Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system

Abstract

Astyanax mexicanus is a well-established and widely used fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. The establishment of A. mexicanus as an emergent model organism for understanding the evolutionary basis of development and behavior has been accelerated by an increasing availability of genomic approaches to identify genotype-phenotype associations. Despite important progress in the deployment of new technologies, deep mechanistic insights into A. mexicanus evolution and development have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter fused to eGFP expressed eGFP ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, we injected fish with a Cntnap2-mCherry construct that labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype to phenotype gap.

Cavefish brain atlases reveal functional and anatomical convergence across independently evolved populations

“…Transgenic technology has recently been applied to A. mexicanus, allowing tissue labeling of specific tissues with genetic precision ( 21 , 22 ). To broadly label the brain, we generated transgenic A. mexicanus surface and Molino cavefish harboring Huc: GCaMP6s, which has previously been reported to label most neurons in zebrafish ( 24 ).…”

“…Like zebrafish, larval A. mexicanus are transparent and amenable to whole-brain imaging in intact fish ( 21 , 22 ). In this study, we combined morphometric analysis, imaging of neural circuits, and whole-brain activity mapping to generate standard brains for river-dwelling surface fish and three independently evolved populations of cavefish.…”

Eye morphogenesis in the blind Mexican cavefish

“…After injection with the appropriate enzyme, transposase or meganuclease, into the 1st cell of the embryo, the construct is randomly inserted one or several times in the genome. This method is technically simple and works in Astyanax (Elipot et al, 2014; Hinaux et al, 2015; Stahl et al, 2019). It has however several drawbacks: the correct identification of most if not all the regulatory sequences driving expression is necessary and often difficult; the insertion of the transgene is random and its expression depends on the insertion site; the insertion of the construct can disrupt coding or regulatory sequences.…”

Non‐model organisms and unique approaches are needed for the future of evo‐devo

“…This ability to make stable transgenic lines is a key feature that can greatly expand the tractability of a new model system, allowing functional genetic studies. In this Special Issue, Stahl et al use the Tol2 transposase system to generate stable transgenic lines in the Mexican tetra, Astynax mexicanus , a cavefish model that is an excellent system for studying the evolution of morphological diversity, behavior and physiology. This advancement, which also uses vectors originally developed in zebrafish, will allow for the speedy generation of new A mexicanus transgenic lines, while also facilitating cross‐species genetic comparisons.…”

“…While this slow development can be used to a researcher's advantage, allowing the teasing apart of early development processes, it will also be a great challenge when developing tools for this model, such as cell‐labeling techniques and ex‐ovo culture methods, which will need to be geared towards maintaining cell labels and embryo viability over an extended period of development. Additionally, Stahl et al relate a challenge that is likely close to the heart of all evo‐devo researchers, in pointing out the need to build community resources such as stock centers and enable widespread resource sharing to facilitate the expansion of the use of emerging model systems to more labs.…”