Date Published: June 24, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Jill L. O. de Jong, Leonard I. Zon.
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.
In the past few decades, the zebrafish has emerged as an outstanding vertebrate animal model for studying developmental hematopoiesis (reviewed in [1, 2]). In this same time frame, the understanding of the biology of adult hematopoietic stem cells has also blossomed, predominantly due to hematopoietic transplantation experiments performed in mice (reviewed by Orkin and Zon in ). To capitalize on the advantages of the zebrafish model (small size, high fecundity, rapid maturation, external fertilization, and the ability to perform large-scale genetic and chemical screens), a zebrafish hematopoietic transplantation assay was needed.
None of the transplantation experiments described to this point took into account any aspect of immunologic matching, as isogenic and congenic fish lines were not available. This fact highlights another significant difference between murine and zebrafish marrow transplants, namely that murine donors and recipients are congenic and hence immunologically identical. In contrast, although many commonly used zebrafish lines (e.g., AB, Tubingen, and wik) have been repeatedly incrossed through decades of laboratory use, attempts to generate truly isogenic or congenic zebrafish lines have largely failed due to inbreeding depression such that these fish lines could no longer be maintained . In addition, sex skewing of clutches, whereby a generation of siblings was all the same sex, has also hindered the ability to maintain highly inbred fish lines. Despite this disadvantage, significant progress has still been made developing hematopoietic transplantation methods in the zebrafish over the past decade, as described above.
Comparing the function of two HSC populations involves a competitive hematopoietic transplantation assay where both populations engraft in the same transplant recipient (reviewed by Purton and Scadden in ). This experimental design is required when mutant marrow cells from one donor are hypothesized to have defective hematopoietic engraftment. The mutant cells are transplanted into the recipient together with a radio-protective dose of wild-type marrow cells. If the mutant HSCs are defective, the wild-type HSCs will out-compete them, and the donor chimerism of the recipient will highly favor the wild-type donor cells. Without these wild-type HSCs to rescue the recipient, lack of engraftment of the mutant cells would likely result in the recipient’s death, and there would be no blood or marrow cells to evaluate at the end of the experiment. Using a competitive experimental design ensures that all the recipients survive until the end of the experiment and their data are included in the final analyses. In the event that the mutant marrow has normal HSC function, the donor chimerism would reveal an equal mix of engrafted hematopoietic cells from both donors. Immune matching of both donors and the recipient is an essential component of any competitive hematopoietic transplantation assay. Otherwise, one cannot rule out biased immune rejection of one donor’s cells compared to the other, and the engraftment “winner” may merely reflect immunologic differences and not a difference in stem cell biology.
One of the first multimegabase regions of the human genome to be sequenced, the human major histocompatibility complex (MHC) locus, is located on chromosome 6p21.31 and contains over 200 identified genes within a 3.6 × 106 basepair span . The classical class I and class II genes within the MHC region are the central cell surface proteins responsible for determining tissue histocompatibility of an allograft. This gene-dense region also contains a number of other genes important for the immune response, including antigen-processing genes such as proteasome subunit β type (PSMB), complement genes, and the peptide transporters TAP1 and TAP2 [26, 27].
Following up on the adult marrow transplant experiments published in 2004 , subsequent adult transplantation experiments sought to evaluate long-term hematopoietic engraftment greater than 12 weeks after transplant. Having observed poor survival in random donor long-term hematopoietic transplantation experiments (J. L. O. de Jong and L. I. Zon, unpublished data), immune typing of the zebrafish MHC genes was a logical step to ensure that graft rejection and/or GVHD were not contributing to the recipient mortality. In these first hematopoietic transplant experiments with immune matching, the class I MHC genes at the chromosome 19 locus were typed for all the sibling progeny of a single mating pair . Genotyping was achieved by preparing DNA from fin clips of individual fish, then using a panel of PCR primers to amplify MHC gene sequences. The amplified fragments were then sequenced to identify the specific MHC genes present in each individual animal. As expected, there were four MHC haplotypes represented within this family, and approximately 25% of the progeny fell into each of the four genotypes. WKM cells from β-actin-GFP+ donor fish of each MHC genotype were transplanted into GFP-negative siblings of the same MHC genotype and also into unrelated wild-type recipients, presumed to be mismatched. Survival and donor chimerism were significantly improved in the matched recipients compared with the unmatched recipients (Table 1), indicating the importance of immune matching at the chromosome 19 MHC locus for hematopoietic engraftment . These experiments were the first functional evaluation of any zebrafish MHC genes in a transplantation assay.
Survival of zebrafish hematopoietic transplant recipients is often difficult to predict from one experiment to the next. We have implemented a number of changes to the initially published transplantation protocol to address the problem of poor survival after transplant. While lack of histocompatibility may play a role for some animals, a number of other factors also appear to be important. In our experience, younger fish have better survival than older fish, and optimal recipients are approximately 3-4 months of age (J. L. O. de Jong and L. I. Zon, unpublished data). This may be due to colonization of older fish with bacterial or fungal pathogens that overwhelm and kill the immune-compromised host after transplantation. Maintaining excellent water quality is also critically important to recipient survival. We hypothesized that treatment with prophylactic antibiotics for a few days immediately after transplant might improve survival. However, placing transplant recipients “off system” in fish water containing antibiotics paradoxically caused decreased survival, as fish being treated in this way suffered from quickly deteriorating water quality and high ammonia levels (C. Lawrence, personal communication). While it is impractical to keep a therapeutic level of antibiotics in the large volume of water circulating through an entire aquatic system, the ability to maintain water quality at a consistently high standard resulted in improved survival of our transplant recipients, even without antibiotics.
Although HSC transplantation is a commonly used treatment modality for human diseases, including many malignancies, blood disorders, and immune deficiencies, this procedure continues to have high morbidity and mortality. Difficulties include selecting an optimally matched allogeneic donor, prolonged immune suppression with susceptibility to deadly infections, delayed and/or incomplete immune reconstitution, and maximizing the graft-versus-tumor effect while minimizing graft-versus-host disease. A zebrafish model for hematopoietic transplantation permitting in vivo investigation of these challenges would provide a basis to understand the biological mechanisms involved and identify possible solutions to address them.
As a model for human disease, the zebrafish holds numerous advantages. Gaining knowledge of the functional Class I and II genes in the zebrafish will enhance our understanding of basic zebrafish biology, as well as the ability to use this versatile animal model to ask questions about tissue transplantation, including hematopoietic stem cells, other normal tissues and cancers cells. This work will likely inform mammalian biology, improving our understanding of human HSCs, and has the potential to impact the treatment of patients undergoing bone marrow transplantation.