Leonard I Zon

Neural Stem Cell Institute, Rensselaer, New York, United States

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Publications (395)4107.92 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The Ten-ElevenTranslocation-2 (TET2) gene encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors leading to ineffective hematopoiesis. We used genome editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2 m/m zebrafish exhibited normal embryonic and larval hematopoiesis, but developed progressive clonal myelodysplasia as they aged, culminating in MDS at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2 m/m mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow, but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in non-hematopoietic tissues but not in hematopoietic cells. tet2 m/m are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and for small molecule screens in embryos to identify compounds with specific activity against tet2 mutant cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and cellular biology. 12/2014;
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    ABSTRACT: Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.
    Nature Biotechnology 12/2014; · 32.44 Impact Factor
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    ABSTRACT: One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.
    Nat Biotechnol. 10/2014; 26:305-12.
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    ABSTRACT: Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.
    Nucleic Acids Research 09/2014; · 8.81 Impact Factor
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    ABSTRACT: Throughout the lifetime of an individual, hematopoietic stem cells (HSCs) self-renew and differentiate into lineages that include erythrocytes, platelets and all immune cells. HSC transplantation offers a potentially curative treatment for a number of hematopoietic and non-hematopoietic malignancies as well as immune and genetic disorders. Limited availability of immune-matched donors reduces the viable options for many patients in need of HSC transplantation, particularly those of diverse racial and ethnic backgrounds. Due to rapid availability and less stringent immune-matching requirements, umbilical cord blood (UCB) has emerged as a valuable source of transplantable HSCs. A single UCB unit contains a suboptimal number of HSCs for treating larger children or adults and there has thus been great clinical interest in expanding UCB HSCs ex vivo for use in transplantation. In this review we discuss the latest research and future avenues for the therapeutic use of small lipid mediator dmPGE2 to expand HSC numbers for transplantation. Originally identified in a chemical screen in zebrafish, dmPGE2 has now advanced to a phase II clinical trial as a therapy for patients with leukemia and lymphoma who are undergoing UCB transplantation.
    Experimental cell research. 08/2014;
  • Experimental hematology. 08/2014; 42(8S):S15.
  • Experimental hematology. 08/2014; 42(8S):S63.
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    ABSTRACT: Thyroid hormone is a master regulator of differentiation and growth, and its action is terminated by the enzymatic removal of an inner-ring iodine catalyzed by the selenoenzyme type 3 deiodinase (dio3). Our studies of the zebrafish reveal that the dio3 gene is duplicated in this species and that embryonic deiodination is an important determinant of embryo size. Although both dio3 paralogs encode enzymatically active proteins with high affinity for thyroid hormones, their anatomic patterns of expression are markedly divergent and only embryos with knockdown of dio3b, a biallelically expressed selenoenzyme expressed in the developing central nervous system, manifest severe thyroid hormone-dependent growth restriction at 72 hours post fertilization. This indicates that the embryonic deficiency of dio3, once considered only a placental enzyme, causes microsomia independently of placental physiology and raises the intriguing possibility that fetal abnormalities in human deiodination may present as intrauterine growth retardation. By mapping the gene structures and enzymatic properties of all four zebrafish deiodinases, we also identify dio3b as the first multiexon dio3 gene, containing a large intron separating its open reading frame from its selenocysteine insertion sequence (SECIS) element.
    Endocrinology 07/2014; · 4.72 Impact Factor
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    ABSTRACT: During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Objectives: Here we report on a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Methods: Developmentally staged fan mutant and wild type sibling embryos were analyzed using ISH, IHC, AR/Ab staining, and Northern blot analyses. In vitro tissue culture experiments included shRNA analyses, IHC and IF. Y2H studies were used to investigate protein binding. Results: Our results show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate proper nucleolar localization and protein interactions. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Conclusions: Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and future treatments for ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.
    06/2014
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    ABSTRACT: In non-mammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bi-potent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin (Epo), and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.
    Blood 05/2014; · 9.78 Impact Factor
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    ABSTRACT: Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germline mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in two large DBA families. After filtering, one nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29) gene was present in all 5 DBA affected individuals and the obligate carrier, and absent from the unaffected non-carrier parent in one DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious, and resulted in haploinsufficiency of RPS29 expression compared with wildtype RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-rRNA processing defect compared to the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebrafish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for ribosomal RNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germline mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebrafish model.
    Blood 05/2014; · 9.78 Impact Factor
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    ABSTRACT: Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.
    Chemistry & biology 03/2014; · 6.52 Impact Factor
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    ABSTRACT: Neural crest cells (NCCs), a unique cell population originating from the dorsal side of the embryonic neural tube, contribute to a significant portion of the craniofacial skeleton. Aberrant NCC specification, migration, and differentiation can lead to craniofacial defects. In recent years, it has become evident that mutations in a variety of ribosome biogenesis proteins result in distinct craniofacial defects. Objective: Here we investigate the function of the ribosome biogenesis protein Wdr43 in craniofacial development. Method: A forward genetic chemical mutagenesis screen combined with Alcian blue/Alizarin Red staining was used to identify zebrafish mineralized tissue mutants. Result: We identified the zebrafish mutant, fantome (fan), which exhibits severe craniofacial cartilage defects. Positional cloning was used to identify a premature stop codon mutation at amino acid 356 in zebrafish wdr43, the ortholog to yeast Utp5 known to function in ribosome biogenesis. fan mutants exhibit increased apoptosis, reduced cell proliferation, and reduced craniofacial cartilage formation, all of which are partially relieved by targeted depletion of p53. In vitro and in vivo studies were used to demonstrate that Wdr43 is required for the proper subnucleolar localization of a variety of nucleolar proteins including Tcof1, the gene commonly mutated in Treacher-Collins Syndrome. Conclusion: We reveal, for the first time, roles for the ribosome biogenesis gene wdr43 in craniofacial development. We anticipate that the zebrafish fan mutant will be a useful tool for devising effective methods to prevent and/or treat a variety of craniofacial ribosomopathy mutations, including Treacher-Collins Syndrome. This research was supported by NIH/NIDCR R01DE018043 (PCY), NIH/NIGMS R01GM52581 (SJB) and Tufts University School of Dental Medicine, Boston MA.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.
    The American Journal of Human Genetics 03/2014; · 11.20 Impact Factor
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    Elliott J Hagedorn, Leonard I Zon
    Journal of Experimental Medicine 03/2014; 211(3):384-5. · 13.21 Impact Factor
  • Leonard Zon
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    ABSTRACT: Translating basic research findings into therapeutic settings presents many scientific, logistic, and financial challenges for academic researchers. Here, I highlight some key insights for navigating such challenges based on recent clinical trials initiated by basic research from my lab.
    Cell stem cell 02/2014; 14(2):146-8. · 23.56 Impact Factor
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    ABSTRACT: Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC.
    Nature Communications 02/2014; 5:3228. · 10.74 Impact Factor
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    ABSTRACT: Comprehensive functional annotation of vertebrate genomes is fundamental to biological discovery. Reverse genetic screening has been highly useful for determination of gene function, but is untenable as a systematic approach in vertebrate model organisms given the number of surveyable genes and observable phenotypes. Unbiased prediction of gene-phenotype relationships offers a strategy to direct finite experimental resources towards likely phenotypes, thus maximizing de novo discovery of gene functions. Here we prioritized genes for phenotypic assay in zebrafish through machine learning, predicting the effect of loss of function of each of 15,106 zebrafish genes on 338 distinct embryonic anatomical processes. Focusing on cardiovascular phenotypes, the learning procedure predicted known knockdown and mutant phenotypes with high precision. In proof-of-concept studies we validated 16 high-confidence cardiac predictions using targeted morpholino knockdown and initial blinded phenotyping in embryonic zebrafish, confirming a significant enrichment for cardiac phenotypes as compared with morpholino controls. Subsequent detailed analyses of cardiac function confirmed these results, identifying novel physiological defects for 11 tested genes. Among these we identified tmem88a, a recently described attenuator of Wnt signaling, as a discrete regulator of the patterning of intercellular coupling in the zebrafish cardiac epithelium. Thus, we show that systematic prioritization in zebrafish can accelerate the pace of developmental gene function discovery.
    Development 01/2014; 141(1):224-235. · 6.60 Impact Factor
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    ABSTRACT: During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.
    PLoS Genetics 01/2014; 10(1):e1004074. · 8.52 Impact Factor
  • Julien Ablain, Leonard I Zon
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    ABSTRACT: Long restricted to the field of developmental biology, the use of the zebrafish (Danio rerio) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism.
    Trends in cell biology 12/2013; 23(12):584-6. · 12.12 Impact Factor

Publication Stats

23k Citations
4,107.92 Total Impact Points

Institutions

  • 2009–2014
    • Neural Stem Cell Institute
      Rensselaer, New York, United States
    • Beth Israel Deaconess Medical Center
      • Division of Hematology/Oncology
      Boston, MA, United States
  • 1995–2014
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1992–2014
    • Harvard Medical School
      • • Department of Systems Biology
      • • Department of Surgery
      • • Department of Genetics
      • • Department of Pediatrics
      Boston, Massachusetts, United States
    • Istituto Superiore di Sanità
      • Laboratory of Virology
      Roma, Latium, Italy
  • 2013
    • Memorial Sloan-Kettering Cancer Center
      New York City, New York, United States
  • 2009–2013
    • University of California, San Diego
      • • Department of Cellular and Molecular Medicine (CMM)
      • • Section of Cell and Developmental Biology
      San Diego, California, United States
  • 1990–2013
    • Dana-Farber Cancer Institute
      • • Center for Hematologic Oncology
      • • Department of Pediatric Oncology
      Boston, Massachusetts, United States
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, MA, United States
  • 1989–2013
    • Boston Children's Hospital
      • • Manton Center of Orphan Disease Research
      • • Department of Pediatrics
      Boston, Massachusetts, United States
  • 2012
    • Karlsruhe Institute of Technology
      • Institut für Toxikologie und Genetik
      Karlsruhe, Baden-Wuerttemberg, Germany
    • St. Jude Children's Research Hospital
      Memphis, Tennessee, United States
    • University of Chicago
      • Department of Pediatrics
      Chicago, IL, United States
  • 1994–2012
    • Harvard University
      • • Department of Stem Cell and Regenerative Biology
      • • Department of Developmental Biology
      Cambridge, MA, United States
    • Massachusetts General Hospital
      • Diabetes Laboratory
      Boston, MA, United States
  • 2005–2011
    • Brigham and Women's Hospital
      • Department of Pathology
      Boston, MA, United States
    • Riley Hospital for Children
      Indianapolis, Indiana, United States
  • 2007
    • Partners HealthCare
      Boston, Massachusetts, United States
    • University of Pittsburgh
      • School of Medicine
      Pittsburgh, PA, United States
  • 2002
    • French National Centre for Scientific Research
      • Institute for Molecular and Cellular Biology (IBMC)
      Paris, Ile-de-France, France
  • 2001–2002
    • National Human Genome Research Institute
      Maryland, United States
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 2000
    • Wolfson Childrens Hospital
      Jacksonville, Florida, United States
  • 1999
    • University of Massachusetts Medical School
      Worcester, Massachusetts, United States
    • Washington University in St. Louis
      • Department of Genetics
      Saint Louis, MO, United States
    • Boston University
      • Center for Human Genetics
      Boston, MA, United States
  • 1995–1999
    • Northeastern University
      • Department of Biology
      Boston, MA, United States
  • 1997
    • University of Nebraska at Omaha
      • Department of Genetics, Cell Biology and Anatomy
      Omaha, NE, United States
  • 1996
    • Niigata University
      • Department of Biology
      Niigata-shi, Niigata-ken, Japan
  • 1993
    • University of North Carolina at Chapel Hill
      • Department of Chemistry
      Chapel Hill, NC, United States
  • 1990–1991
    • Whitehead Institute for Biomedical Research
      Cambridge, Massachusetts, United States