RESUMO
Free-living flatworms, such as the planarian Schmidtea mediterranea, are extensively used as model organisms to study stem cells and regeneration. The majority of flatworm studies so far focused on broadly conserved genes. However, investigating what makes these animals different is equally informative for understanding its biology and might have biomedical value. We re-analyzed the neoblast and germline transcriptional signatures of the flatworm M. lignano using an improved transcriptome assembly and show that germline-enriched genes have a high fraction of flatworm-specific genes. We further identified the Mlig-sperm1 gene as a member of a novel gene family conserved only in free-living flatworms and essential for producing healthy spermatozoa. In addition, we established a whole-animal electron microscopy atlas (nanotomy) to visualize the ultrastructure of the testes in wild type worms, but also as a reference platform for different ultrastructural studies in M. lignano. This work demonstrates that investigation of flatworm-specific genes is crucial for understanding flatworm biology and establishes a basis for such future research in M. lignano.
Assuntos
Platelmintos/genética , Reprodução/genética , Animais , Células Germinativas , Fatores do Domínio POU/genética , Regeneração/genética , Especificidade da Espécie , Células-Tronco , TranscriptomaRESUMO
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.
Assuntos
Técnicas de Transferência de Genes , Genoma Helmíntico/genética , Platelmintos/genética , Regeneração/genética , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Especificidade de Órgãos/genética , Ovário/metabolismo , Platelmintos/embriologia , Platelmintos/fisiologia , Regiões Promotoras Genéticas/genética , Testículo/metabolismo , Transgenes/genéticaRESUMO
The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.
