Research Resources for Model Amphibians

Salamanders are important vertebrate model organisms in biomedical research. During the previous funding period we continued efforts under the Salamander Genome Project (SGP) to develop genomic and bioinformatic resources for the Mexican axolotl (Ambystoma mexicanum). Specifically, we developed and characterized a BAC library, performed transcriptional studies of regeneration, innovated methods to isolate and sequence DNA from axolotl chromosomes, and built an initial assembly of the axolotl genome, which is 10x larger than the human genome. Considering the pace of our current work, we anticipate entering the next funding period with a set of resources that can greatly accelerate axolotl research efforts. We propose specific aims to leverage, extend, and make these resources available to biomedical researchers: Specific Aim 1. Improve the contiguity of the current genome assembly. The large axolotl genome presents an interesting challenge because existing genome assembly strategies were designed to facilitate the assembly of human-sized genomes. Our current genome assembly will likely approach an N50 exceeding 100 Kbp when we enter the next funding period. While such an assembly provides a useful resource for developing probes and genetic constructs, greater contiguity is needed to characterize and facilitate studies of DNA and epigenetic variations distributed throughout the genome. We propose to use innovative methods to push contiguity to the scale of entire chromosomes. Using nucleated red blood cells, we will manipulate chromatin to capture long-range sequence information, essentially building mate-pair like datasets that allow long distance scaffolding (Hi-C). This information will be integrated with our existing assembly and dense meiotic maps to achieve chromosome scale contiguity. Accomplishment of this Aim will yield a high quality assembly for biomedical researchers. Specific Aim 2. Characterize and annotate coding and non-coding regions of the axolotl genome. After an assembly is developed, a logical next step is to identify and annotate the structure of genes, regulatory regions, and epigenetic variations. In a recent study that comprehensively and deeply examined transcription during limb regeneration, we identified five punctuated episodes of transcription. We hypothesize that punctuated episodes of transcription are regulated epigenetically to initiate and coordinate parallel biological processes during regeneration. We propose whole genome mapping of conserved histone modifications and DNA methylation patterns to characterize the epigenetic landscape of genes that are differentially regulated during these punctuated episodes of transcription. Accomplishment of this Aim will integrate dense transcriptomic datasets with information related to chromatin state, reveal information about gene structure, identify regulatory regions in the axolotl genome, and characterize epigenetic changes during salamander limb regeneration. Specific Aim3. Sequence a second salamander genome. With a highly contiguous genome in sight for the axolotl, we see need to generate DNA sequence for a second salamander species. The red-spotted newt (Notophthalamus viridescens) is the most likely choice because it has been the subject of regeneration studies for many decades and the evolutionary distance to the axolotl is sufficiently deep to distinguish between conserved and unique regulatory elements. Additionally, we have developed genomic tools for the newt including a partial BAC library and meiotic map. We propose to sequence the newt genome to a depth of 16x and use existing transcripts to build an initial assembly. Accomplishment of this Aim will yield the first comparative genomic data for urodele amphibians.