Manipulation of Gene Function in Mexican Cavefish

Summary

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.

Abstract

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. 

Introduction

Since Darwin’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 ‘surface’ 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. mexicanu....

Protocol

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 polymorphic variation within a morpholino target site can lead to ineffective binding. The design is similar to other fish systems, such as zebrafish, and....

Results

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 mammals^47,48. We have previously demonstrated that cave A. mexicanus have increased expression of H.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fish breeding & egg supplies
Fine mesh fish net	Penn Plax	BN4
Fish tank heater	Aqueon	100106108
Egg traps	Custom made	NA	Design and create plastic grate to place at bottom of tank to protect eggs
Glass pipettes	Fisher Scientific	13-678-20C
Pipette bulbs	Fisher Scientific	03-448-21
Agarose	Fisher Scientific	BP160-500
Egg molds	Adaptive Science Tools	TU-1
Morpholino supplies
Control Morpholino	Gene Tools, LLC	Standard control olio
Custom Morpholino	Gene Tools, LLC	NA
Phenol Red	Sigma Aldrich	P0290-100ML
CRISPR supplies
Cas9 Plasmid	AddGene	46757
GoTaq DNA Polymerase	Promega	M3001
KOD Hot Start Taq	EMD Millipore	71-842-3
Primers	Integrated DNA Technologies	Custom
T7 Megascript Kit	Ambion/Thermofisher	AM1333
miRNeasy Kit	Qiagen	217004
mMessage mMachine T3 kit	Ambion/Thermofisher	AM1348
MinElute Kit	Qiagen	28204
Tol2 transgenesis supplies
pCS-zT2TP plasmid	Kawakami et al., 2004	Request from senior author
CutSmart Buffer	New England Biolabs	B7204
NotI-HF Restriction Enzyme	New England Biolabs	R3189
PCR purification Kit	Qiagen	28104
SP6 mMessenger Kit	Ambion/Thermofisher	AM1340
Microinjection supplies
Glass Capillary Tubes	Sutter Instruments	BF100-58-10
Pipette puller	Sutter Instruments	P-97
Picoinjector	Warner Instruments	PLI-100A
Micromanipulator	World Precision Instruments	M3301R
Micromanipulator Stand	World Precision Instruments	M10
Micmanipulator Base	World Precision Instruments	Steel Plate Base, 10 lbs