Adeno-associated viral tools to trace neural development and connectivity across amphibians

Creators: Jaeger, Eliza C.B.¹; Vijatovic, David²; Deryckere, Astrid¹; Zorin, Nikol³; Nguyen, Akemi L.⁴; Ivanian, Georgiy²; Woych, Jamie¹; Arnold, Rebecca C.²; Gurrola, Alonso Ortega¹; Shvartsman, Arik³; Barbieri, Francesca²; Toma, Florina A.²; Cline, Hollis T.⁵; Shay, Timothy F.⁶; Kelley, Darcy B.¹; Yamaguchi, Ayako⁴; Shein-Idelson, Mark³; Tosches, Maria Antonietta¹; Sweeney, Lora B. (Corresponding)²

1. Columbia University
2. Institute of Science and Technology Austria
3. Tel Aviv University
4. University of Utah
5. Scripps Research Institute
6. California Institute of Technology

Abstract

Amphibians, by virtue of their phylogenetic position, provide invaluable insights on nervous system evolution, development, and remodeling. The genetic toolkit for amphibians, however, remains limited. Recombinant adeno-associated viral vectors (AAVs) are a powerful alternative to transgenesis for labeling and manipulating neurons. Although successful in mammals, AAVs have never been shown to transduce amphibian cells efficiently. We screened AAVs in three amphibian species-the frogs Xenopus laevis and Pelophylax bedriagae and the salamander Pleurodeles waltl-and identified at least two AAV serotypes per species that transduce neurons. In developing amphibians, AAVs labeled groups of neurons generated at the same time during development. In the mature brain, AAVrg retrogradely traced long-range projections. Our study introduces AAVs as a tool for amphibian research, establishes a generalizable workflow for AAV screening in new species, and expands opportunities for cross-species comparisons of nervous system development, function, and evolution.

Copyright and License

Acknowledgement

We thank members of the Sweeney, Tosches, Shein-Idelson, Yamaguchi, Kelley, and Cline Labs for their contributions to this project, discussion, and support. We additionally thank the Beckman Institute CLOVER Center and Viviana Gradinaru (Caltech), Kimberly Ritola (UNC NeuroTools), and Flavia Gomez-Leite (ISTA Viral Core) for AAV production and consultation; Andras Simon and Alberto Joven (Karolinska Institute) for feedback; Elizabeth Bagnato-Cohen (Columbia) for project coordination; our animal care and imaging facilities; the amphibian stock centers (NXR, EXRC, and XenopusExpress); and our funding sources: NSF IOS 2110086 (D.B.K., L.B.S., M.A.T., A.Y., and H.T.C.); US-Israel Binational Science Foundation (BSF) 2020702 (M.S.-I.); FTI Strategy Lower Austria Dissertation FT121-D-046 (D.V.); Horizon Europe ERC Starting Grant 101041551 and Special Research Programme (SFB) of the Austrian Science Fund (FWF) project F7814-B (L.B.S.); NIH grant R35GM146973, Rita Allen Foundation Award GA_032522_FE, and CZI Ben Barres Early Career Acceleration Award 2023-331758 (M.A.T.); EMBO Long-Term Fellowship ALTF 874-2021 (A.D.); and NSF GRFP DGE 2036197 (E.C.B.J.).

Contributions

M.A.T. and L.B.S. led and coordinated the project. E.C.B.J., D.V., A.D., H.T.C., T.F.S., D.B.K., A.Y., M.S.-I., M.A.T., and L.B.S. devised the project and implemented experiments. E.C.B.J., A.D., J.W., A.O.G., and M.A.T. collected the Pleurodeles data. D.V., A.L.N., G.I., R.C.A., F.B., F.A.T., A.Y., and L.B.S. collected the Xenopus data. N.Z., A.S., and M.S.-I. collected the Pelophylax data. E.C.B.J., D.V., A.D., N.Z., G.I., M.A.T., and L.B.S. analyzed the data. E.C.B.J., D.V., A.D., M.A.T., and L.B.S. wrote the manuscript. H.T.C., T.F.S., D.B.K., A.Y., and M.S.-I. edited the manuscript and provided critical feedback on the project.

Supplemental Material

Document S1. Figures S1–S6 and Tables S1–S4.

Data Availability

FAMD RStudio code and raw data used for analysis of post-metamorphic P. waltl injection outcomes are available via Dryad repository: https://doi.org/10.5061/dryad.mpg4f4r89. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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