Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system

Abstract

Background Astyanax mexicanus is a well‐established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. Results 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 expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell‐types, a Cntnap2‐mCherry construct 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. Conclusion 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‐phenotype gap.

An Adult Brain Atlas Reveals Broad Neuroanatomical Changes in Independently Evolved Populations of Mexican Cavefish

“…Moreover, transgenic labeling of neurons in the brain permits tracing of neuronal projections, something that is not possible with Nissl staining (deCarvalho et al, 2013; Miyasaka et al, 2014). Whereas transgenic technology has not been widely used in A. mexicanus , recent studies have shown that the Tol2 system, which is widely used in zebrafish, is highly effective in A. mexicanus surface and cavefish (Elipot et al, 2014; Stahl et al, 2019a, b). Future work incorporating these tools would facilitate a highly defined neuroanatomical brain atlas for the A. mexicanus adult brain.…”

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease

“…Collaborative efforts between multiple cavefish laboratories have yielded standardized procedures for Tol2 transgenesis, leading to the ability to produce stable transgenic lines that mark specific cell types Stahl, Peuß, et al, 2019). In addition to visualizing specific cell types, these methods can be extended into multiple areas of functional analysis, including performing comparative fate mapping, comparing brain activity to determine how sensory inputs affect brain activity in cave and surface fish, and activating or silencing different neuronal subtypes to dissect the neural circuits underlying behavioral evolution.…”

Subterranean life: Behavior, metabolic, and some other adaptations of Astyanax cavefish

“…The transgenic zebrafish lines used in that study (Wee et al, 2019) do not exist in Astyanax CF. However, recent advances indicate that it may be now possible to perform this type of experiment in Astyanax fishes Stahl, Peuss et al, 2019).…”