Linda Mannini

Deciphering the molecular machinery of stem cells: a look at the neoblast gene expression profile.

BackgroundMammalian stem cells are difficult to access experimentally; model systems that can regenerate offer an alternative way to characterize stem cell related genes. Planarian regeneration depends on adult pluripotent stem cells - the neoblasts. These cells can be selectively destroyed using X-rays, enabling comparison of organisms lacking stem cells with wild-type worms.ResultsUsing a genomic approach we produced an oligonucleotide microarray chip (the Dj600 chip), which was designed using selected planarian gene sequences. Using this chip, we compared planarians treated with high doses of X-rays (which eliminates all neoblasts) with wild-type worms, which led to identification of a set of putatively neoblast-restricted genes. Most of these genes are involved in chromatin modeling and RNA metabolism, suggesting that epigenetic modifications and post-transcriptional regulation are pivotal in neoblast regulation. Comparing planarians treated with low doses of X-rays (after which some radiotolerant neoblasts re-populate the planarian body) with specimens irradiated with high doses and unirradiated control worms, we identified a group of genes that were upregulated as a consequence of low-dose X-ray treatment. Most of these genes encode proteins that are known to regulate the balance between death and survival of the cell; our results thus suggest that genetic programs that control neoblast cytoprotection, proliferation, and migration are activated by low-dose X-rays.ConclusionThe broad differentiation potential of planarian neoblasts is unparalleled by any adult stem cells in the animal kingdom. In addition to our validation of the Dj600 chip as a valuable platform, our work contributes to elucidating the molecular mechanisms that regulate the self-renewal and differentiation of neoblasts.

A mortalin-like gene is crucial for planarian stem cell viability

In adult organisms, stem cells are crucial to homeostasis and regeneration of damaged tissues. In planarians, adult stem cells (neoblasts) are endowed with an extraordinary replicative potential that guarantees unlimited replacement of all differentiated cell types and extraordinary regenerative ability. The molecular mechanisms by which neoblasts combine long-term stability and constant proliferative activity, overcoming the impact of time, remain by far unknown. Here we investigate the role of Djmot, a planarian orthologue that encodes a peculiar member of the HSP70 family, named Mortalin, on the dynamics of stem cells of Dugesia japonica. Planarian stem cells and progenitors constitutively express Djmot. Transient Djmot expression in differentiated tissues is only observed after X-ray irradiation. DjmotRNA interference causes inability to regenerate and death of the animals, as a result of permanent growth arrest of stem cells. These results provide the first evidence that an hsp-related gene is essential for neoblast viability and suggest the possibility that high levels of Djmot serve to keep a p53-like protein signaling under control, thus allowing neoblasts to escape cell death programs. Further studies are needed to unravel the molecular pathways involved in these processes.

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue.

Two msh/msx-related genes, Djmsh1 and Djmsh2, contribute to the early blastema growth during planarian head regeneration

Regeneration in planarians is an intriguing phenomenon, based on the presence of pluripotent stem cells, known as neoblasts. Following amputation, these cells activate mitotic divisions, migrate distally and undergo differentiation, giving rise to the regeneration blastema. We have identified two msh/msx-related genes, Djmsh1 and Djmsh2, which are expressed in distinct cell populations of the planarian Dugesia japonica and activated, with different patterns, during head regeneration. We demonstrate that RNA interference of Djmsh1 or Djmsh2 generates a delay in the growth of cephalic blastema, interfering with the dynamics of mitoses during its initial formation. Our data also reveal that the activity of the two planarian msh genes is required to regulate Djbmp expression during head regeneration. This study identifies, for the first time, a functional association between muscle segment homeobox (MSH) homeoproteins and BMP signaling during stem cell-based regeneration of the planarian head and provides a functional analysis of how msh genes may regulate in vivo the regenerative response of planarian stem cells.

Expression of a retinal homeobox (Rx) gene during planarian regeneration.

Retinal homeobox (Rx) genes, with representatives in vertebrates and invertebrates, encode fundamental regulators of early eye and brain formation. Here we describe the spatio-temporal expression profile of a candidate planarian orthologue of Rx during regeneration in Dugesia japonica and Schmidtea mediterranea. Although low levels of Rx transcripts were found throughout the body of intact planarians, high levels of Rx expression were specific to regenerating tissue in both head and tail fragments. We also observed that Rx was never expressed in the simple rhabdomeric planarian eyes, supporting the notion that only formation of eyes that use the ciliary type of photoreceptors requires Rx function.

Genetic regulation of planarian head morphogenesis during regeneration

Planarians possess amazing powers of regeneration and can rebuild any lost body part. The regenerative process, based on stem cells (neoblasts), is mediated by the formation of a blastema. During head regeneration, a complete functional brain, as well as the cephalic sensory structures, including eyes, are rebuilt. Recently, the characterization of a number of neural genes has revealed the complexity of the planarian central nervous system (CNS) at both the molecular and structural levels. In this review, special attention is addressed to planarian eye regeneration, and we cloned and characterized several of the genes involved in this process. Our work demonstrated that regulatory genes similar to those found in a variety of organisms, such as Pax6, eya, and six, orchestrate eye regeneration in planarians. However, the strategies that control this process may not be conserved completely, as demonstrated by our RNA interference (RNAi)‐based functional studies. We also performed a comprehensive search of eye‐related genes generating reciprocal suppressive subtractive hybridization (SSH) cDNA libraries, taking advantage from the use of the RNAi‐induced functional ablation of six‐1, a gene expressed only in the photoreceptors, to obtain planarians devoid of eyes. We cloned several genes with a role in a variety of cellular functions, as well as gene products of unknown function. The information gathered from this study provides new resources to better understand the molecular mechanisms of regeneration in planarians.

Chromosomes and genetic organization of rdna in planarians of the genus schmidtea (platyhelminthes, tricladida)

Abstract The genus Schmidtea includes four planarian species characterized by karyological differentiation. Different levels of ploidy also define various biotypes, typically associated with sexual or pseudogamic forms of reproduction. The present paper gives a karyological survey of somatic cells and unfertilized oocytes of specimens collected from natural populations. With the aim of integrating classical karyological analyses with molecular data, the molecular structure of ribosomal DNA (rDNA), in individuals of different species and biotypes is also compared, using restriction enzyme analysis. The results reveal the presence of species‐specific rDNA restriction patterns. In spite of the occurrence of poly‐ploid forms, a high level of structural intraspecific homogeneity of these genes was also observed among different populations.