Department of Genetics, Harvard Medical School
Department of Genetics, Harvard Medical School
Collection OverviewThe Mexican Tetra, Astyanax mexicanus is an excellent model organism to study the developmental and genetic basis of evolution. This species of fish consists of a river-dwelling (surface) population and a number of cave-dwelling populations that evolved from surface fish trapped in caves. Cavefish have distinct morphology and physiology including: absence of eyes, reduced pigment, increased sensory structures, sensitive smell, reduced circadian rhythm, altered blood… Show More glucose regulation, and increased fat accumulation. Surface fish and all cavefish are interfertile allowing for genetic mapping studies that, in combination with a sequenced and annotated genome, have revealed the genetic changes leading to some of the unique cavefish traits. A. mexicanus have been bred in the lab for generations and numerous protocols exist for investigating their biology. The goal of this collection is to demonstrate standardized protocols for breeding, provide broader access to existing methods, and ultimately lower the threshold for researchers interested in working with this species.
Gamete collection and in-vitro fertilization of Astyanax mexicanus
Astyanax mexicanus is an uprising model organism in a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses a cave- and river dwelling population, which are interfertile. This enables genetic mapping of heritable traits that were fixated during adaptation to different environments of these populations. The transition of an emerging model organism from the wild to the lab, however, depends on the ability to establis… Show Moreh a functional breeding protocol. In-vitro fertilization (IVF) has been used in a variety of different model organism to successfully and repeatedly breed animals in the lab. Here we present a protocol to identify suitable parents, collect viable gametes from male and female fish and produce viable offspring using IVF. By acclimatizing A. mexicanus to different light cycles, we were able to shift breeding cycles to any given time of the day. This enables subsequent procedures such as injection of genetic constructs or developmental analysis during normal working hours. In addition, this technique can be used to create hybrids between the cave and surface dwelling populations and thereby enable the study of the genetic bases of phenotypic adaptations to different environments.
Neuromast imaging and free software-based tracking of a mechanosensory behavior in the Mexican cavefish
In perpetual darkness, cave-dwelling populations of Astyanax mexicanus have evolved a series of adaptive morphological and behavioral phenotypes. Vibration attraction behavior is a well-characterized adaptive behavior for foraging in darkness and is associated with increased number of sensory structures (neuromasts) in cavefish. In this article we describe a method for measuring and analyzing vibration attraction behavior and imaging neuromasts. In addition, we present methods for… Show More high throughput monitoring of a series of additional cavefish behaviors including: social interaction, hyperactivity, and repetitive stereotypic behavior. Our method uses free-software and custom-made scripts that can also be applied to a sleep analysis pipeline (see accompanied manuscript, Keene et al). This method provides a practical and cost-effective alternative to commercially available tracking software. Show Less
Raising Astyanax mexicanus for analysis of post-larval phenotypes and whole mount immunohistochemistry
River and cave-adapted populations of A. mexicanus have differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages although it could provide insight into how cavefish survive to adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feedin… Show Moreg regimes. Here we describe how to raise larvae off a recirculating system to two-weeks of age using live rotifers as a nutrient-rich food source that does not require daily replenishment. We demonstrate how to collect post-larval fish from this nursery system and perform whole mount immunostaining and imaging. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults. Show Less
A simple and flexible protocol for whole-mount in situ hybridization for Astyanax embryos
In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of coding genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci association various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant c… Show Morehallenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly visualize differences in gene expression between cave- and surface-dwelling forms, as a means of evaluating candidate genes underlying established QTL, genes of interest arising from next-generation sequencing studies, or other discovery-based approaches. In this report, we present a simple protocol, alongside a flexible checklist, that can be widely adapted for use well beyond our study system. Our hope is that this protocol and checklists can serve as a useful set of resources for the Astyanax community and beyond. Show Less
Automated measurements of sleep and locomotor activity in Mexican cavefish
Sleep is fundamental to animal life, yet little is known about how sleep has evolved throughout the animal kingdom. Genetic and comparative approaches to studying sleep in fish models have provided fundamental insight into the molecular and evolutionary basis of sleep regulation. Across phyla, sleep can be characterized by highly conserved behavioral characteristics including elevated arousal threshold, rebound following deprivation, and consolidated periods of immobility. The Mexican cavefish, … Show MoreAstyanax mexicanus, is a model for studying trait evolution in response to environmental perturbation and we have identified the evolution of sleep loss in multiple, independently evolved cavefish populations compared to river-dwelling surface fish populations. Here, we described a protocol for recording sleep and locomotor activity in A. mexicanus cave and surface fish. An inexpensive infrared-based monitoring system allows for behavioral imaging of individually housed fish for periods of a week or greater. The system can be applied to fish aged four days post fertilization through adulthood. The system can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using a customized macro that quantifies multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in additional fish species including zebrafish and sticklebacks. Show Less
Implementation of Tol2-mediated transgenesis in the 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, depigmentation, sleep loss, hyperphagia, and sensory processing. While diverse cave species have been studied in the laboratory setting, the Mexican cavefish, Astyanax mexicanus, has provided unique insights into biological and molecular processes underlying complex traits, and is well poised as an emerging mod… Show Moreel system. Candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, yet, a lack of transgenic tools has provided a significant impediment to investigating the genetic mechanisms underlying the evolution of these complex traits. Here, we describe methodology for efficient implementation of Tol2-mediated transgenesis, a system commonly used in zebrafish and other fish models, to generate stable transgenic lines of both surface and cave populations. We describe optimized procedures for breeding, embryo collection, and injection of single-cell embryos. Our findings reveal that Tol2 functions efficiently in A. mexicanus at levels roughly equivalent to zebrafish. Transgenic fish can be assessed in the injected F0 fish, or germ-line insertions can be selected in F1's with fluorescent or genetic screening. This approach enables ectopic expression of transgenes, manipulation of specific cells or tissues, and functional studies of neural circuits in A. mexicanus. Together, these applications facilitate investigation of the genetic and neural mechanisms of trait evolution in A. mexicanus. Show Less