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Fishing for Genetic Effects on Social Behavior: Viral-mediated Transgenesis in Stickleback Fish

Graduate #15
Discipline: Biological Sciences
Subcategory: Genetics

Noelle James - University of Illinois at Urbana-Champaign
Co-Author(s): Alison Bell, University of Illinois at Urbana-Champaign, IL



Establishing a causal relationship between genes and social behavior is challenging due to dynamic spatial and temporal gene expression. One gene can have drastically different consequences on behavior when expressed in different parts of the social behavior network (a combination of areas in the fore- and mid-brain). Thus, to determine the causal relationship between a gene of interest and a behavior, it is crucial to have a method for manipulating gene expression at a specific time and location.
We developed one such method, viral-mediated transgenesis, for stickleback fish. Threespine sticklebacks (G. aculeatus) are a classic system for the study of behavior, ecology, & evolution, with a growing genomic toolkit. Our recent studies1 have identified hundreds of differentially expressed genes in the brain following social interactions, but the directionality of causation is unknown. Viral-mediated transgenesis is an appealing method for directly manipulating gene expression in this system because it is fast (4 to 9 days), flexible (it can target specific regions and individual cell types with different promoters), and does not require establishing a stable transgenic line.
Based on work in zebrafish2, we hypothesized that a herpes simplex 1 (HSV1) derived virus with the mCMV, hCMV or hEF1a promoters would drive continuous expression of a target gene. Adult fish were injected with 300nL of either a saline control or suspended virus causing expression of a fluorescent protein. Successful transgenesis was determined via widefield microscopy on whole mount brains. Behavioral recovery was also assessed based on stress levels and mating behaviors ? female fecundity and male nesting.

We successfully altered expression in adult stickleback brains using both the short-term promoter mCMV, showing expression by 4 days post-injection, and the longer-term promoter hCMV, reaching peak expression at 10 days post-injection with continued expression for several weeks afterward. No fluorescence was seen in saline-injected controls. Opercula beat rate, a stress indicator, returned to baseline within 2 hours post-surgery. Behavioral recovery following the injection occurred within 9 days for females and 3 days for males, prior to peak expression of the long-term construct.
Next, we will test for the behavioral consequences of candidate genes (prl, ajap1, cacna1g, nsmfb and trpc4) selected from previous RNAseq datasets for DEGs following an aggressive interaction. Functionally altered expression will be confirmed by qPCR, comparing expression between injected and un-injected brain regions of an individual. Aggression will be assayed prior to transgenesis and at 14 and 21 days. This technique will enable us to demonstrate that gene expression is sufficient to induce behavioral change.
References : 1. Bukhari, S.A. et al. PLoSGenetics 13, e1006840 (2017).
2. Zou, M., De Koninck, P., Neve, R.L. & Friedrich, R.W. Front. Neural Circuits 8, 41 (2014).

ERN Abstract (Noelle James).docx

Funder Acknowledgement(s): This study was supported by an EDGE grant from NSF awarded to Alison Bell PhD. I thank Rachael Neve for assistance in developing and making the viral constructs and Brian James for help with engineering the surgical setup.

Faculty Advisor: Alison Bell, alisonmb@illinois.edu

Role: I did all the research in the abstract from the technique development to the testing and selection of candidate constructs and genes. The packing of the viruses was done by Rachael Neve, as addressed in the acknowledgements.

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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