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

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

Analysis of pigmentation in <i>oca2</i>-injected F0s.

<p>A. Pigmentation in an uninjected surface fish. B. Close up of the dorsal region of the uninjected surface fish from A. C. Pigmentation in a 400 pg <i>oca2</i> exon 9 injected F0 surface fish. D. Close up of pigmentation patch lacking melanin pigmentation from panel C.</p

L-DOPA treated scales.

<p>A. A Pachón cavefish scale. B. A 400 pg <i>oca2</i> exon 9 injected scale from a non-melanin pigmented patch. C. An uninjected surface fish scale. D. The Pachón cavefish scale from A following treatment with L-DOPA. E. The 400 pg <i>oca2</i> exon 9 injected scale from B following treatment with L-DOPA. F. The uninjected surface fish scale from C following treatment with L-DOPA. Insets are close up pictures of a melanophore (in C) and melanin-producing cells (D and E). The scale bar in inset C is 20 uM. The scale bars in insets D and E are 10 uM. Note that dark patches on the unstained scales are tissue and that dark spots on B are bubbles or particles, not melanophores.</p

TALEN‐mediated gene editing of the thrombospondin‐1 locus in axolotl

Abstract Loss‐of‐function genetics provides strong evidence for a gene's function in a wild‐type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long‐standing questions. Here we demonstrate targeted modification of the thrombospondin‐1 (tsp‐1) locus using transcription‐activator‐like effector nucleases (TALENs) and identify a role of tsp‐1 in recruitment of myeloid cells during limb regeneration. We find that while tsp‐1‐edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN‐mediated gene edits