Jill L. O. de Jong

High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

Interaction of retinoic acid and scl controls primitive blood development

Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

Characterization of immune-matched hematopoietic transplantation in zebrafish

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

A Chemical Genetic Screen in Zebrafish for Pathways Interacting with cdx4 in Primitive Hematopoiesis

cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior-posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway.

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.

Histocompatibility and Hematopoietic Transplantation in the Zebrafish

The zebrafish has proven to be an excellent model for human disease, particularly hematopoietic diseases, since these fish make similar types of blood cells as humans and other mammals. The genetic program that regulates the development and differentiation of hematopoietic cells is highly conserved. Hematopoietic stem cells (HSCs) are the source of all the blood cells needed by an organism during its lifetime. Identifying an HSC requires a functional assay, namely, a transplantation assay consisting of multilineage engraftment of a recipient and subsequent serial transplant recipients. In the past decade, several types of hematopoietic transplant assays have been developed in the zebrafish. An understanding of the major histocompatibility complex (MHC) genes in the zebrafish has lagged behind transplantation experiments, limiting the ability to perform unbiased competitive transplantation assays. This paper summarizes the different hematopoietic transplantation experiments performed in the zebrafish, both with and without immunologic matching, and discusses future directions for this powerful experimental model of human blood diseases.

Evaluation of the risk of venous thromboembolism after quadrivalent human papillomavirus vaccination among US females.

After the Food and Drug Administration (FDA) licensed quadrivalent human papillomavirus vaccine (HPV4) in 2006, reports suggesting a possible association with venous thromboembolism (VTE) emerged from the Vaccine Adverse Event Reporting System and the Vaccine Safety Datalink. Our objective was to determine whether HPV4 increased VTE risk. The subjects were 9-26-year-old female members of five data partners in the FDAs Mini-Sentinel pilot project receiving HPV4 during 2006-2013. The outcome was radiologically confirmed first-ever VTE among potential cases identified by diagnosis codes in administrative data during Days 1-77 after HPV4 vaccination. With a self-controlled risk interval design, we compared counts of first-ever VTE in risk intervals (Days 1-28 and Days 1-7 post-vaccination) and control intervals (Days 36-56 for Dose 1 and Days 36-63 for Doses 2 and 3). Combined hormonal contraceptive use was treated as a potential confounder. The main analyses were: (1) unadjusted for time-varying VTE risk from contraceptive use, (2) unadjusted but restricted to cases without such time-varying risk, and (3) adjusted by incorporating the modeled risk of VTE by week of contraceptive use in the analysis. Of 279 potential VTE cases identified following 1,423,399 HPV4 doses administered, 225 had obtainable charts, and 53 were confirmed first-ever VTE. All 30 with onsets in risk or control intervals had known risk factors for VTE. VTE risk was not elevated in the first 7 or 28 days following any dose of HPV in any analysis (e.g. relative risk estimate (95% CI) from both unrestricted analyses, for all-doses, 28-day risk interval: 0.7 (0.3-1.4)). Temporal scan statistics found no clustering of VTE onsets after any dose. Thus, we found no evidence of an increased risk of VTE associated with HPV4 among 9-26-year-old females. A particular strength of this evaluation was its control for both time-invariant and contraceptive-related time-varying potential confounding.

Targeted ITP strategies: Do they elucidate the biology of ITP and related disorders?

An elusive goal in hematology is a detailed understanding of autoimmune blood disorders including idiopathic (immune) thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), and Evans Syndrome. While it is clear that these disorders have an autoimmune pathophysiology, the exact mechanisms are not clear. Agents that target the immune system specifically, such as rituximab and tacrolimus, are currently being used clinically with favorable results. Targeted therapy may be useful not only in treating these difficult diseases, but also may provide clues to the underlying disease pathophysiology. Carefully designed studies using targeted therapy may provide biologic predictors of disease severity, as well as predictors of response to therapy. Pediatr Blood Cancer 2006;47:706–709.

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

Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. Polymorphism within additional components of the MHC pathway, particularly the antigen processing genes, may also shape immune responses. Using transcriptome, exome, and whole-genome sequencing to examine immune gene variation in zebrafish, we uncovered several antigen processing genes not found in the reference genome clustered within a deeply divergent haplotype of the core MHC locus. Our data provide evidence that these previously undescribed antigen processing genes retain ancient alternative sequence lineages, likely derived during the formation of the adaptive immune system, and represent the most divergent collection of antigen processing and presentation genes yet identified. These findings offer insights into the evolution of vertebrate adaptive immunity. 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-β 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.