Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano

Abstract

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

Resilience to aging in the regeneration‐capable flatworm Macrostomum lignano

“…In addition, the previous study used a wild‐type strain which is not available anymore. The current study was performed with the inbred DV1 line, which was used for establishing the genome and transcriptome assemblies (Grudniewska et al., 2016; Wudarski et al., 2017). During the current survival studies, it was, however, shown that the DV1 line has a chromosome duplication, which has unknown effects on the biology of the worms (Wudarski et al., 2017; Zadesenets, Ershov, Berezikov & Rubtsov, 2017).…”

“…The current study was performed with the inbred DV1 line, which was used for establishing the genome and transcriptome assemblies (Grudniewska et al., 2016; Wudarski et al., 2017). During the current survival studies, it was, however, shown that the DV1 line has a chromosome duplication, which has unknown effects on the biology of the worms (Wudarski et al., 2017; Zadesenets, Ershov, Berezikov & Rubtsov, 2017). Another fascinating observation is that M. lignano does not show signs of a decreased regeneration capacity with advancing age, even after up to twelve amputations of the body, which is in strong contrast to mammals (Sharpless & DePinho, 2007).…”

“…Illumina sequencing reads were mapped to the annotated Mlig_3_7 genome assembly (Wudarski et al., 2017) using star v.2.5.2b (Dobin et al., 2013) in the transcriptome quantification mode with following parameters “–outFilterMultimapNmax 30 –outFilterMismatchNmax 5 –quantMode TranscriptomeSAM.” To generate read counts for transcript clusters, the resulting bam files were processed by Corset v.1.06 (Davidson & Oshlack, 2014). EdgeR package and EBSeq‐HMM (Leng et al., 2015) were used to perform differential gene expression analysis.…”

“…The selection of genes and their primers are listed in Table S6. The primers of the target and reference genes were designed with IDT's online PrimerQuest Tool, using the Mlig_3_7 assembly of the genome and transcriptome (Wudarski et al., 2017) and requiring that at least one primer in each pair spanned exon–exon splice junction. Three replicates of 2‐month‐old and three replicates of 26‐month‐old animals were used for RNA extraction.…”

A phylogenetically informed search for an alternative Macrostomum model species, with notes on taxonomy, mating behavior, karyology, and genome size

“…The free‐living flatworm Macrostomum lignano Ladurner, Schärer, Salvenmoser, Rieger 2005 (Rhabditophora, Macrostomorpha) has emerged as a model for a broad range of research topics (Ladurner, Schärer, Salvenmoser, & Rieger, ), including the biology of aging (Mouton, Grudniewska, Glazenburg, Guryev, & Berezikov, ; Mouton et al, ), bioadhesion (Lengerer, Hennebert, Flammang, Salvenmoser, & Ladurner, ; Lengerer et al, ; Wunderer et al, ), regeneration (Egger, Ladurner, Nimeth, Gschwentner, & Rieger, ; Lengerer et al, ), stem cell biology (Grudniewska et al, ; Ladurner et al, ), and sexual selection (Marie‐Orleach, Janicke, Vizoso, David, & Schärer, ; Schärer, Littlewood, Waeschenbach, Yoshida, & Vizoso, ; Sekii et al, ). This research has led to the establishment of many resources and tools that are crucial for a modern genetic and genomic model, including in situ hybridization (Pfister et al, ), RNA interference (Kuales et al, ; Sekii et al, ), gene expression (Arbore et al, ; Grudniewska et al, ; Lengerer et al, ), genome and transcriptome assemblies (Wasik et al, ; Wudarski et al, ), transgenesis (Marie‐Orleach, Janicke, Vizoso, Eichmann, & Schärer, ; Wudarski et al, ), and a clarification of the phylogenetic context (Janssen et al, ; Schärer et al, ). Jointly, these achievements make M. lignano an excellent model to complement the, in several aspects, more established planarian flatworm models (Newmark & Sánchez Alvarado, ; Pellettieri & Sánchez Alvarado, ; Rink, ; Rouhana et al, ).…”