Immune gene variation associated with chromosome-scale differences among individual zebrafish genomes.

Immune genes have evolved to maintain exceptional diversity, offering robust defense against pathogens. We performed genomic assembly to examine immune gene variation in zebrafish. Gene pathway analysis identified immune genes as significantly enriched among genes with evidence of positive selection. A large subset of genes was absent from analysis of coding sequences due to apparent lack of reads, prompting us to examine genes overlapping zero coverage regions (ZCRs), defined as 2 kb stretches without mapped reads. Immune genes were identified as highly enriched within ZCRs, including over 60% of major histocompatibility complex (MHC) genes and NOD-like receptor (NLR) genes, mediators of direct and indirect pathogen recognition. This variation was most highly concentrated throughout one arm of chromosome 4 carrying a large cluster of NLR genes, associated with large-scale structural variation covering more than half of the chromosome. Our genomic assemblies uncovered alternative haplotypes and distinct complements of immune genes among individual zebrafish, including the MHC Class II locus on chromosome 8 and the NLR gene cluster on chromosome 4. While previous studies have shown marked variation in NLR genes between vertebrate species, our study highlights extensive variation in NLR gene regions between individuals of the same species. Taken together, these findings provide evidence of immune gene variation on a scale previously unknown in other vertebrate species and raise questions about potential impact on immune function.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions with complex relationships to mammalian receptors.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

An Exclusive Interview With CRISPR.

This article chronicles a didactic encounter between an ethics-minded physician-scientist and a personified genome editing technology called clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins, commonly abbreviated as CRISPR/Cas, or simply CRISPR. The interview considers clinically and ethically relevant questions about this technology related to patient safety, therapeutic efficacy, equitable access, and global governance of humanity's genetic legacy.

Provider engagement in precision oncology education: an exploratory analysis of online continuing medical education data.

Given the rapid growth in genomic tests and targeted therapeutics, clinicians are likely to benefit from additional precision medicine education. Aim: This study evaluated the engagement and effectiveness of two interactive, case-based educational modules about somatic tumor testing, developed by the Jackson Laboratory, American Medical Association and Scripps Research Translational Institute, titled 'Precision Medicine for Your Practice'. Results: 402 participants enrolled in one or both free online continuing education modules, including physicians, nurses, scientists and genetic counselors and 41% completed module evaluations. Over 90% of respondents reported alignment of program with practice needs and planned to change their practice, including patient communication, identifying candidates for testing and/or interpreting test results. Conclusion: These findings support Precision Medicine for Your Practice as an effective education offering for diverse clinical professionals.

United States National Postdoc Survey results and the interaction of gender, career choice and mentor impact.

The postdoctoral community is an essential component of the academic and scientific workforce, but a lack of data about this community has made it difficult to develop policies to address concerns about salaries, working conditions, diversity and career development, and to evaluate the impact of existing policies. Here we present comprehensive survey results from 7,603 postdocs based at 351 US academic and non-academic (e.g. hospital, industry and government lab) institutions in 2016. In addition to demographic and salary information, we present multivariate analyses on factors influencing postdoc career plans and satisfaction with mentorship. We further analyze gender dynamics and expose wage disparities. Academic research positions remain the predominant career choice, although women and US citizens are less likely than their male and non-US citizen counterparts to choose academic research positions. Receiving mentorship training has a significant positive effect on postdoc satisfaction with mentorship. Quality of and satisfaction with postdoc mentorship also appear to heavily influence career choice.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution.

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-ß 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

The MHC class I genes of zebrafish.

Major histocompatibility complex (MHC) molecules play a central role in the immune response and in the recognition of non-self. Found in all jawed vertebrate species, including zebrafish and other teleosts, MHC genes are considered the most polymorphic of all genes. In this review we focus on the multi-faceted diversity of zebrafish MHC class I genes, which are classified into three sequence lineages: U, Z, and L. We examine the polygenic, polymorphic, and haplotypic diversity of the zebrafish MHC class I genes, discussing known and postulated functional differences between the different class I lineages. In addition, we provide the first comprehensive nomenclature for the L lineage genes in zebrafish, encompassing at least 15 genes, and characterize their sequence properties. Finally, we discuss how recent findings have shed new light on the remarkably diverse MHC loci of this species.

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish.

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.

Erythropoiesis in the absence of adult hemoglobin.

During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and ß globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied.

Human globin knock-in mice complete fetal-to-adult hemoglobin switching in postnatal development.

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult ß-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human ß-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and ß-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-ß-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.

Humanized mouse models of Cooley's anemia: correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology.

beta thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Preclinical transfusion-dependent humanized mouse model of beta thalassemia major.

A preclinical humanized mouse model of beta thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult alpha and beta globin genes were replaced with adult human alpha globin genes (alpha2alpha1) and a human fetal to adult hemoglobin (Hb)-switching cassette (gamma(HPFH)deltabeta(0)), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (alpha(2)gamma(2)), with a small amount of human minor adult Hb, HbA2 (alpha(2)delta(2)). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful for the development of new transfusion and iron chelation regimens, the study of iron homeostasis in disease, and testing of cellular and genetic therapies for the correction of thalassemia.

Humanized Mouse Model of Cooley's Anemia.

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.