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Amphibian, Flatworm Genomes Offer Clues to Regeneration

NEW YORK (GenomeWeb) – A pair of new genomic studies have started unraveling the genetic roots of limb or whole-organism regeneration.

For the first of two studies appearing online today in Nature, an international team led by investigators in Germany and Austria focused on the Mexican axolotl, Ambystoma mexicanum, a frequently studied aquatic amphibian capable of replacing lost limbs or tail tissue through regeneration. Although gene editing and other molecular tools have been developed for A. mexicanum, they explained, genomic studies of the axolotl and other salamanders have been tricky due to the size and repeat-rich nature of the genomes.

The researchers used a combination of Pacific Biosciences long read sequencing, Illumina short read sequencing for error correction, Bionano Saphyr de novo optical mapping, and assembly with the MARVEL algorithm to put together a 32.4 billion base axolotl genome assembly for an A. mexicanum strain often used in regeneration studies in the lab.

The resulting assembly contained nearly 99 percent of the non-exonic ultraconserved elements previously described in vertebrate genomes, the authors noted, suggesting its completeness is "comparable to or better than" the Tibetan frog and Xenopus amphibian genomes. That assertion was supported with RNA sequence data from nearly two dozen tissue types, which supported the completeness of the axolotl genome.

From these data, the researchers also identified 23,251 predicted protein-coding genes and some 18.6 billion bases of repetitive sequences. They subsequently delved into developmental and regenerative features in the axolotl genome.

Along with loss of the Pax3 developmental gene and alterations affecting the paralogous gene Pax7, for example, the team narrowed in on transcripts and/or microRNAs that appear to be over-represented in regenerating limb tissue.

"Taken together, these data point to a potential role in limb regeneration for several coding and non-coding sequences that have been lost or diverged rapidly in amniotes," senior author Eugene Myers, a researcher at the Max Planck Institute of Molecular Cell Biology and Genetics, and his colleagues wrote. "Future investigations of such sequences are likely to be a fruitful avenue for understanding the evolution of regeneration capabilities."

Myers was also co-corresponding author on a related Nature study, in which he and colleagues from Germany and the UK sequenced and analyzed the genome of the planarian flatworm Schmidtea mediterranea.

Again using a PacBio long read sequencing and MARVEL assembly — this time in combination with Dovetail Chicago HiRise scaffolding and a new DNA isolation protocol — the researchers put together a 782 million base de novo genome assembly with genomic DNA from an inbred S. mediterranea strain.

They noted that S. mediterranea had previously been sequenced, but that existing assemblies are quite fragmented, in part due to problems successfully isolating DNA from the organisms, which are used to study everything from regeneration, stem cell biology, and aging to the evolution of parasitism in related flatworms.

"Our S. mediterranea genome assembly represents a major improvement over existing S. mediterranea assemblies, and to our knowledge, is the first long-range contiguous assembly of the genome of a non-parasitic flatworm species," co-senior authors Myers and Jochen Rink, and their colleagues wrote.

The team's analysis of the genome revealed rampant repeat sequences, including a previously unappreciated class of giant retroelements. And when they annotated the genome using planarian transcriptome sequences and compared it to flatworm genomes sequenced previously, the investigators uncovered gene losses involving hundreds of otherwise conserved genes in the planarians.

The planarians appeared to be missing components of spindle assembly checkpoint pathways centered on the MAD1/MAD2 or BUB1-3 genes, for example, although they maintain the ability to pause mitotic cell division in response to spindle damage. Based on results from their subsequent RNA interference experiments, the researchers attributed this ability to independent RZZ gene complex activity.

"The demonstration of [spindle assembly checkpoint] function in the likely absence of MAD1 and MAD2 suggests that our genetic and mechanistic understanding of SAC function is incomplete," the authors concluded. "Further studies on planarians and other 'non-traditional' model organisms are needed to understand the basis and mechanism of these cellular functions."

In a related News and Views feature, Yale University researchers Grant Parker Flowers and Craig Crews predicted that the "new genome assemblies, when combined with the sudden ease of genetic manipulation using new genome-editing tools, will make it possible to do experiments that were previously unimaginable in model organisms such as planarians and salamanders."