The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

Targeting insulin-like growth factor 1 leads to amelioration of inflammatory demyelinating disease.

In patients with multiple sclerosis (MS) and in mice with experimental autoimmune encephalomyelitis (EAE), proliferating autoreactive T cells play an important role in the pathogenesis of the disease. Due to the importance of these myelin-specific T cells, these cells have been therapeutic targets in a variety of treatments. Previously we found that Lenaldekar (LDK), a novel small molecule, could inhibit exacerbations in a preclinical model of MS when given at the start of an EAE exacerbation. In those studies, we found that LDK could inhibit human T cell recall responses and murine myelin responses in vitro. In these new studies, we found that LDK could inhibit myelin specific T cell responses through the insulin-like growth factor-1 receptor (IGF-1R) pathway. Alteration of this pathway led to marked reduction in T cell proliferation and expansion. Blocking this pathway could account for the observed decreases in clinical signs and inflammatory demyelinating disease, which was accompanied by axonal preservation. Our data indicate that IGF-1R could be a potential target for new therapies for the treatment of autoimmune diseases where autoreactive T cell expansion is a requisite for disease.

Discovery of biologically active oncologic and immunologic small molecule therapies using zebrafish: overview and example of modulation of T cell activation.

Zebrafish models continue to gain popularity as in vivo models for drug discovery. Described in this overview are advantages and challenges of zebrafish drug screening, as well as a novel in vivo screen for immunomodulatory compounds using transgenic, T cell reporting zebrafish larvae designed for discovery of compounds targeting T cell leukemia. This assay system allows rapid screening of large numbers of compounds while avoiding the pitfalls of assays based on cell cultures, which lack biologic context and are afflicted by genomic instability. The rationale for this approach is based on similarities of immature normal T cells and developmentally arrested, malignant lymphoblasts in mammalian species. The screening algorithm has been used to identify a nontoxic compound with activity in both acute leukemia models and models of multiple sclerosis, demonstrating the utility of this screening procedure.

DiGeorge syndrome gene tbx1 functions through wnt11r to regulate heart looping and differentiation.

DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1(-/-) mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1(-/-) mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1(-/-) mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1(-/-) mutants. In addition, both wnt11r (-/-) mutants and alcama morphants have heart looping and differentiation defects similar to tbx1(-/-) mutants. Strikingly, heart looping and differentiation in tbx1(-/-) mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.

Genomic amplification of an endogenous retrovirus in zebrafish T-cell malignancies.

Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH), a technique to detect numeric variations between different DNA samples, we examined genomes from zebrafish (Danio rerio) T-cell leukemias of three cancer-prone lines. In all malignancies tested, we identified recurring amplifications of a zebrafish endogenous retrovirus. This retrovirus, ZFERV, was first identified due to high expression of proviral transcripts in thymic tissue from larval and adult fish. We confirmed ZFERV amplifications by quantitative PCR analyses of DNA from wild-type fish tissue and normal and malignant D. rerio T cells. We also quantified ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplification and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERV's phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.

Tbl3 regulates cell cycle length during zebrafish development.

The regulation of cell cycle rate is essential for the correct timing of proliferation and differentiation during development. Changes to cell cycle rate can have profound effects on the size, shape and cell types of a developing organ. We previously identified a zebrafish mutant ceylon (cey) that has a severe reduction in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we find that the cey phenotype is due to absence of the gene transducin (beta)-like 3 (tbl3). The tbl3 homolog in yeast regulates the cell cycle by maintaining rRNA levels and preventing p53-induced cell death. Zebrafish tbl3 is maternally expressed, but later in development its expression is restricted to specific tissues. Tissues expressing tbl3 are severely reduced in cey mutants, including HSPCs, the retina, exocrine pancreas, intestine, and jaw cartilage. Specification of these tissues is normal, suggesting the reduced size is due to a reduced number of differentiated cells. Tbl3 MO injection into either wild-type or p53-/- mutant embryos phenocopies cey, indicating that loss of tbl3 causes specific defects in cey. Progression of both hematopoietic and retinal development is delayed beginning at 3 day post fertilization due to a slowing of the cell cycle. In contrast to yeast, reduction of Tbl3 causes a slowing of the cell cycle without a corresponding increase in p53 induced cell death. These data suggest that tbl3 plays a tissue-specific role regulating cell cycle rate during development.

Zebrafish screen identifies novel compound with selective toxicity against leukemia.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

A model 450 million years in the making: zebrafish and vertebrate immunity.

Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis.

Human T cell expansion and experimental autoimmune encephalomyelitis inhibited by Lenaldekar, a small molecule discovered in a zebrafish screen.

Immune-mediated diseases [multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE)] are driven by proliferating, highly activated autoreactive T-cells that are unresponsive to in vivo immunoregulatory mechanisms. The compound Lenaldekar (LDK) was identified in a zebrafish screen by inhibiting T-cell expansion. By monitoring mitogen- and antigen-driven proliferation, we found that LDK inhibited human and murine T-cell expansion in a non-cytolytic manner. This suppressive activity directly correlated with the degree of activation/proliferation of the T-cells. In testing LDK in an EAE model of MS, exacerbations were suppressed in treated animals. Therefore, LDK represents a novel therapeutic approach to T-cell-mediated autoimmune diseases.

Trans-centromere effects on meiotic recombination in the zebrafish.

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.

The zebrafish as a model for cancer.

For the last three decades significant parts of national science budgets, and international and private funding worldwide, have been dedicated to cancer research. This has resulted in a number of important scientific findings. Studies in tissue culture have multiplied our knowledge of cancer cell pathophysiology, mechanisms of transformation and strategies of survival of cancer cells, revealing therapeutically exploitable differences to normal cells. Rodent animal models have provided important insights on the developmental biology of cancer cells and on host responses to the transformed cells. However, the rate of death from some malignancies is still high, and the incidence of cancer is increasing in the western hemisphere. Alternative animal models are needed, where cancer cell biology, developmental biology and treatment can be studied in an integrated way. The zebrafish offers a number of features, such as its rapid development, tractable genetics, suitability for in vivo imaging and chemical screening, that make it an attractive model to cancer researchers. This Primer will provide a synopsis of the different cancer models generated by the zebrafish community to date. It will discuss the use of these models to further our understanding of the mechanisms of cancer development, and to promote drug discovery. The article was inspired by a workshop on the topic held in July 2009 in Spoleto, Italy, where a number of new zebrafish cancer models were presented. The overarching goal of the article is aimed at raising the awareness of basic researchers, as well as clinicians, to the versatility of this emerging alternative animal model of cancer.

Characterization of the zebrafish T cell receptor beta locus.

Zebrafish (Danio rerio) has become an increasingly important model for immunological study. Its immune system is remarkably similar to that of mammals and includes both the adaptive and innate branches. Zebrafish T cells express functional T cell receptors (TCR), and all four TCR loci are present within the genome. Using 5'-rapid amplification of cDNA ends, we cloned and sequenced zebrafish TCRbeta transcripts. TCRbeta VDJ coding joints demonstrate conservation of mechanisms used by other vertebrate species to increase junctional diversity. Using the sequences obtained, along with previously published data, we comprehensively annotated the zebrafish TCRbeta locus. Overall, organization of the locus resembles that seen in mammals. There are 51 V segments, a single D segment, 27 Jbeta1 segments, a single Jbeta2 segment, and two constant regions. This description of the zebrafish TCRbeta locus has the potential to enhance immunological research in zebrafish and further our understanding of mammalian TCR repertoire generation.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish.

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Enhanced transcription of complement and coagulation genes in the absence of adaptive immunity.

A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1(-/-)) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1(-/-) zebrafish may possess an "enhanced" innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1(-/-) kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1(-/-) intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1(-/-) mice indicate increased transcription of the complement C4 gene in the Rag1(-/-) intestine.

Disease modeling in zebrafish: cancer and immune responses--a report on a workshop held in Spoleto, Italy, July 20-22, 2009.

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).

Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.

Generation of mature T lymphocytes requires an intact hematopoietic stem cell compartment and functional thymic epithelium. We used the zebrafish (Danio rerio) to isolate mutations that affect the earliest steps in T lymphopoiesis and thymic organogenesis. Here we describe the results of a genetic screen in which gynogenetic diploid offspring from heterozygous females were analyzed by whole-mount in situ hybridization for the expression of rag-1. To assess immediately if a global defect in hematopoiesis resulted in the mutant phenotype, alpha-embryonic globin expression was simultaneously assayed for multilineage defects. In this report, we present the results obtained with this strategy and show representative mutant phenotypes affecting early steps in T-cell development and/or thymic epithelial cell development. We discuss the advantage of this strategy and the general usefulness of the zebrafish as a model system for vertebrate lymphopoiesis and thymic organogenesis.

Immunology and zebrafish: spawning new models of human disease.

The zebrafish has emerged as a powerful new vertebrate model of human disease. Initially prominent in developmental biology, the zebrafish has now been adopted into varied fields of study including immunology. In this review, we describe the characteristics of the zebrafish, which make it a versatile model, including a description of its immune system with its remarkable similarities to its mammalian counterparts. We review the zebrafish disease models of innate and adaptive immunity. Models of immune system malignancies are discussed that are either based on oncogene over-expression or on our own forward-genetic screen that was designed to identify new models of immune dysregulation.

Evolutionarily conserved and divergent regions of the autoimmune regulator (Aire) gene: a comparative analysis.

During T cell differentiation, medullary thymic epithelial cells (MTEC) expose developing T cells to tissue-specific antigens. MTEC expression of such self-antigens requires the transcription factor autoimmune regulator (Aire). In mammals, defects in aire result in multi-tissue, T cell-mediated autoimmunity. Because the T cell receptor repertoire is randomly generated and extremely diverse in all jawed vertebrates, it is likely that an aire-dependent T cell tolerance mechanism also exists in nonmammalian vertebrates. We have isolated aire genes from animals in all gnathostome classes except the cartilaginous fish by a combination of molecular techniques and scanning of expressed sequence tags and genomic databases. The deduced amino acid sequences of Aire were compared among mouse, human, opossum, chicken, Xenopus, zebrafish, and pufferfish. The first of two plant homeodomains (PHD) in human Aire and regions associated with nuclear and cytoplasmic localization are evolutionarily conserved, while other domains are either absent or divergent in one or more vertebrate classes. Furthermore, the second zinc-binding domain previously named Aire PHD2 appears to have greater sequence similarity with Ring finger domains than to PHD domains. Point mutations in defective human aire genes are generally found in the most evolutionarily conserved regions of the protein. These findings reveal a very rapid evolution of certain regions of aire during vertebrate evolution and support the existence of an aire-dependent mechanism of T cell tolerance dating back at least to the emergence of bony fish.