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Researchers Assemble Newt Transcriptome with Eye to Understanding Tissue Regeneration

NEW YORK (GenomeWeb News) – A study in Genome Biology is taking stock of the transcripts expressed in the red spotted newt, Notophthalmus viridescens, providing the foundation for more comprehensive studies of the processes underlying tissue regeneration.

Researchers from the Max-Planck Institute and the University of Dayton coupled RNA sequencing with mass spectrometry-based proteomic profiling to assess transcript patterns in adult newt tissues as well as tissues sampled during the amphibian's development or tissue regeneration.

Using this information, they were able to put together a newt transcriptome containing tens of thousands of annotated transcripts. Among them was a specific set of proteins that appears specific to an amphibian order called urodeles, which includes the newt and its regeneration-competent relatives.

"[W]e describe the first comprehensive de novo assembled transcriptome of Notophthalmus viridescens combined with large-scale experimental validation of coding sequences," said study leaders Thomas Braun and Thilo Borchardt, with the Max-Planck Institute for Heart and Lung Research, and the University of Dayton's Panagiotis Tsonis and their co-authors.

They argued that the newly generated data should not only be "an indispensable resource for a better understanding of the regenerative events in newts," but will also "facilitate the identification of molecules, conserved or urodele-specific, that control this fascinating process."

The newt can bounce back from injuries that would permanently damage most other creatures, regenerating central nervous system tissue and even whole limbs. Such capabilities have piqued researchers' interest in N. viridescens and other tissue-regenerating urodelians such as salamanders.

Still, the intimidating sizes of the newt and salamander genomes have rendered genomic analyses of regeneration tricky. So while the red spotted newt "represents an excellent model organism to study regenerative processes," Braun, Borchardt, Tsonis and co-authors explained, "mechanistic insights into molecular processes driving regeneration have been hindered by paucity and poor annotation of coding nucleotide sequences."

To get around complications associated with this corpulent genome size, the team focused its attention on parts of the genome expressed during different stages of newt development.

Using a combination of Sanger, Illumina, and Roche 454 sequencing technologies, the researchers sequenced RNA from a variety of undamaged adult newt tissues. They also did similar analyses on tissues taken during the animal's embryonic or larval stages and during regeneration of heart, limb, and eye tissues after injury.

After comparing several assembly approaches, the group came up with a hybrid transcriptome assembly built from a collection of pre-assembled sequence sets. From there, they went on to annotate newt genes, using mass spec, RT-PCR, and microarray analyses to validate transcripts and get clues as to where specific genes are expressed in the newt.

All told, the team annotated some 56 percent of the 120,922 transcripts in the newt transcriptome. These included 826 proteins found only in the urodelian amphibians so far, fueling speculation that at least some of these proteins participate in regeneration-related processes in the newts, salamanders, and closely related animals.

"[B]ioinformatic analysis disclosed several new protein families exclusive to urodelian amphibians," the study's authors noted, "of which some contain known domains from public databases, but also entirely new clusters of proteins sharing sequence motifs not known in other species."

"We reason that some of the proprietary newt proteins might play important roles in regeneration processes unique for urodeles," they added.

Consistent with that notion, their preliminary RT-PCR experiments uncovered expression shifts for a few of the urodeles-specific proteins during regeneration at different sites in the newt body.

Data generated for the study is included in a clearinghouse for newt genomic and proteomic data called Newt-omics, which Borchardt, Braun, and others described online in Nucleic Acids Research last fall.

This transcript-focused database makes it possible to "store, retrieve, link, and visualize sequences, proteins, and expression data," study authors said, and is expected to support ongoing analyses of the animal in the future.