Biomedical subjects

Leonard I Zon

Publications and source records attributed to Leonard I Zon.

At least 19 recordsLinked to original sources

PubMed2026

Functional characterization of the 9q34.13 locus identifies RAPGEF1 as a candidate gene modulating risk for melanoma and nevi via RAS activation.

Genome-wide association studies identified a melanoma- and nevus count-associated locus on chromosome band 9q34.13. Fine-mapping and melanocyte expression data collectively suggest two potential risk genes with opposite associations with risk: higher levels of Rap guanine nucleotide exchange factor 1 (RAPGEF1) and lower levels of uridine-cytidine kinase 1 (UCK1). Colocalization analyses and conditional transcriptome-wide association studies (TWASs) suggest multiple causal cis-regulatory sequence variants in partial linkage disequilibrium (LD) to each other. Melanocyte capture-HiC and CRISPR inhibition demonstrated regulatory interactions between fine-mapped variants and the RAPGEF1 and UCK1 promoters. Focusing on RAPGEF1, we demonstrate that RAPGEF1 expression promotes melanocyte growth and drives colony formation of human immortalized melanocytes. Following treatment with human epidermal growth factor (EGF), RAPGEF1 overexpression activated both RAP1 and RAS. Further, we show that RAPGEF1 expression is significantly enriched in melanomas that lack strongly activating RAS-MAPK pathway mutations, which suggests that RAPGEF1 may promote oncogenic RAS-MAPK pathway signaling in melanomas. Furthermore, in these tumors, we provide preliminary evidence to support the prognostic relevance of RAPGEF1 expression in individuals whose melanomas lack RAS or BRAF mutations. Together with other recent studies, these data suggest that germline variation influencing RAS activation may play a key role in nevus development and melanoma risk.

GWAS↗

PubMed2026

Functional characterization of the 9q34.13 locus identifies RAPGEF1 as modulating risk for melanoma and nevi via RAS activation.

Genome-wide association studies identified a melanoma- and nevus count-associated locus on chromosome band 9q34.13. Fine-mapping and melanocyte expression data collectively suggest two potential causal genes with opposite association with risk: higher levels of Rap guanine nucleotide exchange factor 1 (RAPGEF1) and lower levels of uridine-cytidine kinase 1 (UCK1). Colocalization analyses and conditional TWAS suggest multiple causal cis-regulatory sequence variants in partial linkage disequilibrium (LD) to each other. Melanocyte capture-HiC and CRISPR-inhibition demonstrated regulatory interactions between fine-mapped variants and the RAPGEF1 and UCK1 promoters. Focusing on RAPGEF1, we demonstrate RAPGEF1 expression promotes melanocyte growth and drives malignant transformation of human immortalized melanocytes. Following treatment with human EGF, RAPGEF1 overexpression activated both RAP1 and RAS. Further, we show RAPGEF1 expression is significantly enriched in melanomas lacking strongly activating RAS-MAPK mutations, suggesting that RAPGEF1 may promote oncogenic RAS-MAPK signaling in melanomas. Furthermore, in these tumors, we provide preliminary evidence to support the prognostic relevance of RAPGEF1 expression in patients lacking RAS or BRAF mutations. Together with other recent studies, these data suggest that germline variation influencing RAS activation may play a key role in nevus development and melanoma risk.

Journal Article↗

PubMed2007

A mutation in separase causes genome instability and increased susceptibility to epithelial cancer.

Proper chromosome segregation is essential for maintenance of genomic integrity and instability resulting from failure of this process may contribute to cancer. Here, we demonstrate that a mutation in the mitotic regulator separase is responsible for the cell cycle defects seen in the zebrafish mutant, cease&desist (cds). Analysis of cds homozygous mutant embryos reveals high levels of polyploidy and aneuploidy, spindle defects, and a mitotic exit delay. Carcinogenesis studies demonstrated that cds heterozygous adults have a shift in tumor spectrum with an eightfold increase in the percentage of fish bearing epithelial tumors, indicating that separase is a tumor suppressor gene in vertebrates. These data strongly support a conserved cross-species role for mitotic checkpoint genes in genetic stability and epithelial carcinogenesis.

Animals↗

PubMed2006

Cdx4 dysregulates Hox gene expression and generates acute myeloid leukemia alone and in cooperation with Meis1a in a murine model.

HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, CDX2, and CDX4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. We report here that CDX4 is expressed normally in early hematopoietic progenitors and is expressed aberrantly in approximately 25% of acute myeloid leukemia (AML) patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system and dysregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture, and generate a partially penetrant, long-latency AML in bone marrow transplant recipients. Coexpression of the Hox cofactor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure-function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains but not the putative Pbx cofactor interaction motif. Together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia.

Adult↗

PubMed2006

A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

Alkaline Phosphatase↗

PubMed2006

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens.

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.

Amino Acid Sequence↗

PubMed2006

BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis.

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.

Animals↗

PubMed2006

Mitoferrin is essential for erythroid iron assimilation.

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

Anemia↗

PubMed2006

The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis.

The hox genes play a central role in organogenesis and are implicated in the formation of hematopoietic stem cells (HSCs). The cdx genes encode homeodomain transcription factors that act as master regulators of the hox genes. In zebrafish, mutations in cdx4 cause a severe, but not complete, deficit in embryonic blood cells. Here, we report the expression and function of cdx1a, a zebrafish Cdx1 paralogue. Using morpholino-mediated knockdown of cdx1a in a cdx4 mutant background, we show that a deficiency in both cdx genes causes a severe perturbation of hox gene expression and a complete failure to specify blood. The hematopoietic defect in cdx-deficient embryos does not result from a general block in posterior mesoderm differentiation as endothelial cells and kidney progenitors are still formed in the doubly deficient embryos. In addition, cdx-deficient embryos display a significant reduction in runx1a(+) putative HSCs in the zebrafish equivalent to the aorta-gonad-mesonephros (AGM) region. Overexpressing hoxa9a in cdx-deficient embryos rescues embryonic erythropoiesis in the posterior mesoderm as well as the formation of HSCs in the AGM region. Taken together, these results suggest that the cdx-hox pathway plays an essential role in the formation of both embryonic erythroid cells and definitive HSCs during vertebrate embryogenesis.

Amino Acid Sequence↗

PubMed2006

Homing sweet homing: odyssey of hematopoietic stem cells.

The lineage relationship between the blood cells found in the developmentally successive hematopoietic organs has remained elusive. In this issue of Immunity, Murayama et al. (2006) track the migration of nascent hematopoietic stem cells in zebrafish from their site of origin to a newly described intermediate location.

Animals↗

PubMed2006

A chemical genetic screen for cell cycle inhibitors in zebrafish embryos.

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

Animals↗

PubMed2005

Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism.

The genetic basis of vertebrate morphological evolution has traditionally been very difficult to examine in naturally occurring populations. Here we describe the generation of a genome-wide linkage map to allow quantitative trait analysis of evolutionarily derived morphologies in the Mexican cave tetra, a species that has, in a series of independent caves, repeatedly evolved specialized characteristics adapted to a unique and well-studied ecological environment. We focused on the trait of albinism and discovered that it is linked to Oca2, a known pigmentation gene, in two cave populations. We found different deletions in Oca2 in each population and, using a cell-based assay, showed that both cause loss of function of the corresponding protein, OCA2. Thus, the two cave populations evolved albinism independently, through similar mutational events.

Albinism↗

PubMed2005

A critical role for calponin 2 in vascular development.

Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.

Amino Acid Sequence↗

PubMed2005

Zebrafish to humans: evolution of the alpha3-chain of type IV collagen and emergence of the autoimmune epitopes associated with Goodpasture syndrome.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.

Animals↗

PubMed2005

Hematopoietic stem cell fate is established by the Notch-Runx pathway.

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo.

Animals↗

PubMed2005

A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility.

A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family. crb mutant embryos have defects in mitotic progression and spindle formation, and exhibit genome instability. Regulation of cyclin B levels by bmyb appears to be the mechanism of mitotic accumulation in crb. Carcinogenesis studies reveal increased cancer susceptibility in adult crb heterozygotes. Gene-expression signatures associated with loss of bmyb in zebrafish are also correlated with conserved signatures in human tumor samples, and down-regulation of the B-myb signature genes is associated with retention of p53 function. Our findings show that zebrafish screens can uncover cancer pathways, and demonstrate that loss of function of bmyb is associated with cancer.

Animals↗

PubMed2005

Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis.

Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly.

5-Aminolevulinate Synthetase↗

PubMed2005

Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.

Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.

Animals↗