ABSTRACT
The ability to conditionally inactivate genes is instrumental for fine genetic analysis of all biological processes, but is especially important for studies of biological events, such as regeneration, which occur late in ontogenesis or in adult life. We have constructed and tested a fully conditional gene trap vector, and used it to inactivate tbx5a in the cardiomyocytes of larval and adult zebrafish. We observe that loss of tbx5a function significantly impairs the ability of zebrafish hearts to regenerate after ventricular resection, indicating that Tbx5a plays an essential role in the transcriptional program of heart regeneration.
Subject(s)
Heart/physiology , Myocytes, Cardiac/metabolism , Regeneration , T-Box Domain Proteins/metabolism , Transcriptome , Zebrafish/metabolism , Animals , T-Box Domain Proteins/genetics , Zebrafish/geneticsABSTRACT
Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function.
Subject(s)
Conserved Sequence/genetics , Epitopes/genetics , Amino Acid Sequence , Animals , Antibodies/genetics , Proteins/genetics , Sequence Alignment/methods , ZebrafishABSTRACT
OBJECTIVE: The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. APPROACH AND RESULTS: Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. CONCLUSIONS: Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.
Subject(s)
Angiogenic Proteins/metabolism , Endothelial Cells/metabolism , Neovascularization, Physiologic , Proto-Oncogene Protein c-fli-1/metabolism , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Angiogenic Proteins/genetics , Animals , Animals, Genetically Modified , Apoptosis , Binding Sites , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Genotype , Morpholinos/metabolism , Mutation , Phenotype , Promoter Regions, Genetic , Proto-Oncogene Protein c-fli-1/genetics , Signal Transduction , Time Factors , Transcription Factors/genetics , Transcription, Genetic , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2 kb cHS4 insulators derived from the chicken ß-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.
