Name: manipulation of gene function in mexican cavefish
Uploaded: 2019-04-22T13:00:00
Description: Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

The authors thank Sunishka Thakur for her assistance in genotyping and imaging the oca2 mutant fish depicted in Figure 2. This work was supported by National Science Foundation (NSF) award 1656574 to A.C.K., NSF award 1754321 to J.K. and A.C.K., and National Institutes of Health (NIH) award R21NS105071 to A.C.K. and E.R.D.

1. Morpholino oligo design
NOTE: Sequences for A. mexicanus are available through National Center of Biotechnology Information (NCBI) Gene and NCBI SRA (https://www.ncbi.nlm.nih.gov), as well as from the Ensembl genome browser (https://www.ensembl.org). When designing a morpholino for use in both surface- and cave-dwelling forms, it is critical to identify any genetic variation between the morphs at this stage, so these genetic regions can be avoided as targets for morpholinos. Any poly...

Here, we provided a methodology for manipulating gene function using morpholinos, CRISPR/Cas9 gene editing, and transgenesis methodology. The wealth of genetic technology and the optimization of these systems in zebrafish will likely allow for the transfer of these tools into A. mexicanus with ease52. Recent findings have used these approaches in A. mexicanus, but they remain underutilized in the investigation of diverse morphological, developmental, and behavioral traits in this...

Since Darwin&#8217;s Origin of Species1, scientists have gained profound insights into how traits are shaped evolutionarily in response to defined environmental and ecological pressures, thanks to cave organisms2. The Mexican tetra, A. mexicanus, consists of eyed ancestral &#8216;surface&#8217; populations that inhabit rivers throughout Mexico and southern Texas and of at least 29 geographically isolated populations of derived cave morphs inhabiting the Sierra del Abra and other areas of Northeast Mexico3. A number of cave-associated traits have been identified in A. mexicanus, including altered oxygen consumption, depigmentation, loss of eyes, and altered feeding and foraging behavior4,5,6,7,8,9. A. mexicanus presents a powerful model for investigating mechanisms of convergent evolution due to a well-defined evolutionary history, a detailed characterization of ecological environment, and the presence of independently evolved cave populations10,11. Many of the cave-derived traits that are present in cavefish, including eye loss, sleep loss, increased feeding, loss of schooling, reduced aggression, and reduced stress responses, have evolved multiple times through independent origins, often utilizing different genetic pathways between caves8,12,13,14,15. This repeated evolution is a powerful aspect of the A. mexicanus system and can provide insight into the more general question of how genetic systems may be perturbed to generate similar phenotypes.&#160;
While the application of genetic technology for the mechanistic investigation of gene function has been limited in many fish species (including A. mexicanus), recent advances in the zebrafish provide a basis for genetic technology development in fish16,17,18,19,20. Numerous tools are widely used in zebrafish to manipulate gene expression, and the implementation of these procedures have long been standardized. For example, the injection of morpholino oligos (MOs) at the single-cell stage selectively blocks RNA and prevents translation for a brief temporal window during development21,22. In addition, gene-editing approaches, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nuclease (TALEN), allow for the generation of defined deletions or, in some cases, insertions through a recombination in genomes19,20,23,24. Transgenesis is used to manipulate stable gene expression or function in a cell-type specific manner. The Tol2 system is used effectively to generate transgenic animals by coinjecting transposase mRNA with a Tol2 DNA plasmid containing a transgene25,26. The Tol2 system utilizes the Tol2 transposase of medaka to generate stable germline insertions of transgenic construct17. Generating Tol2 transgenics involves coinjecting a plasmid containing a transgene flanked by Tol2 integration sites and mRNA for Tol2 transposase17. This system has been used to generate an array of transgenic lines in zebrafish and its use has recently expanded to additional emergent model systems, including cichlids, killifish, the stickleback, and, more recently, the Mexican cavefish27,28,29,30.&#160;
While the cavefish is a fascinating biological system for elucidating mechanisms of trait evolution, its full capability as an evolutionary model has not been fully harnessed. This has partially been due to an inability to manipulate genetic and cellular function directly31. Candidate genes regulating complex traits have been identified using quantitative trait loci (QTL) studies, but the validation of these candidate genes has been difficult32,33,34. Recently, transient knockdown using morpholinos, gene editing using CRISPR and TALEN systems, and the use of Tol2-mediated transgenesis have been used to investigate the genetic basis underlying a number of traits35,36,37,38. The implementation and standardization of these techniques will allow for manipulations that interrogate the molecular and neural underpinnings of biological traits, including the manipulation of gene function, the labeling of defined cell populations, and the expression of functional reporters. Whereas the successful implementation of these genetic tools to manipulate gene or cellular function has been demonstrated in emergent model systems, detailed protocols are still lacking in A. mexicanus.&#160;
A. mexicanus provide critical insight into the mechanisms of evolution in response to a changing environment and present the opportunity to identify novel genes regulating diverse traits. A number of factors suggest that A. mexicanus is an extremely tractable model for applying established genomic tools currently available in established genetic models, including the ability to easily maintain fish in the laboratories, large brood size, transparency, a sequenced genome, and defined behavioral assays39. Here, we describe a methodology for the use of morpholinos, transgenesis, and gene editing in surface and cave populations of A. mexicanus. The broader application of these tools in A. mexicanus will allow for a mechanistic investigation into the molecular processes underlying the evolution of developmental, physiological, and behavioral differences between cavefish and surface fish.&#160;

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manipulation of gene function in mexican cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

Multiple populations of cave-dwelling A. mexicanus show reduced sleep and increased wakefulness/activity relative to their surface-dwelling conspecifics14. Hypocretin/orexin (HCRT) is a highly conserved neuropeptide, which acts to increase wakefulness, and aberrations in the HCRT pathway cause narcolepsy in humans and other mammals47,48. We have previously demonstrated that cave A. mexicanus have increased expression of H...

Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

Manipulation of Gene Function in Mexican Cavefish

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

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