Disease Modeling in Zebrafish: Cancer and Immune Responses—A Report on a Workshop Held in Spoleto, Italy, July 20–22, 2009

Abstract

The growing interest in using zebrafish for genetic and functional dissection of malignancy and infection was highlighted by the second international workshop on Zebrafish Models of Cancer and the Immune Response in Spoleto, Italy (July 20–22, 2009). The overarching theme of the state-of-the-art reports featured the unique suitability of zebrafish for in vivo monitoring of fundamental biologic and pathologic processes. For example, in vivo imaging was employed for the first demonstration of direct development of hematopoietic stem cells from hemogenic epithelium and for visualization of T-cell homing and interaction with thymic epithelial cells. In addition, in vivo monitoring was instrumental for developing disease models of solid tumors, leukemia, and of inflammatory conditions, and for assessing the efficacy of small molecule drugs under physiologic and pathologic conditions. The success of zebrafish small molecule screens was underscored by the identification of prostaglandin E2 (PGE2) as an efficient inducer of stem cell expansion that led to the initiation of the first human trial on the efficacy of PGE2 in bone marrow transplantation. Further, zebrafish models of infectious diseases such as tuberculosis have been established that are now amenable to high-throughput in vivo drug screens, a much-needed development in the fight against drug-resistant microorganisms. The success of this workshop and the rapidly growing field of cancer and the immune response in zebrafish have spawned follow-up meetings in Boston (June 2010) and Edinburgh (2011).

Introduction

The highly successful zebrafish workshop on infectious disease and cancer in zebrafish in Leiden (The Netherlands) in 2007¹ strongly motivated the authors of this report to organize a follow-up meeting. Prompted by the prediction that similarities between defense mechanisms against microbes and cancer cells can reveal new insights into specific determinants of innate immune responses, this workshop focused on cancer models and infection studies. Due to the amenability of zebrafish to large-scale forward and reverse genetic screens, this model organism is ideal for discovery of novel gene functions in disease processes at a throughput level that cannot be matched by rodent models. Further, owing to its small size and optical transparency, disease manifestations and resulting immune responses can be studied at the whole-organism level. Particularly advantageous in this context are fluorescence multicolor labeling techniques that allow tagging of the players in disease processes (e.g., cancer cells, immune cells, and microbes) for easy detection in vivo. In the following report the oral presentations at the meeting are summarized chronologically.

Cancer Studies

In recent years, the zebrafish has made great strides as a laboratory model organism for cancer research. The striking similarity of fish and human tumors at the molecular and histopathological level coupled with the ease of performing tumor transplantation and to observe tumor invasiveness in vivo have bolstered the rationale for using zebrafish as a cancer model system. The presentations on cancer studies particularly focused on cancer and hematopoiesis, tumor models obtained using forward and reverse genetic screens, xenografts, and in vitro and in vivo tumor models.

Melanoma models

Five of the 12 cancer talks scheduled for the first day of the meeting dealt with fish melanoma models. M. Mione (Milan, Italy) focused on different transgenic lines expressing the oncogene Harvey-RAS (H-RAS)V12, and presented work implicating cellular senescence and ubiquitination in modulating the oncogenic activity of H-RAS. Oncogene-induced senescence is a powerful tumor-suppressive mechanism, which restrains the proliferation of oncogene-expressing cells. In zebrafish, oncogene-induced senescence is associated with increased ubiquitination of the ras oncogene.

E. Patton (Edinburgh, United Kingdom) gave the European Association for Cancer Research–sponsored lecture on chemical and genetic control of melanocyte and melanoma development. She illustrated the power of a multiple organism approach (yeast, fish, and cancer cells) for screening chemical compound libraries. Here the zebrafish was used to identify compounds that affected pigmentation in development. Subsequently, a library of yeast genetic deletion mutants was screened with the most promising compounds, to identify their mechanisms of action, a process called chemical profiling. One of the active compounds appears to selectively kill developing and adult melanocytes and affects nevus growth in the zebrafish.

M. Schartl (Wuerzburg, Germany) reminded the audience that the spontaneous melanomas discovered in the late 1920s in certain strains of Xiphophorus generated the first animal model of cancer that clearly illustrated the genetic nature of the disease. In addition, molecular studies in this system have uncovered for the first time molecules that are implicated in the pathogenesis of melanoma and are also of relevance for the human disease, for instance, signal transducers and activator of transcription (STAT)5, osteopontin, and the importance of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway. The nuclear translocation of Stat5 seems to be a key event (mediated by the oncogenic epidermal growth factor (Egf) receptor, Xmrk) in the activation of antiapoptotic and pro-proliferative pathways. To make use of genetic tools, transgenic medaka expressing the same oncogene in pigment cells have been generated. Here, tumors develop also in the presence of functional p53, whose absence affects the size, but not the timing of melanoma development.

A. Hurlstone (Manchester, United Kingdom) presented the melanoma models developed in his lab and based on H-RAS oncogene expression driven by the mitfa promoter. One of the models displays melanoma onset in the embryo and has been used to screen known drugs for their ability to prevent the phenotype. Combining drugs targeting different pathways downstream of ras, as well as inhibiting a novel putative drug target identified through transcriptome profiling of zebrafish melanoma with morpholino and small molecule inhibitors, prevented melanoma onset.

C. Santoriello (Milan, Italy) described a melanoma model based on the powerful transactivation complex Gal4-UAS. Here, expression of the H-RAS oncogene is driven by the kit-a promoter, a melanoblast-specific transcription factor. The most striking feature of this line is the increase in number/size and abnormal migration of melanophores as early as 72 h postfertilization, making the line ideal for large-scale chemical screens. Data presented by Santoriello suggest that oncogenic H-RAS may use endocytic trafficking of small GTPases to transform melanocytes and give rise to melanomas.

In the first keynote lecture detailed below, L. Zon (Boston) presented an update on the modifier genes of a melanoma model developed in his lab. Using an overexpression screen in transgenic melanoma, a new human disease modifier (histone methyltransferase) that participates in melanoma was found.

Cancer and hematopoiesis

In the keynote talk of this session, L. Zon (Boston) introduced Cancer and Stem Cell Biology in the zebrafish. During embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) region and are capable of self-renewal and production of all mature blood lineages. HSCs in the adult are governed by cell-intrinsic transcriptional regulators maintained by extracellular signals from the niche. The understanding of these signals and their integration with transcriptional complexes likely defines the set of genes involved in self-renewal. Through a chemical genetics screen, the Zon lab found that prostaglandin E2 is a potent stimulator of HSC self-renewal. The effect of prostaglandin E2 is mediated by phosphorylation of β-catenin, activating the Wnt pathway and increasing self-renewal as assayed by competitive transplantation. Using a competitive repopulation assay in the mouse and zebrafish, PGE2 was shown to increase engraftment three- to fourfold. In addition, mice were engrafted more efficiently with PGE2-treated human cord blood. A clinical trial is about to start in which the competitive advantage of PGE2-treated over untreated cord blood in the transplantation setting will be explored. Taken together, these studies demonstrate the integration of developmental pathways and transcriptional regulators in the homeostasis and response of HSCs and cancer cells.

J. Kanki (Boston) investigated the in vivo role of the human nucleolar phosphoprotein nucleophosmin (hNPM1) in hematopoiesis. Mutations in hNPM1 (hNPMc) are found in 30% of patients with acute myeloid leukemia, but despite its frequency and its distinctive clinical and biological features, in vitro and in vivo models of hNPMc transformation are lacking. Overexpressing mutant hNPMc in zebrafish embryos, Kanki's group found an increase in HSCs/progenitor cell and myeloid precursors associated with a block in myeloid differentiation that was not seen with hNPM1 overexpression. Interestingly, expression of hNPMc in p53 mutant embryos resulted in increased numbers of mpx expressing cells, suggesting that hNPMc in zebrafish can activate p53-dependent mechanisms specifically affecting myelopoiesis.

J. Berman (Halifax, Canada) engineered a transgenic zebrafish harboring the human acute myeloid leukemia-associated NUP98-HOXA9 translocation. In this transgenic system, cell survival is dysregulated and hematopoiesis is reprogrammed with upregulated expression of myeloid-specific genes and downregulated erythroid-specific genes. These changes in transcription affected both primitive and definitive hematopoiesis, including the recently described erythro-myeloid progenitors. The mechanism underlying NUP98-HOXA9-mediated leukemogenesis may involve the observed antiapoptotic effect and impairment of terminal myeloid differentiation.

Tumor models obtained using forward and reverse genetic screens

Using forward genetics J. Amatruda (Dallas, TX) identified a mutant zebrafish line with high incidence of testicular germ cell tumors. Homozygous males develop tumors consisting of undifferentiated spermatogonia by 4 months of age, whereas heterozygous males develop tumors around 8 months. A combination of haplotype- and high-resolution mapping identified an underlying mutation in a conserved component of an embryonic signaling pathway. Altered function of the pathway was demonstrated in the zebrafish tumors and corroborated in clinically annotated human germ cell tumors of diverse histologic subtypes, confirming the relevance of the zebrafish model for understanding germ cell tumorigenesis.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role during development and oncogenesis, but functional analysis of PTEN deficiency in a living organism has been hampered by embryonic lethality. Using TILLING, J. den Hertog (Utrecht, The Netherlands) inactivated both zebrafish pten genes. This revealed the redundant function of ptena and ptenb. Although individuals with single gene inactivation are viable and fertile, double knockout mutants display developmental defects after 3 days postfertilization. Double mutants exhibit enhanced cell proliferation and enhanced cell survival after γ-irradiation compared with siblings. These pten mutants represent a unique model to screen for genetic or chemical suppressors of pten loss of function and to investigate the role of PTEN during tumorigenesis in vitro as well as in vivo.

Xenograft models

Recently, several groups have established xenograft tumor models in zebrafish. B.E. Snaar-Jagalska (Leiden, The Netherlands) developed a novel engraftment model in zebrafish embryo to study Ewing's sarcoma (EWS) progression. EWS is a soft tissue cancer with poor prognosis when metastatic or after relapse. Investigation of metastatic tumor cell migration is technically demanding (whole animal imaging) and expensive (instrumentation, animals) in rodents, making zebrafish embryo models an attractive alternative. Snaar-Jagalska reported that human EWS cells survive, proliferate, migrate, and induce angiogenesis after xenografting into zebrafish embryos. This system establishes a sensitive in vivo vertebrate model for identifying mechanisms of EWS progression and for development of new anticancer compounds in a time- and cost-effective manner.

In vitro and in vivo liver tumor models

Hepatocellular carcinoma (HCC) is a prevalent and deadly cancer for which efficient treatment is unavailable. Comparative functional genomics had previously demonstrated the molecular conservation between human and zebrafish carcinogen-induced liver tumors, providing the rationale for a zebrafish HCC model. S. He (Leiden, The Netherlands) established a stable zebrafish liver cell line expressing human RAF-1-ER fusion. After tamoxifen-mediated hRAF-1 activation, zMEK-ERK cascade promoted growth-factor-independent proliferation and apoptosis inhibition, reproduced in vivo after cell implantation into zebrafish embryos. Gene expression analysis revealed a subset of RAF-1 regulated genes, involved in tumor progression. A subset of these genes is also regulated in human HCC, suggesting that this inducible zebrafish model is useful for studying the molecular basis of HCC.

To investigate the function of c-MYC and its underlying mechanism in liver cancer formation, Z. Li (Singapore) established a transgenic zebrafish model to conditionally overexpress mouse c-Myc in the liver. Using the Tet-on system, mouse c-Myc was expressed in the liver by the lfabp (liver fatty acid binding protein) promoter in the presence of doxycycline. Histological analysis showed that overexpression of mouse c-Myc in zebrafish caused liver hyperplasia that progressed to hepatocellular adenoma upon prolonged c-Myc expression. Withdrawal of doxycycline greatly reduced c-Myc expression and decreased liver size. These observations indicate the potential of c-Myc transgenic zebrafish as a model to study liver cancer progression.

A model for neurofibromatosis

T. Look (Boston) gave the meeting's second keynote lecture. In the tradition of tumor modeling in his lab, he reported the first zebrafish model of Neurofibromatosis 1 that was developed together with J. Epstein (Philadelphia). NF1 is the most common inherited human cancer syndrome that confers a predisposition to a number of central and peripheral nervous system tumors. Exploiting the main strengths of the zebrafish, the two groups performed the first in vivo analysis of nf1 loss using time-lapse microscopy. Observing oligodendrocyte precursor cells (OPCs) in the spinal cord of embryos (using the olig2::enhanced green fluorescent protein (EGFP) + transgenic line), they showed that nf1 knockdown individuals have increased numbers and enhanced migration of OPCs. The latter was due to shortening of OPC pausing during migration. The observed phenotypes may explain some of the features of the human disease (e.g., increased invasiveness of NF1-deficient gliomas). NF1 guanosine triphosphatase activating protein (GAP)-related domain overexpression by itself rescued both phenotypes, demonstrating the requirement for GAP function in developing organisms. This zebrafish model provides a platform for genetic and chemical perturbations to suppress the nf1-deficient phenotype.

Models of T acute lymphoblastic leukemia

N. Trede (Salt Lake City, UT) used a T-cell indicator transgenic line to identify new molecules with activity against human T acute lymphoblastic leukemia (T-ALL). After screening a 50,000-compound chemical library, one compound was identified that not only eliminated immature T cells in living zebrafish larvae, but also killed human T-ALL lines. This compound (C3) is orally available and well tolerated by mice. C3 is also highly T-cell selective with dual activity in the AKT/mTOR axis by an as of yet unidentified mechanism.

D.M. Langenau (Boston) reported the use of clonal zebrafish lines for transplantation of T-ALL. The advantage of this system is that recipient fish do not need to be immunosuppressed with irradiation, preventing conditioning-related deaths. In this system, leukemia-initiating cells are quite high (around 1%). High-throughput cell transplantation experiments were described that allow large numbers of animals to be assessed for engraftment from a single leukemic cell. This system highlights the unprecedented opportunity to interrogate functional pathways involved in leukemia self-renewal in a vertebrate.

L. Rudner and K. Frazer (Salt Lake City, UT) reported the first zebrafish screen to identify heritable predisposition to T-ALL. One recessive and two dominant mutants were discussed in detail. The recessive mutant exhibits p53-independent resistance to irradiation-induced apoptosis at embryonic stages. Penetrance of the dominant mutants was greatly increased in homozygous individuals (from 5% to 40%). Leukemias are transplantable and, similarly to Langenau's report, serial transplantations led to increased aggressiveness. They developed and used a number of techniques, including array comparative genomic hybridization and methylated DNA immunoprecipitation, to elucidate genomic, genetic, and epigenetic changes that accompany development of zebrafish T-ALL and increased aggressiveness in serial transplantation. Mapping of one dominant mutant was successful and is ongoing for the remaining T-ALL lines. These models serve as platforms to define multiple types of genetic events associated with the complex in vivo evolution of this important class of human cancers.

Immunity and Infectious Disease

Inflammatory bowel disease

Chronic inflammation, such as inflammatory bowel disease, is widely believed to predispose individuals to developing cancer. Many of the genes connected with inflammation and cancer also have critical roles as cell fate regulators during development. The lab of P. Crosier (Auckland, New Zealand) has developed chemical colitis models in zebrafish larvae to explore the interaction between intestinal development and inflammation. These models, which resemble human disease states, provide convenient in vivo systems to study how genetic and environmental factors contribute to colitis. Homeodomain transcription factors of the Cdx family have critical functions in defining intestinal identity and have also been implicated in colon cancer. The Crosier lab is currently investigating the role of zebrafish cdx genes in responses of the intestinal epithelium to pro-inflammatory stress. Knockdown studies demonstrated their association with intestinal phenotypes as well as inflammatory gene expression.

S. Brugman (Rotterdam, The Netherlands) developed a novel oxazolone-induced enterocolitis model for adult zebrafish, characterized by an influx of granulocytes, epithelial damage, Goblet-cell depletion, and pro-inflammatory cytokine induction. Different antibiotic treatments showed that components of the intestinal microbiota drive the development of enterocolitis and directly affect the composition of the mucosal infiltrate and the severity of intestinal inflammation.

Many inflammatory diseases are caused by a failure of the resolution of neutrophilic inflammation. The lab of S. Renshaw (Sheffield, United Kingdom) is working to combine mutants, transgenes, and pharmacological agents in the zebrafish model to seek an understanding of the molecular mechanisms that drive the resolution of inflammation. Several mutants showing prolonged inflammation after tail injury are currently under investigation. Inflammation resolution was reduced in larvae treated with a pan-caspase inhibitor and improved with apoptosis-promoting drugs, indicating a role for apoptosis in the resolution process in vivo. A compound screen is ongoing to identify other drugs that promote inflammation resolution in the zebrafish model and that might be used in treatment of patients with respiratory diseases.

Immune response to infection

Due to the clear separation of innate immunity from adaptive responses, zebrafish embryos and larvae are useful for dissecting the innate host factors involved in infectious disease processes. A time-course transcriptome profiling study of the response of 1-day-old embryos to Salmonella typhimurium infection by the group of A.H. Meijer (Leiden, The Netherlands) revealed specific regulation of Toll-like receptor 5 (TLR5) and downstream signaling components, including negative regulators of the TLR pathway. Knockdown analysis showed a conserved function for tlr5 in the recognition of Salmonella flagellin and showed that the response to infection is partially dependent on the TLR adaptor myd88. In addition, novel markers for macrophages were identified that are expressed downstream of transcription factor pu.1 and that are currently used to develop transgenic lines. In a joint study with A.M. van der Sar (Amsterdam, The Netherlands) the two groups investigated the specificity of transcriptome responses to acute and chronic mycobacterial infections. Although expression signatures that mark the end stages of disease caused by chronic or acute strains were found to be remarkably similar, specific gene groups responded differently during the early stage of infection. These genes may determine the chronic or acute disease outcome and are useful leads for further investigation. Granuloma formation, the hallmark feature of chronic tuberculosis, occurs not only in adult zebrafish but also in larvae in the absence of adaptive immunity. A substantial overlap between expression signatures of infected zebrafish larvae and adults indicated a major contribution of the innate component of the immune system in the response to mycobacterial infection.

H.P. Spaink (Leiden, The Netherlands) showed that embryo infections can be performed in high-throughput mode to allow chemical compound screens. An automated injection system was coupled to the Copas XL biosorter (Union Biometra) to allow sorting of infected larvae with simultaneous screening of disease symptoms by flow-through laser scanning profiling. To increase screening throughput they employed the common carp, a close relative of the zebrafish that can yield hundreds of thousands of embryos from a single female by in vitro fertilization. By determination of a shotgun sequence of the carp genome and RNA deep sequencing, they showed that the hallmarks of the innate immune responses to mycobacterial infection in carp and zebrafish are remarkably similar.

C. Kim (Orono, ME) used the zebrafish model to study the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR), innate immunity, and Pseudomonas aeruginosa infection. Mutations in the CFTR gene are the root cause of cystic fibrosis. Zebrafish cftr morphant embryos infected with P. aeruginosa showed an increased respiratory burst and expression of pro-inflammatory cytokines compared with controls as well as an increased bacterial burden and higher mortality rate. The increased bacterial burden in cftr morphants was specific for P. aeruginosa compared with several other bacterial infections. The ability of P. aeruginosa to form bacterial biofilms might contribute to the host's inability to clear the infection.

The first fungal infection model in zebrafish embryos was developed by the lab of G.J. Lieschke (Parkville, Australia). They established a model for Penicillium marneffei infection, an opportunistic human pathogenic fungus that shows temperature-dependent dimorphism with hyphal growth at 28°C and growth as a yeast at 33°C. Therefore, an ectothermic host like the zebrafish allows using temperature shifts to identify how morphology affects pathogenicity. Imaging studies indicated that phagocytosis of P. marneffei spores by leukocytes is a critical step in establishing infection. P. marneffei infection strongly stimulated emergency hematopoiesis, a process shown to depend on the gcsfr1 receptor.

The Zebrafish Immune and Hematopoietic System

Immune cells and receptors

The group of P. Herbomel (Paris, France) has made optimal use of the transparency of zebrafish embryos to film the development and behavior of leukocytes during zebrafish development. With lineage tracing they have shown that over half of the myeloid progenitors from the anterior lateral plate mesoderm become tissue neutrophils. They are characterized by expression of mpx, lyz, and nephr and are not fully differentiated until 40 h postfertilization. Differentiated neutrophils appear in mesenchyme and subepidermal tissues throughout zebrafish larvae and show extensive patrolling behavior in contrast to macrophages in the epidermis that do not move much. As opposed to the short-lived neutrophils of adult mammals, neutrophils in zebrafish larvae appear long-lived and still expand when in tissues. Imaging analysis of the response to infection showed that intravenously injected bacteria do not adhere to neutrophils, but are predominantly phagocytosed by macrophages.

I. Hess (Freiburg, Germany) focused on lymphocyte development. To study the process of thymopoiesis in vivo the Boehm group generated an ikaros::EGFP transgenic line using modified bacterial artificial chromosome (BAC) technology. This line allows for tracing the migratory patterns of hematopoietic cells to the thymic anlage. Morpholino knockdown of foxn1 leads to aberrant migration of ikaros::EGFP cells to the thymus, indicating that the differentiation of thymic epithelial cells and the interaction of stromal and lymphoid cells are important for normal immigration of prothymocytes into the thymus and subsequent T-cell development. In cxcl12a/cxcl12b/ccl25a morphants thymus homing is impaired, indicating that these chemokines play an important role in this process.

J. Yoder (Raleigh, NC) gave an update on the form, function, and phylogenetics of novel immune-type receptors (nitrs) in zebrafish and other bony fish. Natural killer (NK) cells are still not definitively characterized in zebrafish, but Nitrs have been proposed to be the functional equivalents of mammalian NK receptors. All Nitrs possess one extracellular immunoglobulin domain of the variable (V) type; like NK receptors, most Nitrs possess short intracellular functional motifs permitting their classification as inhibitory or activating. It is likely that all teleost species encode Nitrs that function in innate immunity to regulate cell-mediated cytotoxicity. Comparison of the zebrafish and medaka genomes indicates a rapid evolution with species-specific expansion and diversification of different families of nitrs.

J.P. Levraud (Paris, France) is studying the cytokine receptor family B (CRFB) proteins involved in signaling by viro-induced interferons. The fish viro-induced interferons have been named ifnΦ and form two distinct classes, with zebrafish ifnf1 and ifnf4 as class I members and ifnf2 and ifnf3 belonging to class II. Overexpression of all four IFNs induced expression of antiviral genes such as viperin and increased the resistance of zebrafish larvae to viral challenge. The two class I Ifns were found to signal through a dimer of Crfb1 and Crfb5, whereas the two class II Ifns were found to rely on a dimer of Crfb5 with another member of the Crfb family.

Hematopoiesis

Runx1 is an essential transcription factor for definitive HSC development. M. Vega Flores (Auckland, New Zealand) used runx1::EGFP transgenic lines to trace the origin of HSCs. As runx1 is transcribed from two promoters, P1 and P2, two transgenic lines were generated. The runx1P1::EGFP line displays fluorescence in the posterior blood island, where definitive erythro-myeloid progenitors develop. The runx1P2::EGFP line marks definitive HSCs in the AGM, which later populate the pronephros and thymus. This suggests that runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. Time-lapse imaging showed runx1P2::EGFP cells to emerge from mCherry-labeled endothelial cells in a compound runx1P2::EGFP/kdrl::nls-mCherry transgenic line, demonstrating for the first time of the direct generation of definitive HSCs from the hemogenic endothelium in a living embryo.

C. Hall (Auckland, New Zealand) focused on the demand-driven or emergency hematopoiesis that occurs during infection. Injection of S. typhimurium bacteria into the hindbrain of 2-day-old zebrafish embryos led to a rapid infiltration of lyz::DsRED2 expressing leukocytes, and 1.5 to 2 days later lyz::DsRED2-marked cells emerged de novo throughout the AGM region. Demand-driven myelopoiesis was found to occur at the expense of lymphopoiesis and to depend on definitive blood progenitors expressing runx1. Demand-driven myelopoiesis also required the gcsfr receptor, but Myd88-dependent signaling was dispensable for the response. Understanding HSC function and the immune system is critically important for successful long-term transplantation of hematopoietic cells, tumors, or any allogeneic tissue.

J.L.O. de Jong (Boston) characterized the genes at putative major histocompatibility complex (MHC) loci at chromosomes 1 and 19 and tested their functional significance by performing immune-matched and mismatched kidney marrow transplants. The data indicated that matching the donor and recipient MHC haplotypes increased engraftment and percentage of donor chimerism compared with MHC-mismatched recipients. Thus, the MHC loci at chromosomes 1 and 19 appear to be functionally important for immune matching in the setting of transplantation. MHC immune-matching will likely facilitate long-term transplantation experiments in zebrafish that have previously not been possible.

Concluding Remarks

From the above it is clear that the zebrafish has many advantages to offer as a model for the studies of immune-related diseases. Although the parallels between immune responses against cancer cells and microbes are still poorly understood, it is clear that the tools developed for the study of the progression of infectious disease and cancer will be equally beneficial for both fields. The growing number of scientists who now use zebrafish in their research in these areas promises many new exciting discoveries in the near future. Therefore, a follow-up meeting is already scheduled to take place in 2011 after the European zebrafish meeting in Edinburgh.

Acknowledgments

We wish to thank The Company of Biologists (through their journal Disease Models & Mechanisms), the European Association for Cancer Research, the European Commission Projects ZF-TOOLS (LSHG-CT-2006-037220) and ZF-CANCER (HEALTH-F2-2008-201439), and the COST Action BM0804 for their support that made this meeting possible.

Disclosure Statement

No competing financial interests exist.

Author Statement

We wish to confirm that we have given all speakers whose work is mentioned in the article the opportunity to make changes to the text. In particular, we asked them to verify that unpublished data mentioned in the report will not compromise future publications. We received a number of amendments that were all incorporated in the final text.