Gene order data from a model amphibian (Ambystoma): new perspectives on vertebrate genome structure and evolution

BACKGROUND: Because amphibians arise from a branch of the vertebrate evolutionary tree that is juxtaposed between fishes and amniotes, they provide important comparative perspective for reconstructing character changes that have occurred during vertebrate evolution. Here, we report the first comparative study of vertebrate genome structure that includes a representative amphibian. We used 491 transcribed sequences from a salamander (Ambystoma) genetic map and whole genome assemblies for human, mouse, rat, dog, chicken, zebrafish, and the freshwater pufferfish Tetraodon nigroviridis to compare gene orders and rearrangement rates. RESULTS: Ambystoma has experienced a rate of genome rearrangement that is substantially lower than mammalian species but similar to that of chicken and fish. Overall, we found greater conservation of genome structure between Ambystoma and tetrapod vertebrates, nevertheless, 57% of Ambystoma-fish orthologs are found in conserved syntenies of four or more genes. Comparisons between Ambystoma and amniotes reveal extensive conservation of segmental homology for 57% of the presumptive Ambystoma-amniote orthologs. CONCLUSION: Our analyses suggest relatively constant interchromosomal rearrangement rates from the euteleost ancestor to the origin of mammals and illustrate the utility of amphibian mapping data in establishing ancestral amniote and tetrapod gene orders. Comparisons between Ambystoma and amniotes reveal some of the key events that have structured the human genome since diversification of the ancestral amniote lineage

Variation in salamander tail regeneration is associated with genetic factors that determine tail morphology

Very little is known about the factors that cause variation in regenerative potential within and between species. Here, we used a genetic approach to identify heritable genetic factors that explain variation in tail regenerative outgrowth. A hybrid ambystomatid salamander (Ambystoma mexicanum x A. andersoni) was crossed to an A. mexicanum and 217 offspring were induced to undergo metamorphosis and attain terrestrial adult morphology using thyroid hormone. Following metamorphosis, each salamander’s tail tip was amputated and allowed to regenerate, and then amputated a second time and allowed to regenerate. Also, DNA was isolated from all individuals and genotypes were determined for 187 molecular markers distributed throughout the genome. The area of tissue that regenerated after the first and second amputations was highly positively correlated across males and females. Males presented wider tails and regenerated more tail tissue during both episodes of regeneration. Approximately 66–68% of the variation in regenerative outgrowth was explained by tail width, while tail length and genetic sex did not explain a significant amount of variation. A small effect QTL was identified as having a sex-independent effect on tail regeneration, but this QTL was only identified for the first episode of regeneration. Several molecular markers significantly affected regenerative outgrowth during both episodes of regeneration, but the effect sizes were small (\u3c4%) and correlated with tail width. The results show that ambysex and minor effect QTL explain variation in adult tail morphology and importantly, tail width. In turn, tail width at the amputation plane largely determines the rate of regenerative outgrowth. Because amputations in this study were made at approximately the same position of the tail, our results resolve an outstanding question in regenerative biology: regenerative outgrowth positively co-varies as a function of tail width at the amputation site

Rediscovering the Axolotl as a Model for Thyroid Hormone Dependent Development

The Mexican axolotl (Ambystoma mexicanum) is an important model organism in biomedical research. Much current attention is focused on the axolotl\u27s amazing ability to regenerate tissues and whole organs after injury. However, not forgotten is the axolotl\u27s equally amazing ability to thwart aspects of tissue maturation and retain juvenile morphology into the adult phase of life. Unlike close tiger salamander relatives that undergo a thyroid hormone regulated metamorphosis, the axolotl does not typically undergo a metamorphosis. Instead, the axolotl exhibits a paedomorphic mode of development that enables a completely aquatic life cycle. The evolution of paedomorphosis allowed axolotls to exploit relatively permanent habitats in Mexico, and preadapted axolotls for domestication and laboratory study. In this perspective, we first introduce the axolotl and the various meanings of paedomorphosis, and then stress the need to move beyond endocrinology-guided approaches to understand the axolotl\u27s hypothyroid state. With the recent completion of the axolotl genome assembly and established methods to manipulate gene functions, the axolotl is poised to provide new insights about paedomorphosis and the role of thyroid hormone in development and evolution

Ion Channel Signaling Influences Cellular Proliferation and Phagocyte Activity During Axolotl Tail Regeneration

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, KV2.1, KV2.2, L-type CaV channels and H/K ATPases) or completely (GlyR, GABAAR, KV1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K+ channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H+ pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair

A de novo reference transcriptome for Bolitoglossa vallecula, an Andean mountain salamander in Colombia

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Arenas Gomez, C. M., Woodcock, M. R., Smith, J. J., Voss, S. R., & Delgado, J. P. A de novo reference transcriptome for Bolitoglossa vallecula, an Andean mountain salamander in Colombia. Data in Brief, 29, (2020): 105256, doi:10.1016/j.dib.2020.105256.The amphibian order Caudata, contains several important model species for biological research. However, there is need to generate transcriptome data from representative species of the primary salamander families. Here we describe a de novo reference transcriptome for a terrestrial salamander, Bolitoglossa vallecula (Caudata: Plethodontidae). We employed paired-end (PE) illumina RNA sequencing to assemble a de novo reference transcriptome for B. vallecula. Assembled transcripts were compared against sequences from other vertebrate taxa to identify orthologous genes, and compared to the transcriptome of a close plethodontid relative (Bolitoglossa ramosi) to identify commonly expressed genes in the skin. This dataset should be useful to future comparative studies aimed at understanding important biological process, such as immunity, wound healing, and the production of antimicrobial compounds.This work was funded by a research grant from COLCIENCIAS 569 (GRANT 027-2103) and CODI (Programa Sostenibilidad) 2013–2014 of the University of Antioquia. A PhD fellowship to the first author, Claudia Arenas was funded by the COLCIENCIAS 567 Grant. We thank the lab of Juan Fernando Alzate from the University of Antioquia for their help in developing our bioinformatic methodological approach. We thank Andrea Gómez and Melisa Hincapie for their help in animal collection and husbandry

MapToGenome: A Comparative Genomic Tool that Aligns Transcript Maps to Sequenced Genomes

Efforts to generate whole genome assemblies and dense genetic maps have provided a wealth of gene positional information for several vertebrate species. Comparing the relative location of orthologous genes among these genomes provides perspective on genome evolution and can aid in translating genetic information between distantly related organisms. However, large-scale comparisons between genetic maps and genome assemblies can prove challenging because genetic markers are commonly derived from transcribed sequences that are incompletely and variably annotated. We developed the program MapToGenome as a tool for comparing transcript maps and genome assemblies. MapToGenome processes sequence alignments between mapped transcripts and whole genome sequence while accounting for the presence of intronic sequences, and assigns orthology based on user-defined parameters. To illustrate the utility of this program, we used MapToGenome to process alignments between vertebrate genetic maps and genome assemblies 1) self/self alignments for maps and assemblies of the rat and zebrafish genome; 2) alignments between vertebrate transcript maps (rat, salamander, zebrafish, and medaka) and the chicken genome; and 3) alignments of the medaka and zebrafish maps to the pufferfish (Tetraodon nigroviridis) genome. Our results show that map-genome alignments can be improved by combining alignments across presumptive intron breaks and ignoring alignments for simple sequence length polymorphism (SSLP) marker sequences. Comparisons between vertebrate maps and genomes reveal broad patterns of conservation among vertebrate genomes and the differential effects of genome rearrangement over time and across lineages

Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs

Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs

A Linkage Map for the Newt \u3cem\u3eNotophthalmus viridescens\u3c/em\u3e: Insights in Vertebrate Genome and Chromosome Evolution

Genetic linkage maps are fundamental resources that enable diverse genetic and genomic approaches, including quantitative trait locus (QTL) analyses and comparative studies of genome evolution. It is straightforward to build linkage maps for species that are amenable to laboratory culture and genetic crossing designs, and that have relatively small genomes and few chromosomes. It is more difficult to generate linkage maps for species that do not meet these criteria. Here, we introduce a method to rapidly build linkage maps for salamanders, which are known for their enormous genome sizes. As proof of principle, we developed a linkage map with thousands of molecular markers (N=2349) for the Eastern newt (Notophthalmus viridescens). The map contains 12 linkage groups (152.3–934.7cM), only one more than the number of chromosome pairs. Importantly, this map was generated using RNA isolated from a single wild caught female and her 28 offspring. We used the map to reveal chromosome-scale conservation of synteny among N. viridescens, A. mexicanum (Urodela), and chicken (Amniota), and to identify large conserved segments between N. viridescens and Xenopus tropicalis (Anura). We also show that met1, a major effect QTL that regulates the expression of alternate metamorphic and paedomorphic modes of development in Ambystoma, associates with a chromosomal fusion that is not found in the N. viridescens map. Our results shed new light on the ancestral amphibian karyotype and reveal specific fusion and translocation events that shaped the genomes of three amphibian model taxa. The ability to rapidly build linkage maps for large salamander genomes will enable genetic and genomic analyses within this important vertebrate group, and more generally, empower comparative studies of vertebrate biology and evolution

Genomics of a metamorphic timing QTL: \u3ci\u3emet1\u3c/i\u3e maps to a unique genomic position and regulates morph and species-specific patterns of brain transcription

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect QTL (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele specific chromosome rearrangement on linkage group 2 that uniquely brought functionally-associated genes into linkage. Further, we found that \u3e 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation

Probability of Regenerating a Normal Limb After Bite Injury in the Mexican Axolotl (\u3cem\u3eAmbystoma mexicanum\u3c/em\u3e)

Multiple factors are thought to cause limb abnormalities in amphibian populations by altering processes of limb development and regeneration. We examined adult and juvenile axolotls (Ambystoma mexicanum) in the Ambystoma Genetic Stock Center (AGSC) for limb and digit abnormalities to investigate the probability of normal regeneration after bite injury. We observed that 80% of larval salamanders show evidence of bite injury at the time of transition from group housing to solitary housing. Among 717 adult axolotls that were surveyed, which included solitary-housed males and group-housed females, approximately half presented abnormalities, including examples of extra or missing digits and limbs, fused digits, and digits growing from atypical anatomical positions. Bite injury likely explains these limb defects, and not abnormal development, because limbs with normal anatomy regenerated after performing rostral amputations. We infer that only 43% of AGSC larvae will present four anatomically normal looking adult limbs after incurring a bite injury. Our results show regeneration of normal limb anatomy to be less than perfect after bite injury