest a highly conserved mechanism of early erythrocyte development in lower and higher vertebrates.