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...

The authors have nothing to disclose.

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, ...

Cave animals provide a compelling system for investigating diverse morphological, developmental, and behavioral traits. But one of the limiting factors in establishing this model system is a lack of genetic tools that would allow us to manipulate gene function. Here we demonstrate three different approaches to manipulate gene function in the Mexican cave fish, Astyanax mexicanus.These tools will enable the investigation of the genes underlying natural variations between surface fish and cave fish and how they relate to phenotypes. Bethany Stahl, a talented postdoc in my laboratory will demonstrate this procedure. Before beginning the procedure, fill a 100 milliliter Petri dish with warm 3%agarose dissolved in fish system water and carefully place an egg injection mold into the agarose at a 45 degree angle, before slowly lowering the mold into the agarose to avoid trapping air under the mold.When the agarose has solidified, carefully remove the mold then store the plate at four degrees Celsius for up to one week. Then pull needles from borosilicate glass capillaries and an electrode needle puller according to the manufacturer's guidelines. On the day of the injection, use a glass pipette to transfer single cell eggs into the injection plate, filling up to five rows of the injection plate with 30 to 40 eggs per row.Keep the eggs hydrated with a small amount of fish system water. Thaw Morpholino on ice and prepare the injection solution so that 400 picograms of Morpholino is injected per egg by using a long Microloader pipette tip or adding a two to four microliter bolus to the end. When all of the needles have been loaded, use forceps to trim the excess length from the injection needle tips and mount the first needle into a micromanipulator connected to a picoliter microinjector.Then, place the injection dish under a dissecting microscope and use the micromanipulator to penetrate each egg with the needle before injecting one nanoliter of Morpholino injection solution directly into the yolk. For CRISPR injection, mix 25 picograms of guide RNA with 300 picograms of CRISPR-associated protein 9 messenger RNA and inject two nanoliters of the resulting RNA solution into each embryo as demonstrated. For Tol2 transposase and Tol2 flanked plasmid injection, combine 25 nanograms per microliter of Tol2 messenger RNA with 25 nanograms per microliter of Tol2 plasmid construct and phenol red in RNase-free water.Then inject one nanoliter of solution into each embryo as just demonstrated. To screen the Morpholino-injected animals at four days post injection, depending on the gene of interest, visualize the fish larvae under a stereo microscope to observe the phenotype of the injected animals or video record to measure behavior. To screen for CRISPR indels, transfer CRISPR-injected embryos into PCR tubes and extract the DNA according to standard DNA extraction protocols for polymerase chain reaction or PCR analysis.To assess for mutagenesis, run five microliters of the PCR product on a 3%agarose gel at 70 volts for three hours. Wild-type, non-mutagenized DNA will result in a distinct band, while mutant DNA will result in a smeary band. Cave A mexicanus injected with a control Morpholino exhibits significantly more locomotor activity and reduced sleep over a 24-hour period, compared to surface fish injected with a scrambled Morpholino.The injection of the hypocretin orexin Morpholino has little effect on sleep in surface fish compared to control-injected fish. In contrast, hypocretin orexin knockdown via Morpholino injection has a significant effect on sleep in cave-dwelling fish, providing a direct link between hypocretin orexin expression and sleep loss. CRPSR-mediated mutagenesis of the albinism gene OCA2 in surface fish results in some individuals homozygous for the wild-type OCA2 positive allele, and is associated with harboring pigmentation, whereas individuals that have two copies of the CRISPR-mutated OCA2 negative allele are albino, providing a direct link between the mutant OCA2 gene and albinism in cave fish.Select F0's positive for the Tol2 transmitted transgene to back cross to the wild type to generate stable F1 lines. This enables live calcium imaging for uncovering differences in neuronal activity, mediating behavioral changes in the cave environment, laying the groundwork for the expression of many additional transgenes to characterize and manipulate gene function in A mexicanus. The most important things to remember are to load the eggs tightly onto the plate, to trim the needle properly, and to optimize the microinjection.After a successful genetic knockdown, transgene integration, or CRISPR-mediated gene editing, these fish can be used for developmental, behavioral, and brain activity analyses.

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8.8K visualizações.  Florida Atlantic University. Os animais das cavernas fornecem um sistema convincente para investigar diversos traços morfológicos, de desenvolvimento e comportamentais. Mas um dos fatores limitantes no estabelecimento desse sistema modelo é a falta de ferramentas genéticas que nos permitam manipular a função genética. Aqui demonstramos três abordagens diferentes para manipular a função genética no peixe-caverna mexicano, Astyanax mexicanus.Essas ferramentas permitirão a investigação dos genes subjace...