RESUMO
Planaria, particularly Schmidtea mediterranea and Dugesia japonica, are now established as research organisms in many laboratories across the life sciences community. Planarians are cheap and easy to keep in the lab. This chapter provides techniques and guidelines for establishing and maintaining a planarian colony. We provide sections on food preparation, housing, feeding, cleaning, culture expansion by amputation, and recognizing and responding to culture problems.
Assuntos
Planárias/crescimento & desenvolvimento , Planárias/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , LaboratóriosRESUMO
Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.
Assuntos
MicroRNAs/genética , Planárias/genética , RNA Interferente Pequeno/genética , Regeneração/genética , Animais , Sequência de Bases , Clonagem Molecular , MicroRNAs/metabolismo , Dados de Sequência Molecular , Planárias/metabolismo , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNARESUMO
We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.