The genetic basis of behavior in the blind Mexican cavefish, Astyanax mexicanus

In recent years, considerable progress has been made towards understanding the genetic basis of the evolution of morphological traits. In contrast, relatively little is known about how behavioral traits evolve. Astyanax mexicanus, a species of fish that exists in both surface and cave forms, is an ideal system to study behavioral evolution. Surface and cave morphs of Astyanax mexicanus differ in a variety of morphological and behavioral traits. They are interfertile, allowing for genetic analysis of the evolution of these traits. Finally, Astyanax mexicanus exists in multiple, independently evolved cave populations, providing an excellent system for studying convergent evolution

Fundamental research questions in subterranean biology

Five decades ago, a landmark paper inSciencetitledThe Cave Environmentheralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.Peer reviewe

Genome Editing Using TALENs in Blind Mexican Cavefish, <i>Astyanax mexicanus</i>

<div><p><i>Astyanax mexicanus</i>, a teleost fish that exists in a river-dwelling surface form and multiple cave-dwelling forms, is an excellent system for studying the genetic basis of evolution. Cavefish populations, which independently evolved from surface fish ancestors multiple times, have evolved a number of morphological and behavioral traits. Quantitative trait loci (QTL) analyses have been performed to identify the genetic basis of many of these traits. These studies, combined with recent sequencing of the genome, provide a unique opportunity to identify candidate genes for these cave-specific traits. However, tools to test the requirement of these genes must be established to evaluate the role of candidate genes in generating cave-specific traits. To address this need, we designed transcription activator-like effector nucleases (TALENs) to target two genes that contain coding changes in cavefish relative to surface fish and map to the same location as QTL for pigmentation, <i>oculocutaneous albinism 2</i> (<i>oca2</i>) and <i>melanocortin 1 receptor</i> (<i>mc1r</i>). We found that surface fish genes can be mutated using this method. TALEN-induced mutations in <i>oca2</i> result in mosaic loss of melanin pigmentation visible as albino patches in F₀ founder fish, suggesting biallelic gene mutations in F₀s and allowing us to evaluate the role of this gene in pigmentation. The pigment cells in the albino patches can produce melanin upon treatment with L-DOPA, behaving similarly to pigment cells in albino cavefish and providing additional evidence that <i>oca2</i> is the gene within the QTL responsible for albinism in cavefish. This technology has the potential to introduce a powerful tool for studying the role of candidate genes responsible for the evolution of cavefish traits.</p></div

Genome Editing Using TALENs in Blind Mexican Cavefish, Astyanax mexicanus

Astyanax mexicanus, a teleost fish that exists in a river-dwelling surface form and multiple cave-dwelling forms, is an excellent system for studying the genetic basis of evolution. Cavefish populations, which independently evolved from surface fish ancestors multiple times, have evolved a number of morphological and behavioral traits. Quantitative trait loci (QTL) analyses have been performed to identify the genetic basis of many of these traits. These studies, combined with recent sequencing of the genome, provide a unique opportunity to identify candidate genes for these cave-specific traits. However, tools to test the requirement of these genes must be established to evaluate the role of candidate genes in generating cave-specific traits. To address this need, we designed transcription activatorlike effector nucleases (TALENs) to target two genes that contain coding changes in cavefish relative to surface fish and map to the same location as QTL for pigmentation, oculocutaneous albinism 2 (oca2) and melanocortin 1 receptor (mc1r). We found that surface fish genes can be mutated using this method. TALEN-induced mutations in oca2 result in mosaic loss of melanin pigmentation visible as albino patches in F0 founder fish, suggesting biallelic gene mutations in F0s and allowing us to evaluate the role of this gene in pigmentation. The pigment cells in the albino patches can produce melanin upon treatment with L-DOPA, behaving similarly to pigment cells in albino cavefish and providing additional evidence that oca2 is the gene within the QTL responsible for albinism in cavefish. This technology has the potential to introduce a powerful tool for studying the role of candidate genes responsible for the evolution of cavefish traits.This article is from PLoS One 10 (2015): e0119370, doi:10.1371/journal.pone.0119370. Posted with permission.</p

Percentage of surviving embryos at 9–12 hours post injection.

<p>The number of total and surviving embryos were calculated for control and injected embryos in the morning after three sets of injections for each TALEN pair. mRNA is the total amount of mRNA injected per embryo. Percentage survival was calculated by dividing the number of living embryos by the total number of embryos. Survival of embryos for each trial can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.s002" target="_blank">S1 Table</a>.</p><p>Percentage of surviving embryos at 9–12 hours post injection.</p

Analysis of mutagenesis in <i>oca2</i> TALEN-injected F₀ fish.

<p>A. Genotyping gel of uninjected control and embryos injected with 400 pg and 600 pg of the <i>oca2</i> TALEN targeting exon 9. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 9 from 10 individual control embryos or individual injected embryos, and half of the PCR product was digested (D) with <i>Bsr</i>I. Wild type DNA digests completely with <i>Bsr</i>I whereas alleles with mutations induced by the TALEN pair are resistant to restriction digest, indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site, and may still be sensitive to restriction digest. B. Genotyping gel of uninjected control and embryos injected with 800 pg of the <i>oca2</i> TALEN targeting exon 9. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 9 from individual control embryos or individual injected embryos and half of the PCR product was digested (D) with <i>Bsr</i>I. Wild type DNA digests completely with <i>Bsr</i>I, whereas alleles with mutations induced by the TALEN pair are resistant to restriction digestion, as indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site, and may still be sensitive to restriction digest. C. Sequence of a wild type surface fish exon 9 and of five clones from the restriction enzyme resistant band from an <i>oca2</i>-injected individual. The dashed lines indicate missing nucleotides, and the lower case letters indicate mismatches. The total number of base pairs less than the wild type sequence is indicated to the right of each clone. Note that two of the five clones were identical. D. Genotyping gel of uninjected control and embryos injected with 400 pg total mRNA of the <i>oca2</i> TALENs targeting either side of exon 21. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 21 from 10 pooled control embryos or individual injected embryos. A band approximately 100 base pairs lower than the wild type band (arrow) indicates mutant alleles in injected embryos. The percentage of mutant alleles is indicated below the band. E. Sequence of a wild type surface fish around exon 21 and of five clones from 100 base pair smaller band from an <i>oca2</i>-injected individual. The dashed lines indicate missing nucleotides. The lower case letters are intron sequence, and the upper case letters are exon sequence. Sequence between the TALEN pairs is not shown and is indicated by the three dots. The total number of base pairs less than the wild type sequence is indicated to the right of each clone. Note that the starred clone contains a large deletion (321 bp) and an insertion (209 bp). F. Sequence of a wild type surface fish exon 9 and of four mutant clones from the restriction enzyme resistant band from pools of F₁s from a cross between an <i>oca2</i> exon 9 400 pg-injected F₀ individual and a wildtype surface fish. The dashed lines indicate missing nucleotides, and the red letters indicate mismatches. The total number of base pairs less than the wild type or the mutation compared to wildtype sequence is indicated to the right of each clone. Note that two of the four clones were identical.</p

Analysis of mutagenesis in <i>mc1r</i> TALEN F₀ fish.

<p>A. Genotyping gel of uninjected control and 800 pg-injected embryos. A portion of the <i>mc1r</i> genomic region was amplified by PCR from individual embryos, and half of the PCR product was digested (D) with <i>Bss</i>SI. Wild type DNA digests completely with <i>Bss</i>SI whereas alleles with mutations induced by the TALEN pair are resistant to restriction digest, indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site and may still be sensitive to restriction digest. B. Sequence of a wild type surface fish and sequence of four clones from the restriction enzyme resistant band from a <i>mc1r</i>-injected individual. The dashed lines indicate missing nucleotides, the lower case letters indicate mismatches, and the sequence below the last clone is additional nucleotide sequence. The total number of base pairs more or less than the wild type sequence is indicated to the right of each clone.</p

<i>mc1r</i> and <i>oca2</i> TALEN targeting design.

<div><p>A. Diagram of the <i>mc1r</i> annotated coding sequence with the location and sequences of the Pachón, Japonés and Yerbaniz mutations highlighted. The transmembrane domains are indicated in light gray. The two base pair deletion in Pachón is indicated by dashed lines. The single nucleotide change in the Japonés and Yerbaniz populations is in red. The sequence targeted by the TALEN is indicated below. The TALEN 1 binding site is in green and the TALEN 2 binding site is in blue. The spacer region is gray. The underlined sequence is the <i>Bss</i>SI restriction enzyme recognition sequence used for genotyping. The gene structure is based on the <i>Astyanax</i> genome sequence database and Gross et al. 2009 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.ref019" target="_blank">19</a>].</p> <p>B. Diagram of the <i>oca2</i> gene. Boxes indicate exons and lines indicate introns. The empty boxes are UTR and the closed boxes are coding sequence. The slanted lines indicate a region of the genome left out because the distance is unknown. Note that the small scale of the figure resulted in some exons not being to scale and the size of some introns being so small that they appear to be one continuous exon. The exon 24 Pachón and the exon 21 Molino deletions are indicated in red. The amount of intronic sequence deleted in these populations is currently unknown. TALENs were designed targeting exon 9 and the either end of exon 21. The TALEN 1 binding sites are in green and the TALEN 2 binding sites are in blue. The spacer regions are gray. The underlined sequence in the exon 9 TALEN is the <i>Bsr</i>I restriction enzyme recognition sequence used for genotyping. The exon sequence is capitalized and the intronic sequence in the two exon 21 TALENs is lower case. The genomic structure is based on the <i>Astyanax</i> genome sequence database and Protas et al. 2006 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.ref017" target="_blank">17</a>]. Both gene structures were generated using <a href="http://wormweb.org/exonintron" target="_blank">http://wormweb.org/exonintron</a> and then modified.</p></div