Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish

Tang, Qin; Moore, John C.; Ignatius, Myron S.; Tenente, Inês M.; Hayes, Madeline; Garcia, Elaine G.; Yordán, Nora Torres; Bourque, Caitlin; He, Shuning; Blackburn, Jessica S.; Look, A. Thomas; Houvras, Yariv; Langenau, David M.

doi:10.1038/ncomms10358

Cited by 83 publications

(82 citation statements)

References 66 publications

(94 reference statements)

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“…rag2-kRAS G12D and mylpfa-myf5 constructs were linearized with XhoI, phenol:chloroform-extracted, ethanol-precipitated, re-suspended in 0.5× Tris-EDTA + 0.1 M KCl, and injected into one-cell stage embryos of the respective backgrounds, as previously described (Langenau et al, 2007; Tenente et al, 2014). We and others have used the mylpfa promoter to drive transgene expression in differentiated muscle cells both in the stable and mosaic transgenic setting (Ju et al, 2003; Ignatius et al, 2012; Storer et al, 2013; Tang et al, 2016) confirming that mylpfa transgene expression is confined to differentiated ERMS cells.…”

Section: Methodsmentioning

confidence: 56%

“…The model has also been used to identify functional heterogeneity in molecularly defined cell types, including isolation of myf5:GFP+ TPCs (Ignatius et al, 2012). In total, the zebrafish kRAS G12D ERMS model has emerged as one of the most relevant for discovering pathways that drive cancer growth in human RMS (Chen et al, 2013a, 2014; Ignatius et al, 2012; Kashi et al, 2015; Langenau et al, 2007, 2008; Le et al, 2013; Storer et al, 2013; Tang et al, 2016)…”

Section: Introductionmentioning

confidence: 99%

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Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Tenente

Hayes

Ignatius

et al. 2017

eLife

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Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.DOI: http://dx.doi.org/10.7554/eLife.19214.001

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Section: Methodsmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Tenente

Hayes

Ignatius

et al. 2017

eLife

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“…4D), further suggesting the existence of a myogenic population of cells distinct from (or possibly derived from) the Pax7-expressing satellite-like cell population. Additionally, we further investigated the presence of myoblast-like cells by performing injury in adult zebrafish carrying two transgenes: - 80.0myf5:GFP zf37 ( myf5 -driven GFP, hereafter ‘ myf5:GFP ’; Chen et al, 2007) and myog:H2B-mRFP ( myog -driven H2B (nuclear)-mRFP; Tang et al, 2016) (Fig. 4E).…”

Section: Resultsmentioning

confidence: 99%

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

Berberoglu

Gallagher

Morrow

et al. 2017

Developmental Biology

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Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.

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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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Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish

Cited by 83 publications

References 66 publications

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

T Cell Immune Deficiency in zap70 Mutant Zebrafish

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