Zebrafish model for allogeneic hematopoietic cell transplantation not requiring preconditioning

Hess, Isabell; Iwanami, Norimasa; Schorpp, Michael; Boehm, Thomas

doi:10.1073/pnas.1219847110

Cited by 31 publications

(21 citation statements)

References 30 publications

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“…6D). This sharp decline in the engraftment ratio at 5000-10,000 transgenic WKM cells closely resembles that reported previously for limiting dilution transplantation experiments (de Jong et al, 2011;Hess et al, 2013). Thus, luciferase imaging offers high sensitivity and a new opportunity for detecting the progeny of engrafted stem cells in freely moving recipient animals.…”

Section: Ubiquitous and Tissue-specific Luciferase-based Transgenic Zsupporting

confidence: 86%

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

et al. 2013

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The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology.

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Section: Ubiquitous and Tissue-specific Luciferase-based Transgenic Zsupporting

confidence: 86%

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

et al. 2013

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“…Because their embryos are transparent and contain relatively few HSCs 6,11 , the zebrafish ( Danio rerio ) is a unique developmental system to observe native haematopoietic development in vivo . In Tg(cd41:eGFP) or Tg(Runx1 +23 :eGFP) zebrafish embryos, GFP + haematopoietic stem and progenitor cells (HSPCs) appear in the VDA between 33 h post fertilization (hpf) and 54 hpf, with peak budding at 48 hpf 6,12,13 .…”

mentioning

confidence: 99%

Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development

et al. 2016

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Haematopoietic stem cells (HSCs) arise in the developing aorta during embryogenesis. The number of HSC clones born has been estimated through transplantation, but experimental approaches to assess the absolute number of forming HSCs in a native setting have remained challenging. Here, we applied single-cell and clonal analysis of HSCs in zebrafish to quantify developing HSCs. Targeting creERT2 in developing cd41:eGFP+ HSCs enabled long-term assessment of their blood contribution. We also applied the Brainbow-based multicolour Zebrabow system with drl:creERT2 that is active in early haematopoiesis to induce heritable colour barcoding unique to each HSC and its progeny. Our findings reveal that approximately 21 HSC clones exist prior to HSC emergence and 30 clones are present during peak production from aortic endothelium. Our methods further reveal that stress haematopoiesis, including sublethal irradiation and transplantation, reduces clonal diversity. Our findings provide quantitative insights into the early clonal events that regulate haematopoietic development.

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“…zap70 y442 mutants are immunocompromised and accept allografts. One of the principal phenotypic characteristics of immunocompromised mice and zebrafish is the loss of adaptive immune responses, resulting in an enhanced ability to engraft nonimmune-matched tissue without the need for preconditioning (21,34,35). To assess the ability of zap70 y442 mutant fish to accept allografts, we orthotopically transplanted muscle from Tg(alpha: actin-RFP) fish by intramuscular injection into 2-to 4-month-old zap70 y442 mutant fish (26,36).…”

Section: Resultsmentioning

confidence: 99%

T Cell Immune Deficiency in zap70 Mutant Zebrafish

Moore

Mulligan

Yordán

et al. 2016

Molecular and Cellular Biology

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j ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP ؉ thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency. Z AP70 [zeta-chain (TCR) associated protein kinase, 70 kDa], is an important mediator of T cell differentiation and function. ZAP70 is a cytoplasmic protein kinase consisting of two aminoterminal Src homology 2 (SH2) domains and a carboxy-terminal kinase domain that plays a key role in regulating the T cell receptor (TCR) phosphorylation cascade. TCRs are activated by interacting with peptide antigens bound to major histocompatibility complexes (MHCs), causing the recruitment of CD4 or CD8 costimulatory receptors. The CD4 and CD8 receptors are bound by intracellular lymphocyte-specific protein kinase (LCK), which, in turn, phosphorylates the TCR immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3. Phosphorylated ITAMs recruit ZAP70 by binding its SH2 domains with high affinity. LCK phosphorylates ITAM-bound ZAP70, releasing it from an autoinhibited conformation and promoting its catalytic activity. Further phosphorylation of the TCR-associated scaffolding proteins by ZAP70 allows for the recruitment of additional signaling molecules and ultimately results in T cell activation, proliferation, and differentiation (1).Human ZAP70 deficiencies result in severe combined immune deficiency (SCID) with specific defects in T cell maturation (2-4). Patients with inactivating mutations in ZAP70 lack mature CD8 ϩ cytotoxic T cells and produce nonfunctional CD4 ϩ helper T cells. ZAP70 null CD4 ϩ T cells exit the thymus, yet they have dysfunctional T cell signaling and cannot mount effective T cell responses. Zap70 mutant mice also have T cell deficiencies, but they exhibit key differences compared with humans (5, 6). Zap70 mutant mice have a more severe block in thymocyte maturation, with T cells arresting at the CD4 ϩ /CD8 ϩ corti...

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Zebrafish model for allogeneic hematopoietic cell transplantation not requiring preconditioning

Cited by 31 publications

References 30 publications

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development

T Cell Immune Deficiency in zap70 Mutant Zebrafish

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