Leonard I Zon

Howard Hughes Medical Institute, Ашбърн, Virginia, United States

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Publications (456)4798.58 Total impact

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    ABSTRACT: The “cancerized field” concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAFV600E mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.
    No preview · Article · Jan 2016 · Science
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    ABSTRACT: Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.
    Full-text · Article · Jan 2016 · Developmental Cell
  • Xiaoying Bai · Leonard I. Zon
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    ABSTRACT: Vertebrate blood derives from mesoderm. Blood formation (hematopoiesis) occurs in successive waves. The primitive wave predominantly generates red cells to transiently provide oxygen for the rapidly growing embryo. The definitive wave produces hematopoietic stem cells (HSCs) that ultimately differentiate into all the blood lineages in the adult. Hematopoiesis is tightly regulated by complex interactions between extrinsic and intrinsic factors. Transcription factors play intrinsic roles to define HSC generation and lineage differentiation. The process of hematopoiesis is highly conserved throughout vertebrate evolution. The genetic manipulation of animal models, such as mouse and zebrafish, has identified many essential genes controlling normal and malignant hematopoiesis.
    No preview · Chapter · Dec 2015
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    ABSTRACT: Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.
    Full-text · Article · Oct 2015 · Cell stem cell
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    ABSTRACT: Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)GFP(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-) GFP(+/+) HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.
    No preview · Article · Sep 2015 · Blood
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    ABSTRACT: The vertebrate heart develops from two distinct lineages of cardiomyocytes that arise from the first and second heart fields (FHF and SHF, respectively). The FHF forms the primitive heart tube, while adding cells from the SHF allow elongation at both poles of the tube. Initially seen as an exclusive characteristic of higher vertebrates, recent work has demonstrated the presence of a distinct FHF and SHF in lower vertebrates, including zebrafish. We found that key transcription factors that regulate septation and chamber formation in higher vertebrates, including Tbx5 and Pitx2, influence relative FHF and SHF contributions to the zebrafish heart tube. To identify molecular modulators of heart field migration, we used microarray-based expression profiling following inhibition of tbx5a and pitx2ab in embryonic zebrafish (Mosimann & Panakova, et al, 2015; GSE70750). Here, we describe in more detail the procedure used to process, prioritize, and analyze the expression data for functional enrichment.
    No preview · Article · Sep 2015 · Genomics Data
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    ABSTRACT: The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.
    Full-text · Article · Aug 2015 · Nature Communications
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    Full-text · Dataset · Aug 2015
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    ABSTRACT: Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAFV600E;p53-/- fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (~1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems. Copyright © 2015, American Association for Cancer Research.
    Full-text · Article · Aug 2015 · Cancer Research

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.
    No preview · Article · Jul 2015 · Nature Cell Biology
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    ABSTRACT: Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.
    No preview · Article · Jul 2015 · Nature
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    ABSTRACT: Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l(cas002) embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.Cell Research advance online publication 3 July 2015; doi:10.1038/cr.2015.81.
    Preview · Article · Jul 2015 · Cell Research
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    ABSTRACT: In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.
    Full-text · Article · Jul 2015 · PLoS Genetics
  • X Peng · M Dong · L Ma · X-E Jia · J Mao · C Jin · Y Chen · L Gao · X Liu · K Ma · [...] · T Du · Y Jin · Q Huang · K Li · L I Zon · T Liu · M Deng · Y Zhou · X Xi · S Chen ·
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    ABSTRACT: Controlled self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) are critical for vertebrate development and survival. These processes are tightly regulated by the transcription factors, signaling molecules, and epigenetic factors. Impaired regulations of their function could result in hematological malignancies. Using a large-scale zebrafish N-ethyl-N-nitrosourea (ENU) mutagenesis screening, we identified a line named LDD731, which presented significantly increased HSPCs in hematopoietic organs. Further analysis revealed that the erythoid/myeloid lineages in definitive hematopoiesis were increased while the primitive hematopoiesis was not affected. The homozygous mutation was lethal with a median survival time around 14-15 days post-fertilization. The causal mutation was located by positional cloning in the c-cbl gene, of which the human ortholog, c-CBL, is found frequently mutated in myeloproliferative neoplasms (MPN) or acute leukemia. Sequence analysis showed the mutation in LDD731 caused a histidine-to-tyrosine substitution of the amino acid (aa) codon 382 within the RING finger domain of c-Cbl. Moreover, the myeloproliferative phenotype in zebrafish seemed dependent on the Flt3 (fms-like tyrosine kinase 3) signaling, consistent with that observed in both mice and humans. Our study may shed new lights on the pathogenesis of MPN and provide a useful in vivo vertebrate model for drug screenings for this syndrome.Leukemia accepted article preview online, 24 June 2015. doi:10.1038/leu.2015.154.
    No preview · Article · Jun 2015 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    ABSTRACT: Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition. © 2015 Kim et al.
    Preview · Article · Apr 2015 · Journal of Experimental Medicine
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    ABSTRACT: Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. © 2015 Jing et al.
    Full-text · Article · Apr 2015 · Journal of Experimental Medicine

  • No preview · Article · Apr 2015 · Leukemia Research
  • Julien Ablain · Ellen M Durand · Song Yang · Yi Zhou · Leonard I Zon
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    ABSTRACT: CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Mar 2015 · Developmental Cell
  • Michelle Dang · Leonard I. Zon
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    ABSTRACT: Metastatic melanoma is an aggressive disease that historically has had very limited therapeutic options. FDA approved BRAF and MEK inhibitors are now used to successfully treat mutant BRAF(V) (600E) -driven human melanomas; however, the efficacy is short-lived, and patients generally develop resistance within a year. Immunotherapies are also promising, but predicting responses is still difficult and side effects can be severe. As there is no effective secondary line of treatment for patients with BRAF/MEK-inhibitor drug-resistant melanomas, there is a major unmet clinical need to identify mechanisms of drug resistance and develop novel drug combinations to prevent or delay resistance. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Mar 2015 · Pigment Cell & Melanoma Research

Publication Stats

34k Citations
4,798.58 Total Impact Points

Institutions

  • 1992-2015
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
    • Istituto Superiore di Sanità
      • Department of Haematology, Oncology and Molecular Medicine
      Roma, Latium, Italy
  • 1989-2015
    • Boston Children's Hospital
      • • Division of Genetics
      • • Manton Center of Orphan Disease Research
      • • Department of Pediatrics
      Boston, Massachusetts, United States
  • 1987-2015
    • Harvard University
      • • Department of Chemistry and Chemical Biology
      • • Department of Developmental Biology
      Cambridge, Massachusetts, United States
  • 2014
    • Neural Stem Cell Institute
      Rensselaer, New York, United States
  • 1992-2014
    • Dana-Farber Cancer Institute
      • • Department of Medical Oncology
      • • Department of Pediatric Oncology
      Boston, Massachusetts, United States
  • 1986-2014
    • Harvard Medical School
      • • Department of Pediatrics
      • • Department of Genetics
      • • Department of Medicine
      Boston, Massachusetts, United States
    • University of Massachusetts Boston
      Boston, Massachusetts, United States
  • 2009
    • Beth Israel Deaconess Medical Center
      • Division of Hematology/Oncology
      Boston, MA, United States
  • 2008
    • University of Utah
      • Department of Pediatrics
      Salt Lake City, Utah, United States
  • 2007
    • University of Pittsburgh
      • Department of Medicine
      Pittsburgh, Pennsylvania, United States
  • 2005
    • Brigham and Women's Hospital
      • Department of Pathology
      Boston, Massachusetts, United States
  • 2002-2003
    • Complutense University of Madrid
      • Department of Cellular Biology
      Madrid, Madrid, Spain
    • Salk Institute
      لا هویا, California, United States
  • 2001
    • National Human Genome Research Institute
      베서스다, Maryland, United States
  • 1999-2001
    • Princeton University
      • Department of Molecular Biology
      Princeton, New Jersey, United States
    • Washington University in St. Louis
      • Department of Genetics
      Saint Louis, MO, United States
    • University of Rochester
      • Department of Biology
      Rochester, NY, United States
  • 2000
    • Wolfson Childrens Hospital
      Jacksonville, Florida, United States
  • 1998
    • Kumamoto University
      • Department of Cell Differentiation
      Kumamoto, Kumamoto, Japan
  • 1994
    • Massachusetts General Hospital
      • Diabetes Laboratory
      Boston, MA, United States