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Larval expression patterns for a selection of enhancer trap lines.(a) Horizontal section of et48 showing expression in the photoreceptor layer of the retina. (b) Horizontal section of et19.1, with broad expression in the retina. (c) et10.1, showing specific expression in the pallium. (d) et27, pineal (arrows) and habenulae (arrowheads). (e) et25.2, habenulae (arrows) and scattered cells in the tectum (bottom half of the right image). (f) et206, with expression in many or all habenular neurons, and visible projections (arrowheads) to the interpeduncular nucleus (arrow). (g) et44.1, lateral confocal stack (left) and dorsal confocal stack (right) showing expression in the tectum (arrows). (h) et24.1, expression along the dorsal midline (arrow) within the midbrain. (i) et208, with expression in the thalamus. (j) Lateral confocal stacks of et44.4, with expression in head neuromasts (a single neuromast is shown in the right image). (k) Lateral confocal stacks of et29.4, with expression in the lateral line ganglia. Terminals of lateral line ganglion neurons at a neuromast are shown in the right image. (l) Lateral confocal stacks of et11.1, showing expression in the cristae of the ear. (m) et40.1, with expression in the olfactory pits (all arrows). (n) et56.3, with expression in the subpallium (arrow), olfactory bulb (arrowhead) and olfactory pits (small arrows). (o) Ventral view of et30.2, with expression in the heart (arrow), neurons in the lower lip (small arrows) and unidentified axons (arrowheads). All panels show larvae carrying a GAL4 insertion and UAS:Kaede. Dorsal confocal stacks are shown, unless otherwise indicated. The dotted line indicates the larval outline. For each enhancer trap pattern, a low (left) and a high-magnification (right) image are shown. Scale bars, 200 m in all left panels and 50 m in all right panels.
Source publication
We present a pilot enhancer trap screen using GAL4 to drive expression of upstream activator sequence (UAS)-linked transgenes in expression patterns dictated by endogenous enhancers in zebrafish. The patterns presented include expression in small subsets of neurons throughout the larval brain, which in some cases persist into adult. Through targete...
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While technological innovations are sometimes published in methods papers, theyoften are partof a research paper and thus not apparent to people outside of the specific research area. To enhance the dissemination of generally useful technological advances and transgenic lines, Zebrafish is introducing TechnoFish, which will highlight these innovati...
In this communication, we report the generation of a cre transgenic zebrafish line under an oocyte-specific promoter, zp3. The transgenic line Tg(zp3:cre; krt8:rfp) also contains a co-integrated rfp transgene under the skin epithelial promoter krt8 to allow selection of cre transgenic fish based on RFP fluorescence in the skin. We demonstrated in t...
Citations
... In order to facilitate the segmentation of neurons, brain-wide calcium imaging is generally performed with a nuclear-targeted GCaMP. The morphologies of these neurons can be revealed by a number of approaches, including sparse stochastic genetic labeling (Förster et al. 2017, Kunst et al. 2019, Scott et al. 2007) and targeted photoactivation (Antinucci et al. 2019. Connectivity can be addressed with transsynaptic tracers or viruses (Coomer et al. 2023, Dohaku et al. 2019, Kler et al. 2021, Ma et al. 2019, Satou et al. 2022) and probed with twophoton holographic optogenetics , Förster et al. 2017. ...
The zebrafish visual system has become a paradigmatic preparation for be-havioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 13 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tec-tum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographi-cally organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated mul-tistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior. 255
... All transgenes, including those regulated by apparently ubiquitous promoters, are subject to position effects, a phenomenon where the regulatory context of the genomic locus where the transgene is integrated exerts influence on transgene expression pattern and level (Hans et al., 2009(Hans et al., , 2011Lalonde et al., 2022;Mosimann et al., 2011). With the exception of enhancer trapping (Balciunas et al., 2004;Parinov et al., 2004;Scott et al., 2007), position effects are considered to be a detrimental side effect of transgenesis. Attempts to minimize position effects by incorporating border elements or insulators have had variable success (Caldovic et al., 1999;Grajevskaja et al., 2013). ...
We have generated transgenic lines containing zebrafish-optimized CreER T2 recombinase under the control of a recombinant ubb R promoter consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carp beta-actin2 gene. These lines enable highly efficient tamoxifen-inducible recombination in embryonic, larval and adult zebrafish.
Abstract
The ability to inactivate gene function in an adult organism is essential for studies of biological processes such as regeneration and behavior. This is best achieved by engineering an allele which could be conditionally inactivated using Cre recombinase and subsequently inactivating gene function using a drug-inducible Cre recombinase. Several recent studies clearly demonstrate feasibility of engineering such conditional alleles in zebrafish. Meanwhile, achieving sufficient degree of recombination to induce complete loss of function has remained a major limitation. Herein we address this limitation by engineering a recombinant ubiquitin promoter ubb R consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carp beta-actin2 gene. Using phiC31-mediated targeted integration, we demonstrate that ubb R clearly outperforms both parental promoters as well as currently available ubiquitous CreER T2 driver lines at all embryonic and larval stages tested. Furthermore, the ubb R :CreER T2 driver line we generated enables near-complete inactivation of floxed alleles in adult zebrafish hearts. Finally, we demonstrate that our ubb R promoter retains high activity when integrated at other genomic loci, making it uniquely suitable for robust expression of transgenes at all stages of zebrafish ontogenesis.
Highlights
Used targeted integration to directly compare different CreER T2 drivers
Generated a ubiquitous ubb R :CreER T2 driver line capable of near-complete inactivation of floxed genes in adult zebrafish hearts
Demonstrated that the recombinant ubb R promoter is suitable for robust transgene expression when integrated at different genomic loci
... To construct 14uas: sypb-Gcamp6s, synaptophysin was cloned by from zebrafish genomic and recombined with plasmid 14uas: Gcamp6s. The published transgenic fishline used are Tg (ptf1a:EGFP) (jh1Tg) [69], Tg(uas:kaede) (s1999tTg) [70], and TgBAC(slc6a9:EGFP) (ion85Tg) [71]. Tg(uas:Gcamp6s) (nkUAShspzGCaMP6s13aTg) [72] was a gift from Dr. Jiulin Du. ...
In mammals, retinal direction selectivity originates from GABAergic / cholinergic amacrine cells (ACs) specifically expressing the sox2 gene. However, the cellular diversity of GABAergic / cholinergic ACs of other vertebrate species remains largely unexplored. Here, we identified 2 morphologically and genetically distinct GABAergic/cholinergic AC types in zebrafish, a previously undescribed bhlhe22 + type and a mammalian counterpart sox2+ type. Notably, while sole sox2 disruption removed sox2+ type, the codisruption of bhlhe22 and bhlhe23 was required to remove bhlhe22 + type. Also, both types significantly differed in dendritic arbors, lamination, and soma position. Furthermore, in vivo two-photon calcium imaging and the behavior assay suggested the direction selectivity of both AC types. Nevertheless, the 2 types showed preferential responses to moving bars of different sizes. Thus, our findings provide new cellular diversity and functional characteristics of GABAergic/cholinergic ACs in the vertebrate retina.
... To investigate whether the suppressed neurogenesis in paroxetine-treated embryos affected later neural circuit formation, we examined the retinotectal projections in the optic tecta. We used transgenic line Tg(pou4l-hsp70l:GFP) expressing GFP in excitatory tectal neurons and retinal ganglion cells (RGCs), and line Tg(pou4f3:GAL4;UAS:mCherry) expressing mCherry in RGCs [42][43] . For wild-type larvae, GFP-and mCherry-expressing areas were gradually but signi cantly larger in the optic tecta than those larvae at 72 hpf, according to developmental progression ( Supplementary Fig. S4). ...
Autism spectrum disorder (ASD) is a neurodevelopmental condition caused by various genetic and environmental factors. This disorder has the cardinal symptoms including impaired social behavior involving the amygdala. Antidepressants such as paroxetine in early pregnancy increase the risk of ASD in offspring. However, a comprehensive picture of the underlying pathogenic mechanisms remains elusive. Here, we demonstrate that early exposure of zebrafish embryos to paroxetine suppresses neurogenesis in the optic tectum and the dorsal telencephalon which corresponds to the human amygdala. Paroxetine-treated embryos exhibit impaired growth, with small heads and short body lengths resulting from transient apoptosis. This is reminiscent of the early-onset fetal growth restriction (FGR) associated with ASD. Interestingly, the suppressed neurogenesis in the small heads was found to be restored after the cessation of paroxetine. This was accompanied by extended retinotectal projections, suggesting brain-preferential remodeling. Finally, the paroxetine-treated fish exhibited impaired social behavior, further supporting the correspondence with ASD. Our findings offer new insights into the early neurodevelopmental etiology of ASD.
... With the aid of suitable promoters and the GAL4/UAS system, opsins can be expressed in defined brain regions or cell types. [46][47][48] This capability would enable us to explore the influence of anatomically and/or genetically defined cell assemblies on brainwide activity and animal behavior. ...
We introduce an all-optical technique that enables volumetric imaging of brain-wide calcium activity and targeted optogenetic stimulation of specific brain regions in unrestrained larval zebrafish. The system consists of three main components: a 3D tracking module, a dual-color fluorescence imaging module, and a real-time activity manipulation module. Our approach uses a sensitive genetically encoded calcium indicator in combination with a long Stokes shift red fluorescence protein as a reference channel, allowing the extraction of Ca²⁺ activity from signals contaminated by motion artifacts. The method also incorporates rapid 3D image reconstruction and registration, facilitating real-time selective optogenetic stimulation of different regions of the brain. By demonstrating that selective light activation of the midbrain regions in larval zebrafish could reliably trigger biased turning behavior and changes of brain-wide neural activity, we present a valuable tool for investigating the causal relationship between distributed neural circuit dynamics and naturalistic behavior.
... All experiments were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of the University of Michigan. Transgenic fish lines used were Tg(hb9:mgfpz) (Flanagan-Steet et al., 2005), Tg(1020t:gal4) (Wyart et al., 2009) and Tg(UAS:ChR2-mCherry) (Scott et al., 2007). The sex of the zebrafish embryos analyzed was not determined. ...
... To do this we examined whether swimming could be initiated in embryos by optogenetic activation of the CSF-cNs. We used progeny from crosses of transgenic zebrafish carrying a UAS regulated channelrhodopsin Tg(UAS:ChR2-mCherry) (Scott et al., 2007) with transgenic zebrafish which express Gal4 in CSF-cNs and motor neurons Tg(1020t:gal4) (Wyart et al., 2009). Tg(1020t:gal4; UAS:ChR2-mCherry) embryos could be identified by red fluorescence in the spinal cord. ...
... odf3l2a expression is restricted to the inner ear and lateral line hair cells ( Figure 1D), consistent with the pattern reported for the et10.4 enhancer trap line where the transgenic insertion is 1.5 kilobases (kb) upstream of the odf3l2a gene [47]. saxo2 is also expressed in otic and lateral line hair cells, with additional robust expression in the developing olfactory epithelia ( Figure 1E). ...
Sensory hair cells are the receptors for auditory, vestibular, and lateral line sensory organs in vertebrates. These cells are distinguished by “hair”-like projections from their apical surface collectively known as the hair bundle. Along with the staircase arrangement of the actin-filled stereocilia, the hair bundle features a single, non-motile, true cilium called the kinocilium. The kinocilium plays an important role in bundle development and the mechanics of sensory detection. To understand more about kinocilial development and structure, we performed a transcriptomic analysis of zebrafish hair cells to identify cilia-associated genes that have yet to be characterized in hair cells. In this study, we focused on three such genes—ankef1a, odf3l2a, and saxo2—because human or mouse orthologs are either associated with sensorineural hearing loss or are located near uncharacterized deafness loci. We made transgenic fish that express fluorescently tagged versions of their proteins, demonstrating their localization to the kinocilia of zebrafish hair cells. Furthermore, we found that Ankef1a, Odf3l2a, and Saxo2 exhibit distinct localization patterns along the length of the kinocilium and within the cell body. Lastly, we have reported a novel overexpression phenotype of Saxo2. Overall, these results suggest that the hair cell kinocilium in zebrafish is regionalized along its proximal-distal axis and set the groundwork to understand more about the roles of these kinocilial proteins in hair cells.
... Dr. M. Affolter and Dr. H. Belting, University of Basel, Switzerland) were obtained from adult Tg(mpeg1:Gal4;UAS:Kaede) fish [34,43] with macrophages expressing EGFP or wild-type (AB/TU) fish [44] and were kept at 28°C in a zebrafish-culture medium supplemented with 30 µg/mL 1-phenyl-2-thiourea (PTU). All fish were kept in accordance with Swiss animal-welfare regulations [22,23]. ...
Introduction:
Blood infections from multi-drug-resistant Salmonella pose a major health burden. This is especially true because Salmonella can survive and replicate intracellularly, and the development of new treatment strategies is dependent on expensive and time-consuming in vivo trials. The aim of this study was to develop a Salmonella-infection model that makes it possible to directly observe Salmonella infections of macrophages in vivo and to use this model to test the effect of antimicrobials against intra- and extracellular Salmonella in order to close the gap between in vitro and rodent-infection models.
Methods:
We established suitable Salmonella-infection conditions using genetically engineered zebrafish and Salmonella-expressing fluorescent proteins (green fluorescent protein (GFP) and/or mCherry).
Results:
We detected Salmonella inside and outside zebrafish larvae macrophages. Administration of the cell-impermeable antibiotic tobramycin removed Salmonella residing outside macrophages but did not affect Salmonella in macrophages, whereas ceftriaxone successfully cleared both types of Salmonella. Salmonella inside and outside macrophages experienced substantial DNA damage after administration of fluoroquinolones consistent with the excellent cell penetration of these antibiotics.
Conclusions:
The zebrafish-larvae model enables testing of antimicrobials for efficacy against extra- and intracellular Salmonella in a complex in vivo environment. This model thus might serve for antimicrobial lead optimization prior to using rodent models.
... A relatively simple brain greatly reduces the search space for pinpointing disease-associated changes. But just as importantly, most of these 100,000 neurons are identifiable in the sense that the same neuron, or cluster of neurons, can be identified readily in different fish thanks to extensive libraries of transgenic lines that express reporter genes in particular subsets of neurons [29][30][31]. Using chemogenetic and optogenetic methods, neuronal activity in the same neurons across fish can be monitored and manipulated to resolve how specific connections between neurons regulate physiology and behavior in wildtype and mutant animals. ...
... The Gal4-UAS system has been combined with the high-efficiency generation of random transgene insertions in studies to generate libraries of Gal4 drivers using enhancer trap methods [29,31]. Gaining genetic control over individual groups of CNS neurons is both powerful and useful, since analysis of cellular mechanisms that selectively involve specific neuronal types depends on the ability to express reporters or biosensors in these populations. ...
The last decade has seen a dramatic rise in the number of genes linked to neurological disorders, necessitating new models to explore underlying mechanisms and to test potential therapies. Over a similar period, many laboratories adopted zebrafish as tractable model for studying brain development, defining neural circuits, and performing chemical screens. Here we discuss strengths and limitations of using the zebrafish system to model neurological disorders. The underlying premise for many disease models is the high degree of homology between human and zebrafish genes, coupled with the conserved vertebrate Bauplan and repertoire of neurochemical signaling molecules. Yet we caution that important evolutionary divergences often limit the extent to which human symptoms can be meaningfully modeled in zebrafish. We outline advances in genetic technologies that allow human mutations to be faithfully reproduced in zebrafish. Together with methods that visualize the development and function of neuronal pathways at the single cell level, there is now an unprecedented opportunity to understand how disease-associated genetic changes disrupt neural circuits, a level of analysis that is ideally suited to uncovering pathogenic changes in human brain disorders.
... Since reporter expression can be positively and negatively influenced by neighboring loci in the genome (Amsterdam et al., 1995;Jaenisch et al., 1981;Lalonde et al., 2022;Wilson et al., 1990), an expression construct designed to test an enhancer requires an inert minimal promoter that is not sensitive to other enhancers in the vicinity of the transgene insertion. Several minimal promoters have been applied for transgenesis in zebrafish, including Ef1a, hsp70l, c-fos, krt4, TK, gata2a promoter regions that are several hundred to several kilo-bases in length (Amsterdam et al., 1995;Bessa et al., 2009;Johnson and Krieg, 1995;Kondrychyn et al., 2009;Meng et al., 1997;Ogura et al., 2009;Scott et al., 2007;Shoji and Sato-Maeda, 2008;Weber and Köster, 2013). ...
Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models.
