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The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. Four zinc fingers constitute its DNA binding domain and two zinc fingers are present in the C-terminal protein interaction module. We describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. An intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Both mutations stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers. [ABSTRACT FROM AUTHOR]

In mammals, T cell development critically depends on the IL-7 cytokine signaling pathway. Here we describe the identification of the zebrafish ortholog of mammalian IL-7 based on chromosomal localization, deduced protein sequence, and expression patterns. To examine the biological role of il7 in teleosts, we generated an il7 allele lacking most of its coding exons using CRISPR/Cas9-based mutagenesis. il7-deficient animals are viable and exhibit no obvious signs of immune disorder. With respect to intrathymic T cell development, il7 deficiency is associated with only a mild reduction of thymocyte numbers, contrasting with a more pronounced impairment of T cell development in il7r-deficient fish. Genetic interaction studies between il7 and il7r mutants, and il7 and crlf2(tslpr) mutants suggest the contribution of additional, as-yet unidentified cytokines to intrathymic T cell development. Such activities were also ascertained for other cytokines, such as il2 and il15, collectively indicating that in contrast to the situation in mammals, T cell development in the thymus of teleosts is driven by a degenerate multicomponent network of γc cytokines; this explains why deficiencies of single components have little detrimental effect. In contrast, the dependence on a single cytokine in the mammalian thymus has catastrophic consequences in cases of congenital deficiencies in genes affecting the IL-7 signaling pathway. We speculate that the transition from a degenerate to a nonredundant cytokine network supporting intrathymic T cell development emerged as a consequence of repurposing evolutionarily ancient constitutive cytokine pathways for regulatory functions in the mammalian peripheral immune system. [ABSTRACT FROM AUTHOR]

Recent work on vertebrate hematopoiesis has uncovered the presence of deeply rooted similarities between fish and mammals at molecular and cellular levels. Although small animal models such as zebrafish are ideally suited for genetic and chemical screens, the study of cellular aspects of hematopoietic development in lower vertebrates is severely hampered by the complex nature of their histocompatibility-determining genes. Hence, even when hosts are sublethally irradiated before hematopoietic cell transplantation, stable and long-term reconstitution by allogeneic stem cells often fails. Here, we describe the unexpected observation that transplantation and maintenance of allogeneic hematopoietic stem cells in zebrafish homozygous for the c-mybt25127 allele, carrying a missense mutation (Ile181Asn) in the DNA binding domain can be achieved without prior conditioning. Using this model, we examined several critical parameters of zebrafish hematopoiesis in a near-physiological setting. Limiting dilution analysis suggests that the kidney marrow of adult zebrafish harbors about 10 transplantable hematopoietic stem cells; this tissue also contains thymus-settling precursors that colonize the thymic rudiment within days after transplantation and initiate robust T-cell development. We also demonstrate that c-myb mutants can be stably reconstituted with hematopoietic cells carrying specific genetic defects in lymphocyte development, exemplifying one of the many potential uses of this model in experimental hematology. [ABSTRACT FROM AUTHOR]

Lymphoid organs are integral parts of all vertebrate adaptive immune systems. Primary lymphoid tissues exhibit a remarkable functional dichotomy: T cells develop in specialized thymopoietic tissues located in the pharynx, whereas B cells develop in distinct areas of general hematopoietic areas, such as the kidney or bone marrow. Among secondary lymphoid tissues, the spleen is present in all vertebrates, whereas lymph nodes represent an innovation particular to mammals and some birds. A comparative analysis of anatomical, functional and genomic features thus reveals the core components of adaptive immune systems. Such information has guided recent attempts at reconstructing lymphopoietic functions in vivo and in the future might inspire the development of new strategies for medical interventions restoring and modulating immune functions. [ABSTRACT FROM AUTHOR]

Summary: T cell development occurs in the thymus. The thymic microenvironment attracts hematopoietic progenitors, specifies them toward the T cell lineage, and orchestrates their differentiation and egress into the periphery. The anatomical location of the thymus and the intrauterine development of mouse embryos have so far precluded a direct visualization of the initial steps of thymopoiesis. Here, we describe transgenic zebrafish lines enabling the in vivo observation of thymopoiesis. The cell-autonomous proliferation of thymic epithelial cells, their morphological transformation into a reticular meshwork upon contact with hematopoietic cells, and the multiple migration routes of thymus-settling cells could be directly visualized. The unexpectedly dynamic thymus homing process is chemokine driven and independent of blood circulation. Thymocyte development appears to be completed in less than 4 days. Our work establishes a versatile model for the in vivo observation and manipulation of thymopoiesis. [Copyright &y& Elsevier]

The thymus is essential for T-cell development. Here, we focus on the role of the transcription factor Foxn1 in the development and function of thymic epithelial cells (TECs) of the mouse. TECs are of endodermal origin; they initially express Foxn1 and give rise to orthotopic (thoracic) and additional (cervical) thymi. Using Foxnldirected cytoablation, we show that during embryogenesis, cervical thymi develop a few days after the thoracic lobes, and that bipotent epithelial progenitors of cortical and medullary compartments express Foxn1. We also show that following acute selective near-total ablation during embryogenesis, complete regeneration of TECs does not occur, providing an animal model for human thymic aplasia syndromes. Finally, we address the functional role of Foxn1-negative TECs that arise postnatally in the mouse. Lineage tracing shows that such Foxn1-negative TECs are descendants of Foxn 1-positive progenitors; furthermore, Foxn 1-directed subacute intoxication of TECs by polyglutamine-containing EGFP proteins indicates that a presumptive Foxnl-independent lineage does not contribute to thymopoietic function of the adult thymus. Our findings therefore support the notion that Foxn1 is the essential transcription factor regulating the differentiation of TECs and that its expression marks the major functional lineage of TECs in embryonic and adult thymic tissue. [ABSTRACT FROM AUTHOR]

Abstract: The cis-regulatory regions of many developmental regulators and transcription factors are believed to be highly conserved in the genomes of vertebrate species, suggesting specific regulatory mechanisms for these gene classes. We functionally characterized five notochord enhancers, whose sequence is highly conserved, and systematically mutated two of them. Two subregions were identified to be essential for expression in the notochord of the zebrafish embryo. Synthetic enhancers containing the two essential regions in front of a TATA-box drive expression in the notochord while concatemerization of the subregions alone is not sufficient, indicating that the combination of the two sequence elements is required for notochord expression. Both regions are present in the five functionally characterized notochord enhancers. However, the position, the distance and relative orientation of the two sequence motifs can vary substantially within the enhancer sequences. This suggests that the regulatory grammar itself does not dictate the high evolutionary conservation between these orthologous cis-regulatory sequences. Rather, it represents a less well-conserved layer of sequence organization within these sequences. [Copyright &y& Elsevier]

The evolutionary emergence of vertebrates was accompanied by the invention of adaptive immunity. This is characterized by extraordinarily diverse repertoires of somatically assembled antigen receptors and the facility of antigen-specific memory, leading to more rapid and efficient secondary immune responses. Adaptive immunity emerged twice during early vertebrate evolution, once in the lineage leading to jawless fishes (such as lamprey and hagfish) and, independently, in the lineage leading to jawed vertebrates (comprising the overwhelming majority of extant vertebrates, from cartilaginous fishes to mammals). Recent findings on the immune systems of jawless and jawed fishes (here referred to as lower vertebrates) impact on the identification of general principles governing the structure and function of adaptive immunity and its coevolution with innate defenses. The discovery of conserved features of adaptive immunity will guide attempts to generate synthetic immunological functionalities and thus provide new avenues for intervening with faulty immune functions in humans. [ABSTRACT FROM AUTHOR]

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens. [ABSTRACT FROM AUTHOR]

Human severe combined immunodeficiencies (SCID) are phenotypically and genotypically heterogeneous diseases. Reticular dysgenesis is the most severe form of inborn SCID. It is characterized by absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth. In bone marrow of individuals with reticular dysgenesis, myeloid differentiation is blocked at the promyelocytic stage, whereas erythro- and megakaryocytic maturation is generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelo-lymphoid lineages. The dramatic clinical course of reticular dysgenesis and its unique hematological phenotype have spurred interest in the unknown genetic basis of this syndrome. Here we show that the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) is mutated in individuals with reticular dysgenesis. Knockdown of zebrafish ak2 also leads to aberrant leukocyte development, stressing the evolutionarily conserved role of AK2. Our results provide in vivo evidence for AK2 selectivity in leukocyte differentiation. These observations suggest that reticular dysgenesis is the first example of a human immunodeficiency syndrome that is causally linked to energy metabolism and that can therefore be classified as a mitochondriopathy. [ABSTRACT FROM AUTHOR]