A viral toolbox for conditional and transneuronal gene expression in zebrafish.
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ABSTRACT:
SUBMITTER: Satou C
PROVIDER: S-EPMC9307271 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The unique combination of small size, translucency, and powerful genetic tools makes larval zebrafish a uniquely useful vertebrate system to investigate normal and pathological brain structure and function. While functional connectivity can now be assessed by optical imaging (via fluorescent calcium or voltage reporters) at the whole-brain scale, it remains challenging to systematically determine structural connections and identify connectivity changes during development or disease. To address this, we developed Tracer with Restricted Anterograde Spread (TRAS), a novel vesicular stomatitis virus (VSV)-based neural circuit labeling approach. TRAS makes use of replication-incompetent VSV (VSV?G) and a helper virus (lentivirus) to enable anterograde transneuronal spread between efferent axons and their direct postsynaptic targets but restricts further spread to downstream areas. We integrated TRAS with the Z-Brain zebrafish 3D atlas for quantitative connectivity analysis and identified targets of the retinal and habenular efferent projections, in patterns consistent with previous reports. We compared retinofugal connectivity patterns between wild-type and down syndrome cell adhesion molecule-like 1 (dscaml1) mutant zebrafish and revealed differences in topographical distribution. These results demonstrate the utility of TRAS for quantitative structural connectivity analysis that would be valuable for detecting novel efferent targets and mapping connectivity changes underlying neurological or behavioral deficits.
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Project description:Systems for spatial and temporal control of gene expression are essential for developmental studies and are of particular importance for research in adult model organisms. We present two modified dually inducible TetON systems for tissue-specific conditional control of gene expression in zebrafish based on (i) a tetracycline inducible transcriptional activator (TetActivator) fused to the ligand binding domain of a mutated glucocorticoid receptor (TetA-GBD) and (ii) a TetActivator fused with a domain of the Ecdysone receptor (TetA-EcR). Both systems showed strong induction of tetracycline-responsive promoters upon administration of the appropriate ligands (doxycycline and dexamethasone for TetA-GBD, and doxycycline and tebufenozide for TetA-EcR), and undetectable leakiness when compared with classical TetActivators. Combinations of transgenic lines expressing TetA-GBD specifically in the heart or the CNS with different Tet-responsive transgenic lines allows conditional and tissue-specific control of gene expression in embryos and adults. Importantly, induction is fully reversible and tunable by the doses of drugs used. The TetA-EcR system avoids the possible side effects of dexamethasone and displays improved sensitivity both in zebrafish and in mammalian cells. These results show that dually inducible TetON systems are convenient tools for reversible and very tightly controlled conditional gene expression in zebrafish.
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Project description:The tuba1a gene encodes a neural-specific ?-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a-expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreER(T2) and ?-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to label tuba1a-expressing cells conditionally and permanently via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a-expressing progenitors contribute to multiple cell lineages during development and that tuba1a-expressing Müller glia are retinal progenitors in the adult.
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Project description:Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
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Project description:Mycobacterium abscessus is an emerging human pathogen responsible for lung infections, skin and soft-tissue infections and disseminated infections in immunocompromised patients. It may exist either as a smooth (S) or rough (R) morphotype, the latter being associated with increased pathogenicity in various models. Genetic tools for homologous recombination and conditional gene expression are desperately needed to allow the study of M. abscessus virulence. However, descriptions of knock-out (KO) mutants in M. abscessus are rare, with only one KO mutant from an S strain described so far. Moreover, of the three major tools developed for homologous recombination in mycobacteria, only the one based on expression of phage recombinases is working. Several conditional gene expression tools have recently been engineered for Mycobacterium tuberculosis and Mycobacterium smegmatis, but none have been tested yet in M. abscessus. Based on previous experience with genetic tools allowing homologous recombination and their failure in M. abscessus, we evaluated the potential interest of a conditional gene expression approach using a system derived from the two repressors system, TetR/PipOFF. After several steps necessary to adapt TetR/PipOFF for M. abscessus, we have shown the efficiency of this system for conditional expression of an essential mycobacterial gene, fadD32. Inhibition of fadD32 was demonstrated for both the S and R isotypes, with marginally better efficiency for the R isotype. Conditional gene expression using the dedicated TetR/PipOFF system vectors developed here is effective in S and R M. abscessus, and may constitute an interesting approach for future genetic studies in this pathogen.
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