The neoblasts are the only somatic stem cells in planarians possessing pluripotency, and can give rise to all types of cells, including germline cells. Recently, accumulated knowledge about the transcriptome and expression dynamics of various pluripotent somatic stem cells has provided important opportunities to understand not only fundamental mechanisms of pluripotency, but also stemness across species at the molecular level. The neoblasts can easily be eliminated by radiation. Also, by using fluorescence activated cell sorting (FACS), we can purify and collect many neoblasts, enabling identification of neoblast-related genes by comparison of the gene expression level among intact and X-ray-irradiated animals, and purified neoblasts. In order to find such genes, here we employed the high coverage expression profiling (HiCEP) method, which enables us to observe and compare genome-wide gene expression le-vels between different samples without advance sequence information, in the planarian D. japonica as a model organism of pluripotent stem cell research. We compared expression levels of ~17,000 peaks corresponding to independent genes among different samples, and ob-tained 102 peaks as candidates. Expression analysis of genes identified from those peaks by in situ hybridization revealed that at least 42 genes were expressed in the neoblasts and in neoblast-related cells that had a different distribution pattern in the body than neoblasts. Also, single-cell PCR analysis of those genes revealed heterogeneous expression of some genes in the neoblast population. Thus, using multidimensional gene expression analyses, we were able to obtain a valuable data set of neoblast-related genes and their expression patterns.
COVER PHOTOGRAPH : Low magnification image of planarian flatworms ( Schmidtea mediterranea ) fed with liver paste containing either red, green, yellow, or blue food dye. Uptake of colored liver paste containing in vitro synthesized double‐stranded RNA is readily visualized in the planarian gut. Ingestion and processing of double‐stranded RNA in the planarian gut leads to systemic RNA‐interference. Image credit: Labib Rouhana and Phillip A. Newmark, Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana‐Champaign. From RNA Interference by feeding in vitro‐synthesized double‐stranded RNA to planarians, Labib Rouhana, Jennifer A. Weiss, David J. Forsthoefel, Hayoung Lee, Ryan S. King, Takeshi Inoue, Norito Shibata, Kiyokazu Agata, and Phillip A. Newmark, Developmental Dynamics 242:718–730, 2013.
The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.
Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system “brain” rather than the distributed nervous system of radiatans. Thus, marine planarians have an early cephalized ‘central’ nervous system compared not only with non-bilaterians but also with those parasitic flatworms or freshwater planarians. Here, we investigated the three-dimensional structures of the planarian central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We identified, in marine planarians, both an apparent optic chiasm (a well-known feature in vertebrates) and mushroom bodies (prominent in insects). These results suggest that the marine planarian “brain” provides a valuable ‘missing-link brain’ between those of the protostome and deuterostome, especially in the evolutionary aspects of the nervous system.
The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CB s), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno‐electron microscopic analysis that Dj CBC ‐1, a planarian Me31B/Dhh1/ DDX 6 homologue, which is a component of ribonucleoprotein granules, was localized in CB s in the planarian Dugesia japonica . Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine ( sDMA ) specifically binds to CB s in which histone mRNA is co‐localized. Here, we showed by double immunostaining and RNA interference ( RNA i) that Dj CBC ‐1‐containing CB s and Y12‐immunoreactive CB s are distinct structures, suggesting that CB s are composed of heterogeneous populations. We also found that the Y12‐immunoreactive CB s specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNA i experiments that Y12‐immunoreactive CB s may have anti‐transposable element activity involving the DjPiwiC protein in the neoblasts.
