ABSTRACT
Clonal hematopoiesis results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. We developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in clonal hematopoiesisassociated genes such as asxl1 promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.
Subject(s)
Clonal Hematopoiesis , Hematopoietic Stem Cells/physiology , Inflammation , Myeloid Cells/physiology , Animals , CRISPR-Cas Systems , Cytokines/genetics , Cytokines/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , Frameshift Mutation , Genes, p53 , Inflammation/genetics , Mutation , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , RNA-Seq , Repressor Proteins/genetics , Selection, Genetic , Single-Cell Analysis , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Determining the relative configuration or enantiomeric excess of a substance may be achieved using NMR spectroscopy by employing chiral shift reagents (CSRs). Such reagents interact noncovalently with the chiral solute, resulting in each chiral form experiencing different magnetic anisotropy; this is then reflected in their NMR spectra. The Keplerate polyoxometalate (POM) is a molybdenum-based, water-soluble, discrete inorganic structure with a pore-accessible inner cavity, decorated by differentiable ligands. Through ligand exchange from the self-assembled nanostructure, a set of chiral Keplerate host molecules has been synthesised. By exploiting the interactions of analyte molecules at the surface pores, the relative configuration of chiral amino alcohol guests (phenylalaninol and 2-amino-1-phenylethanol) in aqueous solvent was establish and their enantiomeric excess was determined by 1 H NMR using shifts of ΔΔδ=0.06â ppm. The use of POMs as chiral shift reagents represents an application of a class that is yet to be well established and opens avenues into aqueous host-guest chemistry with self-assembled recognition agents.
Subject(s)
Amino Alcohols , Water , Capsules , Oxides , StereoisomerismABSTRACT
Split hand/foot malformation (SHFM) is a congenital limb deficiency with missing or shortened central digits. Some SHFM genes have been identified but the cause of many SHFM cases is unknown. We used single-nucleotide polymorphism (SNP) microarray analysis to detect copy-number variants (CNVs) in 25 SHFM cases without other birth defects from New York State (NYS), prioritized CNVs absent from population CNV databases, and validated these CNVs using quantitative real-time polymerase chain reaction (qPCR). We tested for the validated CNVs in seven cases from Iowa using qPCR, and also sequenced 36 SHFM candidate genes in all the subjects. Seven NYS cases had a potentially deleterious variant: two had a p.R225H or p.R225L mutation in TP63, one had a 17q25 microdeletion, one had a 10q24 microduplication and three had a 17p13.3 microduplication. In addition, one Iowa case had a de novo 10q24 microduplication. The 17q25 microdeletion has not been reported previously in SHFM and included two SHFM candidate genes (SUMO2 and GRB2), while the 10q24 and 17p13.3 CNVs had breakpoints within genomic regions that contained putative regulatory elements and a limb development gene. In SHFM pathogenesis, the microdeletion may cause haploinsufficiency of SHFM genes and/or deletion of their regulatory regions, and the microduplications could disrupt regulatory elements that control transcription of limb development genes.
Subject(s)
DNA Copy Number Variations , Genetic Association Studies , Limb Deformities, Congenital/genetics , Mutation , Alleles , Chromosome Aberrations , Female , Humans , Limb Deformities, Congenital/diagnosis , Male , Phenotype , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain Reaction , Regulatory Sequences, Nucleic Acid , Reproducibility of Results , Sequence Analysis, DNAABSTRACT
Tissue or cell transplantation is an invaluable technique with a multitude of applications including studying the developmental potential of certain cell populations, dissecting cell-environment interactions, and identifying stem cells. One key technical requirement for performing transplantation assays is the capability of distinguishing the transplanted donor cells from the endogenous host cells and tracing the donor cells over time. The zebrafish has emerged as an excellent model organism for performing transplantation assays, thanks in part to the transparency of embryos and even adults when pigment mutants are employed. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be readily identified and studied during development in vivo. In this chapter, we will discuss the rationale of different types of zebrafish transplantation in both embryos and adults and then focus on four detailed methods of transplantation: blastula/gastrula transplantation for mosaic analysis, hematopoietic stem cell transplantation, chemical screening using a transplantation model, and tumor transplantation.
Subject(s)
Cell Differentiation/genetics , Hematopoietic Stem Cell Transplantation/methods , Transplantation/methods , Zebrafish/genetics , Animals , Blastula/growth & development , Embryo, Nonmammalian , Gastrula/growth & development , Zebrafish/growth & developmentABSTRACT
Zebrafish chemical screening allows for an in vivo assessment of small molecule modulation of biological processes. Compound toxicities, chemical alterations by metabolism, pharmacokinetic and pharmacodynamic properties, and modulation of cell niches can be studied with this method. Furthermore, zebrafish screening is straightforward and cost effective. Zebrafish provide an invaluable platform for novel therapeutic discovery through chemical screening.
Subject(s)
Drug Discovery/methods , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Zebrafish/growth & development , Animals , Embryo, Nonmammalian/drug effects , Humans , Small Molecule Libraries/pharmacology , Zebrafish/geneticsABSTRACT
The c-MYB transcription factor is a key regulator of hematopoietic cell proliferation and differentiation, and dysregulation of c-MYB activity often associates with various hematological disorders. Yet, its pathogenic role remains largely unknown due to lack of suitable animal models. Here, we report a detail characterization of a c-myb-gfp transgenic zebrafish harboring c-Myb hyperactivity (named c-mybhyper). This line exhibits abnormal granulocyte expansion that resembles human myelodysplastic syndrome (MDS) from embryonic stage to adulthood. Strikingly, a small portion of c-mybhyper adult fish develops acute myeloid leukemia-like or acute lymphoid leukemia-like disorders with age. The myeloid and lymphoid malignancies in c-mybhyper adult fish are likely caused by the hyperactivity of c-myb, resulting in the dysregulation of a number of cell-cycle-related genes and hyperproliferation of hematopoietic precursor cells. Finally, treatment with c-myb target drug flavopiridol can relieve the MDS-like symptoms in both c-mybhyper embryos and adult fish. Our study establishes a zebrafish model for studying the cellular and molecular mechanisms underlying c-Myb-associated leukemogenesis as well as for anti-leukemic drug screening.
Subject(s)
Disease Models, Animal , Leukemia, Myeloid, Acute/etiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/etiology , Proto-Oncogene Proteins c-myb/metabolism , Animals , Animals, Genetically Modified , Cell Cycle/genetics , Cell Proliferation/genetics , Hematopoietic Stem Cells/cytology , Humans , ZebrafishABSTRACT
The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Gene Targeting/methods , Genetic Engineering/methods , Animals , Embryonic Development/genetics , Morpholinos/genetics , Organ Specificity/genetics , Zebrafish/geneticsABSTRACT
Zebrafish is an excellent genetic and developmental model for the study of vertebrate development and disease. Its ability to produce an abundance of transparent, externally developed embryos has facilitated large-scale genetic and chemical screens for the identification of critical genes and chemical factors that modulate developmental pathways. These studies can have profound implications for the diagnosis and treatment of a variety of human diseases. Recent advancements in molecular and genomic studies have provided valuable tools and resources for comprehensive and high-resolution analysis of epigenomes during cell specification and lineage differentiation throughout development. In this chapter, we describe two simple methods to evaluate protein-DNA interaction and chromatin architecture in erythrocytes from adult zebrafish. These are chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) and an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). These techniques, together with gene expression profiling, are useful for analyzing epigenomic regulation of cell specification, differentiation, and function during zebrafish development in both normal and disease models.
Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/genetics , Gene Expression Profiling/methods , Genomics/methods , Animals , Erythrocytes/metabolism , Genome , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA/methods , Zebrafish/geneticsABSTRACT
Zebrafish embryonic cell cultures have many useful properties that make them complementary to intact embryos for a wide range of studies. Embryonic cell cultures allow for maintenance of transient cell populations, control of chemical and mechanical cues received by cells, and facile chemical screening. Zebrafish cells can be cultured in either heterogeneous or homogeneous cultures from a wide range of developmental time points. Here we describe two methods with particular applicability to chemical screening: a method for the culture of blastomeres for directed differentiation toward the myogenic lineage and a method for the culture of neural crest cells in heterogeneous cultures from early somitogenesis embryos.
Subject(s)
Blastomeres/cytology , Cell Culture Techniques , Embryo, Nonmammalian/cytology , Zebrafish/growth & development , Animals , Embryonic Development , Neural Crest/cytologyABSTRACT
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 allows 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.
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 mutagenesis screening, we identified a line named LDD731, which presented significantly increased HSPCs in hematopoietic organs. Further analysis revealed that the cells of erythroid/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, the human ortholog of which, 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 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 light on the pathogenesis of MPN and provide a useful in vivo vertebrate model of this syndrome for screening drugs.
Subject(s)
Myeloproliferative Disorders/genetics , Point Mutation , Proto-Oncogene Proteins c-cbl/genetics , Animals , Cell Proliferation , Hematopoiesis , Hematopoietic Stem Cells/physiology , Humans , Myeloproliferative Disorders/etiology , Phenotype , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/chemistry , ZebrafishABSTRACT
BACKGROUND: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. METHODS AND RESULTS: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. CONCLUSION: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.
Subject(s)
Blood Platelets/enzymology , Myosin-Light-Chain Kinase/blood , Thrombosis/enzymology , Zebrafish Proteins/blood , Zebrafish/blood , Animals , Animals, Genetically Modified , Cell Shape , Disease Models, Animal , Fibrinogen/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Knockdown Techniques , Myosin-Light-Chain Kinase/genetics , Oligonucleotide Array Sequence Analysis , Platelet Adhesiveness , Platelet Membrane Glycoprotein IIb/blood , RNA, Messenger/blood , Recombinant Fusion Proteins/blood , Thrombosis/blood , Thrombosis/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Zebrafish (Danio rerio) has been used in the present work to study the fish response to bacterial lipopolysaccharide (LPS) exposure and LPS tolerance. These mechanisms are not completely understood in mammals and, presently, are totally unknown in fish. Zebrafish larval survival was assessed following treatment with various types of LPS at a variety of concentrations to determine the sensitivity of zebrafish to LPS-induced immune activation. In addition, fish pretreated with a sublethal concentration of LPS did not die after exposure to a lethal concentration of LPS demonstrating, for the first time that LPS tolerance also happens in fish. The time interval between pretreatment and secondary exposure as well as the type of pretreatment dictated the strength of protection. Since zebrafish are in intimate contact with microorganisms, the high resistance of fish to LPS suggests that there must be a tight control of the LPS receptor cluster in order to avoid an excess of inflammation. One of these components is CXCR4, which has previously been shown to regulate the signal transduced by TLR4. Treating fish with AMD3100, a specific inhibitor of CXCR4, increased LPS treatment associated mortality. Blocking CXCR4 via chemical or genetic inhibition resulted in a reversion of LPS tolerance, thus further supporting the negative regulatory role of CXCR4 in this inflammatory response. In support of an inhibitory role for CXCR4 in the inflammatory cascade, IL-1 transcript levels were elevated in both unstimulated and LPS stimulated zebrafish Odysseus (CXCR4 deficient mutant) larvae.
Subject(s)
Drug Tolerance , Immune System/drug effects , Lipopolysaccharides/pharmacology , Zebrafish/physiology , Animals , Gene Expression Regulation/drug effects , Larva/drug effects , Lipopolysaccharides/toxicity , Mortality , Receptors, CXCR4/immunology , Survival Analysis , Time Factors , Zebrafish/immunologyABSTRACT
Hematopoietic stem cells (HSCs) have been used extensively as a model for stem cell biology. Stem cells share the ability to self-renew and differentiate into multiple cell types, making them ideal candidates for tissue regeneration or replacement therapies. Current applications of stem cell technology are limited by our knowledge of the molecular mechanisms that control their proliferation and differentiation, and various model organisms have been used to fill these gaps. This chapter focuses on the contributions of the zebra fish model to our understanding of stem cell regulation within the hematopoietic system. Studies in zebra fish have been valuable for identifying new genetic and signaling factors that affect HSC formation and development with important implications for humans, and new advances in the zebra fish toolbox will allow other aspects of HSC behavior to be investigated as well, including migration, homing, and engraftment.
Subject(s)
Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic System/embryology , Zebrafish/embryology , Animals , Animals, Genetically Modified , Cell Movement , Embryonic Stem Cells/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic System/cytology , Hematopoietic System/metabolism , Models, Biological , Mutation , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
The canonical Wnt/beta-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating beta-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/beta-catenin pathway. DOCK4 expression regulates cellular beta-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the beta-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3beta (GSK3beta). This molecular interaction enhances beta-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3beta, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/beta-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/beta-catenin pathway.
Subject(s)
GTPase-Activating Proteins/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Axin Protein , Cell Line , Cytosol/metabolism , GTPase-Activating Proteins/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mice , Repressor Proteins/metabolism , Signal Transduction , Zebrafish , rac1 GTP-Binding Protein/metabolismABSTRACT
The zebrafish has emerged as a powerful genetic model of cancer, but has been limited by the use of stable transgenic approaches to induce disease. Here, a co-injection strategy is described that capitalizes on both the numbers of embryos that can be microinjected and the ability of transgenes to segregate together and exert synergistic effects in forming tumors. Using this mosaic transgenic approach, gene pathways involved in tumor initiation and radiation sensitivity have been identified.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/radiation effects , Gene Transfer Techniques , Microinjections/methods , Neoplasms, Radiation-Induced/genetics , Animals , Animals, Genetically Modified , Cleavage Stage, Ovum , DNA-Binding Proteins/administration & dosage , DNA-Binding Proteins/genetics , Embryo, Nonmammalian , Genes, bcl-2 , Genes, myc , Genes, p53 , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/genetics , Luminescent Proteins/administration & dosage , Luminescent Proteins/genetics , Mutant Proteins/genetics , Nuclear Proteins/administration & dosage , Nuclear Proteins/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Transgenes , Zebrafish/embryology , ras Proteins/genetics , Red Fluorescent ProteinABSTRACT
The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.
Subject(s)
Drug Evaluation, Preclinical/methods , Zebrafish/embryology , Animals , Drug Evaluation, Preclinical/instrumentation , Embryo, Nonmammalian/drug effects , Genotype , Toxicity Tests/instrumentation , Toxicity Tests/methods , Zebrafish/geneticsABSTRACT
Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins.
Subject(s)
Dystrophin/biosynthesis , Dystrophin/chemistry , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cell Membrane/metabolism , Chromosome Mapping , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/metabolism , Databases as Topic , Electrophoresis, Polyacrylamide Gel , Exons , Gene Library , Humans , Immunoblotting , Immunohistochemistry , Models, Genetic , Molecular Sequence Data , Muscle, Skeletal/metabolism , Muscles/metabolism , Phenotype , Polymerase Chain Reaction , Protein Biosynthesis , Sequence Homology, Amino Acid , ZebrafishABSTRACT
Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.
Subject(s)
Genes, Homeobox , Homeodomain Proteins/genetics , Oncogene Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Gene Expression Regulation, Developmental , In Situ Hybridization , Molecular Sequence Data , Multigene Family , Nervous System/embryology , Nervous System/metabolism , Phylogeny , Sequence Homology, Amino AcidABSTRACT
The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP alpha, beta, gamma, and delta using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future.