Publications by authors named "David M Langenau"

70 Publications

PHF6 Expression Levels Impact Human Hematopoietic Stem Cell Differentiation.

Front Cell Dev Biol 2020 4;8:599472. Epub 2020 Nov 4.

Department of Diagnostic Sciences, Ghent University, Ghent, Belgium.

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in , encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established models. Our findings reveal that levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of and its downstream target genes. This functional interaction in developing thymocytes was confirmed using a -deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced or notch1 activation. In summary, our work reveals that appropriate control of expression is important for normal human hematopoiesis and provides clues towards the role of in T-ALL development.
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http://dx.doi.org/10.3389/fcell.2020.599472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672048PMC
November 2020

Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish.

Nat Protoc 2020 09 21;15(9):3105-3128. Epub 2020 Aug 21.

Molecular Pathology Unit, Mass General Research Institute, Charlestown, MA, USA.

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc, il2rgα immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc, il2rgα model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.
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http://dx.doi.org/10.1038/s41596-020-0372-yDOI Listing
September 2020

Zebrafish patient avatars in cancer biology and precision cancer therapy.

Nat Rev Cancer 2020 05 6;20(5):263-273. Epub 2020 Apr 6.

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.

In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment.
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http://dx.doi.org/10.1038/s41568-020-0252-3DOI Listing
May 2020

The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish.

J Leukoc Biol 2020 03 7;107(3):431-443. Epub 2020 Jan 7.

Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles, Brussels, Belgium.

The mononuclear phagocytic system consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate mononuclear phagocytic system subsets from other hematopoietic lineages. Macrophage-expressed gene (Mpeg)1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since then more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation, and function. Whereas the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1 lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1 macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8 myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception is skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the mononuclear phagocytic system in adult zebrafish.
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http://dx.doi.org/10.1002/JLB.1A1119-223RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064944PMC
March 2020

Adult immune compromised zebrafish for xenograft cell transplantation studies.

EBioMedicine 2019 Sep 12;47:24-26. Epub 2019 Aug 12.

Molecular Pathology Unit, Massachusetts General Hospital Research Institute, Charlestown, MA 02129, United States of America; Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, United States of America; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, United States of America; Harvard Stem Cell Institute, Cambridge, MA 02139, United States of America. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2019.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796557PMC
September 2019

Insights into pediatric rhabdomyosarcoma research: Challenges and goals.

Pediatr Blood Cancer 2019 10 21;66(10):e27869. Epub 2019 Jun 21.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts.

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
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http://dx.doi.org/10.1002/pbc.27869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707829PMC
October 2019

Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Cell 2019 06 25;177(7):1903-1914.e14. Epub 2019 Apr 25.

Molecular Pathology Unit, Massachusetts General Hospital Research Institute, Charlestown, MA 02129, USA; Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02139, USA. Electronic address:

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc, il2rga zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
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http://dx.doi.org/10.1016/j.cell.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570580PMC
June 2019

Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM.

Nat Commun 2019 04 23;10(1):1903. Epub 2019 Apr 23.

Molecular Pathology Unit & Cancer Center, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA, 02114, USA.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package.
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http://dx.doi.org/10.1038/s41467-019-09670-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478907PMC
April 2019

Longitudinal monitoring of cancer cell subpopulations in monolayers, 3D spheroids, and xenografts using the photoconvertible dye DiR.

Sci Rep 2019 04 5;9(1):5713. Epub 2019 Apr 5.

Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts, 02129, USA.

A central challenge in cancer biology is the identification, longitudinal tracking, and -omics analysis of specific cells in vivo. To this aim, photoconvertible fluorescent dyes are reporters that are characterized by a set of excitation and emission spectra that can be predictably altered, resulting in a distinct optical signature following irradiation with a specific light source. One such dye, DiR, is an infrared fluorescent membrane probe that can irreversibly undergo such a switch. Here, we demonstrate a method using DiR for the spatiotemporal labeling of specific cells in the context of cancer cell monolayer cultures, 3D tumor spheroids, and in vivo melanoma xenograft models to monitor the proliferation of cellular subpopulations of interest over time. Importantly, the photoconversion process is performed in situ, supporting the pursuit of novel avenues of research in molecular pathology.
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http://dx.doi.org/10.1038/s41598-019-42165-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450962PMC
April 2019

Molecularly distinct models of zebrafish Myc-induced B cell leukemia.

Leukemia 2019 02 20;33(2):559-562. Epub 2018 Dec 20.

Section of Pediatric Hematology-Oncology, Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.

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http://dx.doi.org/10.1038/s41375-018-0328-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365381PMC
February 2019

deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish.

Elife 2018 09 7;7. Epub 2018 Sep 7.

Department of Pathology, Massachusetts General Hospital Research Institute, Boston, Massachusetts.

The tumor-suppressor gene is mutated in >50% of human tumors and Li-Fraumeni patients with germ line inactivation are predisposed to developing cancer. Here, we generated deleted zebrafish that spontaneously develop malignant peripheral nerve-sheath tumors, angiosarcomas, germ cell tumors, and an aggressive Natural Killer cell-like leukemia for which no animal model has been developed. Because the tp53 deletion was generated in syngeneic zebrafish, engraftment of fluorescent-labeled tumors could be dynamically visualized over time. Importantly, engrafted tumors shared gene expression signatures with predicted cells of origin in human tissue. Finally, we showed that 3 enhanced invasion and metastasis in -induced embryonal rhabdomyosarcoma (ERMS), but did not alter the overall frequency of cancer stem cells, suggesting novel pro-metastatic roles for TP53 loss-of-function in human muscle tumors. In summary, we have developed a Li-Fraumeni zebrafish model that is amenable to large-scale transplantation and direct visualization of tumor growth in live animals.
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http://dx.doi.org/10.7554/eLife.37202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128690PMC
September 2018

JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia.

J Exp Med 2018 07 25;215(7):1929-1945. Epub 2018 Jun 25.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of Furthermore, is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from transgenic zebrafish express high levels of and demonstrate resistance to steroids in vivo. These studies establish as a novel oncogene in high-risk T-ALL and implicate overexpression of as a mechanism of steroid resistance in -overexpressing cells.
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http://dx.doi.org/10.1084/jem.20170484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028512PMC
July 2018

Cell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia.

Leukemia 2018 08 18;32(8):1860-1865. Epub 2018 Apr 18.

Department of Pathology, Massachusetts General Hospital Research Institute, Boston, MA, 02114, USA.

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http://dx.doi.org/10.1038/s41375-018-0130-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620784PMC
August 2018

Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma.

Cell Stem Cell 2018 03;22(3):414-427.e6

Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital Research Institute, Boston, MA 02129, USA; Harvard Stem Cell Institute, Cambridge, MA 02139, USA. Electronic address:

Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.
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http://dx.doi.org/10.1016/j.stem.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354590PMC
March 2018

Identification and characterization of T reg-like cells in zebrafish.

J Exp Med 2017 Dec 24;214(12):3519-3530. Epub 2017 Oct 24.

Program in Molecular Medicine and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg-deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg-like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.
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http://dx.doi.org/10.1084/jem.20162084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716030PMC
December 2017

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

Cancer Discov 2017 11 3;7(11):1336-1353. Epub 2017 Oct 3.

Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation. TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683427PMC
November 2017

Preclinical testing of the glycogen synthase kinase-3β inhibitor tideglusib for rhabdomyosarcoma.

Oncotarget 2017 Sep 16;8(38):62976-62983. Epub 2017 Jun 16.

Children's Cancer Therapy Development Institute, Beaverton, OR 97005, USA.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3β is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3β inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models . In this study, we tested the irreversible GSK3β-inhibitor, tideglusib for efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3β inhibitors may not be a viable treatment for aRMS or eRMS.
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http://dx.doi.org/10.18632/oncotarget.18520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609896PMC
September 2017

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

J Exp Med 2017 Oct 6;214(10):2875-2887. Epub 2017 Sep 6.

Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit (), interleukin-2 receptor γ a (), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.
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http://dx.doi.org/10.1084/jem.20170976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626406PMC
October 2017

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma.

Cell Rep 2017 06;19(11):2304-2318

Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Center of Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA; Harvard Stem Cell Institute, Boston, MA 02114, USA. Electronic address:

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.
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http://dx.doi.org/10.1016/j.celrep.2017.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563075PMC
June 2017

Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors.

Zebrafish 2017 08 30;14(4):379-382. Epub 2017 May 30.

1 Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital , Boston, Massachusetts.

The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.
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http://dx.doi.org/10.1089/zeb.2017.1434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549793PMC
August 2017

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

Dev Biol 2017 04 7;424(2):162-180. Epub 2017 Mar 7.

Departments of Molecular Genetics and Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210, USA; Center for Muscle Health and Neuromuscular Disorders, The Ohio State University and Nationwide Children's Hospital, Columbus, OH 43210, USA. Electronic address:

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-like 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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http://dx.doi.org/10.1016/j.ydbio.2017.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437870PMC
April 2017

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma.

Elife 2017 01 12;6. Epub 2017 Jan 12.

Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital, Boston, United States.

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.
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http://dx.doi.org/10.7554/eLife.19214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231408PMC
January 2017

Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.

J Exp Med 2016 11 24;213(12):2575-2589. Epub 2016 Oct 24.

Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129

Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.
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http://dx.doi.org/10.1084/jem.20160378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110017PMC
November 2016

T Cell Immune Deficiency in Mutant Zebrafish.

Mol Cell Biol 2016 Dec 14;36(23):2868-2876. Epub 2016 Nov 14.

Molecular Pathology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

[, ], is required for T cell activation. deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a loss-of-function mutation in zebrafish ( ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, 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 : 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 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study immune deficiency.
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http://dx.doi.org/10.1128/MCB.00281-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108876PMC
December 2016

Allograft Cancer Cell Transplantation in Zebrafish.

Adv Exp Med Biol 2016 ;916:265-87

Molecular Pathology and Cancer Center, Massachusetts General Hospital, Charlestown, MA, USA.

Allogeneic cell transplantation is the transfer of cells from one individual into another of the same species and has become an indispensable technique for studying development, immunology, regeneration and cancer biology. In experimental settings, tumor cell engraftment into immunologically competent recipients has greatly increased our understanding of the mechanisms that drive self-renewal, progression and metastasis in vivo. Zebrafish have quickly emerged as a powerful genetic model of cancer that has benefited greatly from allogeneic transplantation. Efficient engraftment can be achieved by transplanting cells into either early larval stage zebrafish that have not yet developed a functional acquired immune system or adult zebrafish following radiation or chemical ablation of the immune system. Alternatively, transplantation can be completed in adult fish using either clonal syngeneic strains or newly-generated immune compromised zebrafish models that have mutations in genes required for proper immune cell function. Here, we discuss the current state of cell transplantation as it pertains to zebrafish cancer and the available models used for dissecting important processes underlying cancer. We will also use the zebrafish model to highlight the power of cell transplantation, including its capacity to dynamically assess functional heterogeneity within individual cancer cells, visualize cancer progression and evolution, assess tumor-propagating potential and self-renewal, image cancer cell invasion and dissemination and identify novel therapies for treating cancer.
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http://dx.doi.org/10.1007/978-3-319-30654-4_12DOI Listing
September 2016

In Vivo Imaging of Cancer in Zebrafish.

Adv Exp Med Biol 2016 ;916:219-37

Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, 149 13th Street, #6012, Charlestown, MA, 02129, USA.

Zebrafish cancer models have greatly advanced our understanding of malignancy in humans. This is made possible due to the unique advantages of the zebrafish model including ex vivo development and large clutch sizes, which enable large-scale genetic and chemical screens. Transparency of the embryo and the creation of adult zebrafish devoid of pigmentation (casper) have permitted unprecedented ability to dynamically visualize cancer progression in live animals. When coupled with fluorescent reporters and transgenic approaches that drive oncogenesis, it is now possible to label entire or subpopulations of cancer cells and follow cancer growth in near real-time. Here, we will highlight aspects of in vivo imaging using the zebrafish and how it has enhanced our understanding of the fundamental aspects of tumor initiation, self-renewal, neovascularization, tumor cell heterogeneity, invasion and metastasis. Importantly, we will highlight the contribution of cancer imaging in zebrafish for drug discovery.
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http://dx.doi.org/10.1007/978-3-319-30654-4_10DOI Listing
September 2016

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

J Exp Med 2016 05 2;213(6):979-92. Epub 2016 May 2.

Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129 Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114 Harvard Stem Cell Institute, Cambridge, MA 02139

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.
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http://dx.doi.org/10.1084/jem.20152013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886368PMC
May 2016

Clusters of circulating tumor cells traverse capillary-sized vessels.

Proc Natl Acad Sci U S A 2016 May 18;113(18):4947-52. Epub 2016 Apr 18.

Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; Shriners Hospital for Children, Boston, MA 02114

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.
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http://dx.doi.org/10.1073/pnas.1524448113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983862PMC
May 2016

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

Nat Commun 2016 Jan 21;7:10358. Epub 2016 Jan 21.

Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts 02129, USA.

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2(E450fs) (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAF(V600E)-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.
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http://dx.doi.org/10.1038/ncomms10358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735845PMC
January 2016