NEW YORK (GenomeWeb) – Researchers from Cold Spring Harbor Laboratory and elsewhere have sequenced the genome and transcriptome of the flatworm Macrostomum lignano in a bid to better understand stem cell self-renewal and organismal regeneration.
M. lignano is a free-living flatworm that, when injured, can regenerate an almost entirely new organism. After CSHL's Michael Schatz and colleagues assembled the worm's genome using long sequencing reads and assembled and annotated its transcriptome, they used these new datasets to examine gene expression patterns in M. lignano during regeneration.
"M. lignano has a number of properties that make it advantageous as a model for studying stem and germ cell biology, differentiation, regeneration, and perhaps also aspects of neuroscience," Schatz and his colleagues wrote in a paper published today in the Proceedings of the National Academy of Sciences. "Moreover, viewed in comparison with those of other Platyhelminthes, the resource we provide might shed further light on the evolution of the molecular toolkit of regeneration."
A short read-based assembly of the M. lignano genome generated through Illumina sequencing was highly fragmented, the researchers reported, likely because of the genome's high percentage of simple repeats; some 75 percent of the M. lignano genome is made of simple repeats and transposon sequences.
The researchers turned to Pacific Biosciences' SMRT sequencing platform to generate long reads to span these repeats. From this, they generated 21X coverage of reads greater than 10 kilobases in length.
The inclusion of these reads, Schatz and his colleagues reported, improved their assembly substantially.
Meanwhile, they also generated RNA sequencing libraries from whole worms and assembled them to reveal nearly 150,000 putative transcripts of an average 516 basepairs in length.
Within the M. lignano transcriptome assembly, Schatz and his colleagues noted a number of transposons as well as evidence of trans-splicing. Some 7,500 transcripts, they reported, had potential spliced leader sequences at their 5' ends. These transcripts encompassed a range of protein families and also included introns, suggesting that they undergo both trans- and cis-splicing.
M. lignano, the researchers added, shares 6,217 transcripts with the planarian flatworm Schmidtea mediterranea, and encodes about 2,000 genes that are present in humans but lacking in Caenorhabditis elegans or Drosophila melanogaster.
In mammals, five transcription factors — Oct4/Pou5f1, Nanog, Klf4, c-Myc, and Sox2 — have been identified as key pluripotency factors. However, Schatz and his colleagues were only able to uncover one of these, Sox2, in the M. lignano genome. However, despite the other factors apparently being absent in the worm, the researchers found that the main mammalian stem cell pluripotency maintenance pathways like Jak-Stat, Wnt, and MAPK, among others, seem to be conserved.
The M. lignano genome also includes 49 homeobox-containing genes, which the researchers noted are crucial for patterning of the anterior-posterior axis in bilateral animals and organ regeneration in planarians. Thosehomeobox-containing genes include a number not seen in other Platyhelminthes that could play a role in regeneration, like Cdx, Dbx, and Prrx.
To find gene signatures linked to regeneration, the researchers sliced worms between the pharynx and testes and let them regenerate, finding six different synexpression classes of genes that were up- or down-regulated at different times.
Early response genes, they found, included growth factors as well as homologs of genes in the Tgf/Bmp pathway, which is a regulator of mammalian pluripotency. Other upregulated transcripts were involved in cell signaling and cellular organization.
A day or two after amputation, there was an enrichment of transcription factors with zinc-finger domains, Klf transcription factors, and a TNF-like protein. Meanwhile, a Smad4-like transcript, a potential pluripotency determinant, was downregulated, indicating to the researchers that the worm enters a differentiation phase at that time.
"Even though the majority of transcripts measured were not yet annotated, these datasets can provide a valuable resource for future regeneration studies," Schatz and his colleagues added.