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Transsynaptic labeling and transcriptional control of zebrafish neural circuits

Nature Neuroscience volume 28, pages 189–200 (2025)Cite this article

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Abstract

Deciphering the connectome, the ensemble of synaptic connections that underlie brain function, is a central goal of neuroscience research. Here we report the in vivo mapping of connections between presynaptic and postsynaptic partners in zebrafish, by adapting the trans-Tango genetic approach that was first developed for anterograde transsynaptic tracing in Drosophila. Neural connections were visualized between synaptic partners in larval retina, brain and spinal cord and followed over development. The specificity of labeling was corroborated by functional experiments in which optogenetic activation of presynaptic spinal cord interneurons elicited responses in known motor neuronal postsynaptic targets, as measured by trans-Tango-dependent expression of a genetically encoded calcium indicator or by electrophysiology. Transsynaptic signaling through trans-Tango reveals synaptic connections in the zebrafish nervous system, providing a valuable in vivo tool to monitor and interrogate neural circuits over time.

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Fig. 1: Trans-Tango mediates transsynaptic signaling in zebrafish.
Fig. 2: Gal4-dependent trans-Tango labeling of synaptic partners.
Fig. 3: Progression of trans-Tango labeling during neural development.
Fig. 4: Validation of trans-Tango labeling in the retina.
Fig. 5: Trans-Tango confirms predicted postsynaptic partners of retinal ganglion cells.
Fig. 6: Trans-Tango reveals synaptically coupled neurons.

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Data availability

The source data that support the findings of this study are available at Zenodo at https://doi.org/10.5281/zenodo.13716911 (ref. 85), https://doi.org/10.5281/zenodo.13629169 (ref. 89), https://doi.org/10.5281/zenodo.13629206 (ref. 90), https://doi.org/10.5281/zenodo.13629296 (ref. 91), https://doi.org/10.5281/zenodo.13629407 (ref. 92), https://doi.org/10.5281/zenodo.13629638 (ref. 93), https://doi.org/10.5281/zenodo.13629732 (ref. 94), https://doi.org/10.5281/zenodo.13629664 (ref. 95), https://doi.org/10.5281/zenodo.13629565 (ref. 96), https://doi.org/10.5281/zenodo.13629389 (ref. 97) and https://doi.org/10.5281/zenodo.13629333 (ref. 98). Additional raw imaging data are available upon request. We have the rights to publish BioRender figures and Fig. 1a was generated using BioRender. Source data are provided with this paper.

Code availability

The MATLAB and R codes used in the presented data analyses are publicly available at Zenodo at https://doi.org/10.5281/zenodo.13629206 (ref. 90). Modification to code for image registration84 is also provided.

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Acknowledgements

We thank M. Ahrens (HHMI, Janelia), M. Bagnall (Washington University), A. Douglas (The University of Utah), M. Granato (University of Pennsylvania), T. Kaminy (The University of Utah), K. Kwan (The University of Utah), J. Meserve (University of Pennsylvania), T. Mulligan (Johns Hopkins University) and J. Mumm (Johns Hopkins University) for sharing reagents and transgenic lines. We are grateful to E. Horstick for advice on codon optimization and to E. Spikol and A. Abdelfattah (Brown University) for expert guidance on calcium imaging experiments. Special thanks are extended to P. Robison for microscopy support, K. Young (Northwestern University) for technical assistance, and B. Malskis, J. Devine-Brilliant and C. Mathews for animal care. This work was supported by a Hanna H. Gray fellowship (GT15992 to C.E.C.), the National Institute of Neurological Disorders and Stroke (grants R21NS125187 and R21NS125207 to D.L.M.) and the National Institutes of Health Brain Initiative (RF1MH123213 to M.E.H., G.B., J.L. and D.R.).

Author information

Author notes

  1. Cagney E. Coomer

    Present address: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA

  2. Mustafa Talay

    Present address: Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard University, Boston, MA, USA

  3. David L. McLean

    Present address: Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK

Authors and Affiliations

  1. Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA

    Cagney E. Coomer, Daria Naumova, Ji Cheng & Marnie E. Halpern

  2. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Daria Naumova, Ji Cheng & Marnie E. Halpern

  3. Department of Neuroscience, Division of Biology and Medicine, Brown University, Providence, RI, USA

    Mustafa Talay, Bence Zolyomi, Nathaniel J. Snell, Altar Sorkaç & Gilad Barnea

  4. Robert J. and Nancy D. Carney Institute for Brain Science, Brown University, Providence, RI, USA

    Mustafa Talay, Bence Zolyomi, Nathaniel J. Snell, Altar Sorkaç & Gilad Barnea

  5. Department of Embryology, Carnegie Institution for Science, Baltimore, MD, USA

    Jean Michel Chanchu

  6. Max Planck Institute for Biological Cybernetics, Tübingen, Germany

    Ivana Roman, Jennifer Li & Drew Robson

  7. Department of Neurobiology, Northwestern University, Evanston, IL, USA

    David L. McLean

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Contributions

C.E.C., D.N., D.L.M. and M.E.H. conceived the experiments and wrote the manuscript with input from M.T., A.S., I.R., J.L., D.R. and G.B. C.E.C., D.N. and D.L.M. performed all experiments and data analysis. M.T., B.Z., N.J.S., A.S., J.M.C., J.C. and G.B. designed and generated trans-Tango plasmids. C.E.C., I.R., J.L. and D.R. advised on plasmids and collected preliminary data for zebrafish. J.C. applied image registration tools.

Corresponding author

Correspondence to Marnie E. Halpern.

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The authors declare no competing interests.

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Nature Neuroscience thanks Claire Wyart and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Optimization of the trans-Tango ligand.

Six different ligand constructs were injected along with plasmids encoding the trans-Tango receptor, arrestin–TEV and Tol2 transposase RNA into single-cell zebrafish embryos, the progeny of Tg(QUAS:mApple-CAAX) and the Tg(ptf1a:Gal4-VP16) driver line that promotes strong expression of Tg(UAS:GFP) in the hindbrain. a–f, Ligands containing the transmembrane domain of zebrafish nrxn1a and varying lengths of mouse ICAM. a,a′, 10xUAS-E1B:sGCG-nrxn1a produced consistent trans-Tango labeling in the hindbrain (n = 20/25) but also nonspecific labeling (b; n = 15/25). c,c′, 10xUAS-E1B:sGCG-ICAM(1235)-nrxn1a produced consistent hindbrain labeling (n = 17/25) and nonspecific labeling (d; n = 11/25). e,e′, 10xUAS-E1B:sGCG-ICAM-nrxn1a produced fewer trans-Tango-labeled larvae (n = 6/25) and nonspecific labeling (f; n = 5/25). g–l, Ligands containing the transmembrane domain of zebrafish nrxn1b and varying lengths of mouse ICAM. g,g′, 10xUAS-E1B:sGCG-nrxn1b produced consistent trans-Tango labeling (n = 15/25) and substantial nonspecific labeling (h; n = 12/25). i,i′, 10xUAS-E1B:sGCG-ICAM(1235)-nrxn1b produced consistent labeling in the hindbrain (n = 23/25) and minimal nonspecific labeling (j; n = 2/25). k,k′, 10xUAS-E1B:sGCG-ICAM-nrxn1b produced trans-Tango labeling (n = 21/25) and nonspecific labeling (l; n = 14/25). Scale bars, 50 μm. Images are representative of trans-Tango labeling in the indicated number of larvae in five independent experiments for each ligand construct.

Extended Data Fig. 2 Monitoring synaptic connections over time.

a,a′, Dorsal view of mApple-CAAX-labeled neurons in the optic tectum (arrowheads) of a 6 dpf larva bearing Tg(isl2b.2:Gal4, myl7:tagRFP); Tg(UAS:GFP), Tg(QUAS:mApple-CAAX, he1.1:mCherry) and all trans-Tango components. Diffuse red fluorescent protein labeling (asterisk) is due to a secondary marker (my17:RFP) expressed in the heart. b,b′, In the same animal, labeling of the tectal neurons indicated in a (arrowheads) persisted at 2 weeks postfertilization (wpf; n = 10). Scale bars, 50 μm in a,a′ and 100 μm in b,b′. Images are representative of ten larvae from five independent experiments imaged at each time point.

Extended Data Fig. 3 Trans-Tango-mediated ablation of postsynaptic neurons.

a,b, In 5 dpf TgBAC(ptf1a:Gal4-VP16); Tg(UAS:GFP) larvae expressing the bacterial NfsB reductase gene, trans-Tango-labeled mApple-CAAX neurons (arrowheads) in merged (a) and single channel (b) images were no longer detected (c and d) following 1 day of incubation in 10 μM metronidazole (n = 25). c,d, Merged (c) and single channel (d) images. Scale bars, 50 μm. Images are representative of 25 larvae from 5 independent experiments imaged before (a and b) and after (c and d) metronidazole treatment.

Extended Data Fig. 4 Generation of trans-Tango transgenic zebrafish.

Heterozygous fish bearing transgenes for trans-Tango components and Tg(QUAS:mApple-CAAX, he1.1:mCherry) were mated with the TgBAC(pt1fa:Gal4-VP16)jh16 driver line and resultant embryos injected with plasmids for the other trans-Tango reagents and Tol2 RNA. a, RNA in situ hybridization indicates that the expression of UAS:sGCG-ICAM(1235)-Nrxn1b resembles the transcription pattern of the endogenous ptf1a gene99. b,c, Both the Tg(elavl3:hArr-TEV, he1.1:YFP)cd30 (b) and Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) (c) stable lines are expressed broadly throughout the CNS. d,d″, Tg(10xUAS-E1B:sGCG-ICAM(1235)nrxn1b, cryaa:mCherry) larvae showed trans-Tango labeling in the hindbrain (n = 257/312) consistent with that observed when the ligand is introduced by plasmid injection. e,e″, Tg(elavl3:hArr-TEV, he1.1:YFP) larvae had similar trans-Tango labeling in the hindbrain (n = 132/164). f,f″, mApple-CAAX labeling was not observed in the hindbrain (n = 0/276) with the receptor line Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) or when receptor plasmid was injected at a sub-threshold concentration (10 ng μl−1) along with the ligand and arrestin–TEV plasmids (25 ng µl−1; n = 25; g,g′). h,h′, However, the same concentration of receptor plasmid was effective at producing extensive mApple-CAAX labeling in the hindbrain (n = 21/25) when injected into embryos bearing Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) along with the ligand and arrestin–TEV plasmids (25 ng µl−1). Scale bars, 20 µm for a–c and 50 μm for d–h. Images are representative of five independent experiments for each stable line.

Source data

Extended Data Fig. 5 Trans-Tango labeling in stable transgenic lines.

Robust mApple-CAAX labeling in Tg(isl2b.2:Gal4, myl7:tagRFP) 6 dpf larvae bearing both the ligand and arrestin–TEV stable transgenes and Tg(UAS:GFP) and Tg(QUAS:mApple-CAAX) reporter lines. a,a′, Control larvae from injections of plasmids for all trans-Tango components (n = 10). b,b′, Tg(10xUAS-sGCG-ICAM(1235)-nrxn1b, cryaa:mCherry) larvae injected with the receptor and arrestin–TEV constructs (n = 10). c,c′, Tg(elavl3:hArr:TEV, he1.1:YFP) larvae that had been injected with the receptor and ligand constructs (n = 10). d,d′, Larvae transgenic for both the ligand and arrestin–TEV transgenes that had been injected with the trans-Tango receptor construct (n = 10). e, Uninjected controls (n = 10). f, On average, 45 neurons were labeled with mApple-CAAX in larvae that bore both the ligand and arrestin–TEV stable transgenes compared to those from injections of all trans-Tango components (on average, 11 neurons per sample). In the boxplots the center line represents the median, the box limits indicate top and bottom quartiles, and the whiskers extend to the 1.5× quartile range. g, Image registration of mApple-CAAX-labeled neurons in the optic tectum and hindbrain of ten Tg(isl2b.2:Gal4-VP16, myl7:tagRFP); Tg(UAS:GFP); Tg(10xUAS-sGCG-ICAM(1235)-nrxn1b, cryaa:mCherry); Tg(elavl3:hArr:TEV, he1.1:YFP) larvae injected with the receptor construct. mApple-CAAX-labeled neurons are pseudocolored differently for each larva and dashed circles indicate some cells that were labeled in multiple samples. Scale bars, 20 μm. Double asterisk (**) represents the statistically significant increase (P < 0.0001) in trans-Tango labeled neurons when using the ligand and arrestin stable lines compared to plasmid only and stable lines individually using a one-way ANOVA test on Prism.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1 and 2.

Source data

Source Data Extended Data Fig. 4

Statistical data from Prism used to calculate P value and statistical significance.

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Coomer, C.E., Naumova, D., Talay, M. et al. Transsynaptic labeling and transcriptional control of zebrafish neural circuits. Nat Neurosci 28, 189–200 (2025). https://doi.org/10.1038/s41593-024-01815-z

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