A Dual Platform Approach to Transcript Discovery for the Planarian Schmidtea Mediterranea to Establish RNAseq for Stem Cell and Regeneration Biology

Martin J. Blythe, Damian Kao, Sunir Malla, Joanna Rowsell, Ray Wilson, Deborah Evans, Jamie Jowett, Amy Hall, Virginie Lemay, Sabrina Lam, A. Aziz Aboobaker

The use of planarians as a model system is expanding and the mechanisms that control planarian regeneration are being elucidated. The planarian Schmidtea mediterranea in particular has become a species of choice. Currently the planarian research community has access to this whole genome sequencing project and over 70,000 expressed sequence tags. However, the establishment of massively parallel sequencing technologies has provided the opportunity to define genetic content, and in particular transcriptomes, in unprecedented detail. Here we apply this approach to the planarian model system. We have sequenced, mapped and assembled 581,365 long and 507,719,814 short reads from RNA of intact and mixed stages of the first 7 days of planarian regeneration. We used an iterative mapping approach to identify and define de novo splice sites with short reads and increase confidence in our transcript predictions. We more than double the number of transcripts currently defined by publicly available ESTs, resulting in a collection of 25,053 transcripts described by combining platforms. We also demonstrate the utility of this collection for an RNAseq approach to identify potential transcripts that are enriched in neoblast stem cells and their progeny by comparing transcriptome wide expression levels between irradiated and intact planarians. Our experiments have defined an extensive planarian transcriptome that can be used as a template for RNAseq and can also help to annotate the S. mediterranea genome. We anticipate that suites of other 'omic approaches will also be facilitated by building on this comprehensive data set including RNAseq across many planarian regenerative stages, scenarios, tissues and phenotypes generated by RNAi.

Full article here.

Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development

Collins, J., Hou, X., Romanova, E., Lambrus, B., Miller, C., Saberi, A., Sweedler, J., Newmark, P.

Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

Full article.

Schmidtea mediterranea: a model system for analysis of motile cilia.

Rompolas P, Patel-King RS, King SM.

Cilia are cellular organelles that appeared early in the evolution of eukaryotes. These structures and the pool of about 600genes involved in their assembly and function are highly conserved in organisms as distant as single-cell protists, like Chlamydomonas reinhardtti, and humans (Silflow and Lefebvre, 2001). A significant body of work on the biology of cilia has been produced over the years, with the help of powerful model organisms including C. reinhardtti, Caenorhabditis elegans, sea urchins, and mice. However, specific limitations of these systems, especially regarding the ability to efficiently study gene loss-of-function, warrant the search for a new model organism to study cilia and cilia-based motility. Schmidtea mediterranea is a species of planarian (Class: Tubellaria) with a well-defined monostratified ciliated epithelium, which contributes to the motility of the organism, in addition to other more specialized ciliary structures. The use of S. mediterranea as an experimental system to study stem cell biology and regeneration has led to a recently sequenced genome and to the development of a wide array of powerful tools including the ability to inhibit gene expression via RNA interference. In addition, we have developed and describe here a number of methods for analyzing motile cilia in S. mediterranea. Overall, S. mediterranea is a highly versatile, easy to maintain, and genetically tractable organism that provides a powerful alternative model system for the study of motile cilia.

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The TALE Class Homeobox Gene Smed-prep Defines the Anterior Compartment for Head Regeneration

Daniel A. Felix, A. Aziz Aboobake

Planaria continue to blossom as a model system for understanding all aspects of regeneration. They provide an opportunity to understand how the replacement of missing tissues from preexisting adult tissue is orchestrated at the molecular level. When amputated along any plane, planaria are capable of regenerating all missing tissue and rescaling all structures to the new size of the animal. Recently, rapid progress has been made in understanding the developmental pathways that control planarian regeneration. In particular Wnt/beta-catenin signaling is central in promoting posterior fates and inhibiting anterior identity. Currently the mechanisms that actively promote anterior identity remain unknown. Here, Smed-prep, encoding a TALE class homeodomain, is described as the first gene necessary for correct anterior fate and patterning during planarian regeneration. Smed-prep is expressed at high levels in the anterior portion of whole animals, and Smed-prep(RNAi) leads to loss of the whole brain during anterior regeneration, but not during lateral regeneration or homeostasis in intact worms. Expression of markers of different anterior fated cells are greatly reduced or lost in Smed-prep(RNAi) animals. We find . . .

Full article here.

Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa

José María Martín-Durána, Enrique Amayab and Rafael Romero.

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are . . .

Full article here.

Autophagy and apoptosis in planarians

Cristina Gonzalez-Estevez1 and Emili Salo

Adult planarians are capable of undergoing regeneration and body remodelling in order to adapt to physical damage or extreme environmental conditions. Moreover, most planarians can tolerate long periods of starvation and during this time, they shrink from an adult size to, and sometimes beyond, the initial size at hatching. Indeed, these properties have made them a classic model to study stem cells and regeneration. Under such stressful conditions, food reserves from the gastrodermis and parenchyma are first used up and later the testes, copulatory organs and ovaries are digested. More surprisingly, when food is again made available to shrunken individuals, they grow back to adult size and all their reproductive structures reappear. These cycles of growth and shrinkage may occur over long periods without any apparent impairment to the individual, or to its future maturation and breeding capacities. This plasticity resides in a mesoderm tissue known as . . .

Link to full article.

Flow cytometry methods for the study of cell-cycle parameters of planarian stem cells.

Hara Kang; Alejandro Sánchez Alvarado

Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo.

Link to full article.

Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia

Jochen C. Rink, Kyle A. Gurley, Sarah A. Elliott, Alejandro Sánchez Alvarado

The Hedgehog (Hh) signaling pathway plays multiple essential roles during metazoan development, homeostasis, and disease. Although core protein components are highly conserved, the variations in Hh signal transduction mechanisms exhibited by existing model systems (Drosophila, fish, and mammals) are difficult to understand. We characterized the Hh pathway in planarians. Hh signaling is essential for establishing the anterior/posterior axis during regeneration by modulating wnt expression. Moreover, RNA interference methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable Hh signaling defects; however, these proteins are essential for planarian ciliogenesis. Our study expands the understanding of Hh signaling in the animal kingdom and suggests an ancestral mechanistic link between Hh signaling and the function of cilia.

Link to full article.

Retinoic acid as a regulator of planarian morphogenesis

Ermakova, O.N., et al.

The effect of retinoic acid on regeneration of two species of asexual planarian races, Girardia tigrina and Schmidtea mediterranea, was studied. It was established that retinoic acids at physiological concentrations (10−7–10−10 M) inhibit the regeneration of the head part of planarians but have no effect on tail blastema growth. It is shown that regeneration of the head part is inhibited as a result of arrest of the cell cycle of neoblasts, proliferating stem cells, during the transition from the G 1/G 0 to the S phase. Thus, the morphogenetic role of retinoic acids in planarians, primitive bilaterally symmetrical animals, has been demonstrated.

Full article here.

Figuring out the Heads or Tails Decision in Regeneration

. . . To study how planaria regrow their bodies from a sliver of tissue, Christian Petersen, a postdoctoral fellow in Whitehead Member Peter Reddien's lab, looked at two of the planaria's genes, wntP-1 and wntP-2. These genes act to control cell to cell communication in a process called the Wnt signaling pathway, which is involved in many biological events, including development and growth. Petersen and Reddien had theorized that at least some portion of the Wnt pathway plays a role in regeneration.

In earlier work with planaria, Petersen and Reddien showed that a Wnt-related gene, called Smed-beta-catenin-1 (beta catenin), is necessary for planaria to regenerate a tail instead of a head after tail amputation. This ability to grow a head when the head is cut off and grow a tail when the tail is cut off is known as "regeneration polarity". In this experiment, Petersen cut the head, tail or both the head and tail off of planaria and observed where wntP-1 gene expression occurred. . .

Read the rest here.

High-resolution profiling and discovery of planarian small RNAs

Friedländer, M.; Hirst, M.; Marra, M.; Nusbaum, C.; et al.

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 . . .

Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea

Yi-Chien Lu , Magda Smielewska, Dasaradhi Palakodeti, Michael T. Lovci, Stefan Aigner, Gene W. Yeo and Brenton R. Graveley

MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed . . .

Abstract and full article available here.

Molecular Analysis of Stem Cells and Their Descendants during Cell Turnover and Regeneration in the Planarian Schmidtea mediterranea

Molecular Analysis of Stem Cells and Their Descendants during Cell Turnover and Regeneration in the Planarian Schmidtea mediterranea

George T. Eisenhoffer, Hara Kang, and Alejandro Sanchez Alvarado

In adult planarians, the replacement of cells lost to physiological turnover or injury is sustained by the proliferation and differentiation of stem cells known as neoblasts. Neoblast lineage relationships and the molecular changes that take place during differentiation into the appropriate cell types are poorly understood. Here we report the identification and characterization of a cohort of genes specifically expressed in neoblasts and their descendants. We find that genes with severely downregulated expression after irradiation molecularly define at least three discrete subpopulations of cells. Simultaneous BrdU labeling and . . .

The article in PDF format can be accessed here.

Planarian PTEN homologs regulate stem cells and regeneration through TOR signaling

Planarian PTEN homologs regulate stem cells and regeneration through TOR signaling

Néstor J. Oviedo, Bret J. Pearson, Michael Levin, and Alejandro Sánchez Alvarado

We have identified two genes, Smed-PTEN-1 and Smed-PTEN-2, capable of regulating stem cell function in the planarian Schmidtea mediterranea. Both genes encode proteins homologous to the mammalian tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Inactivation of Smed-PTEN-1 and -2 by RNA interference (RNAi) in planarians disrupts regeneration, and leads to abnormal outgrowths in both cut and uncut animals followed soon after by death (lysis). The resulting phenotype is characterized by hyperproliferation of neoblasts (planarian stem cells), tissue disorganization and a significant accumulation of postmitotic cells with impaired differentiation capacity. Further analyses revealed . . .

The article is available here.

Congratulations to Phil Newmark from the University of Illinois who was named HHMI Investigator! From the article:

According to those making the award, Newmark has been “instrumental in establishing planarians as a model system for studying regeneration at the molecular level. … (He) is exploring a central question that has long intrigued biologists – what are the signals or cues that tell a cell to become a germ cell? He hopes to answer that question by studying the ways planarians develop and regenerate their germ cells.”

“The department is delighted and proud of Dr. Newmark’s selection as an HHMI investigator,” said cell and developmental biology professor and department head Andrew Belmont. “The vast majority of scientists devote their careers to making steady but incremental progress on previously established lines of research. In contrast, a very select, very small group of scientists instead pioneer completely new approaches and methodologies, which then serve a much broader community. Dr. Newmark falls into the latter class of scientists, and it is tremendously satisfying to see the HHMI reward him for his vision, risk-taking and hard work,” Belmont said.

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Beta-Catenin Defines Head Versus Tail Identity During Planarian Regeneration and Homeostasis
Kyle A. Gurley, Jochen C. Rink, Alejandro Sánchez Alvarado*

After amputation, freshwater planarians properly regenerate a head or tail from the resulting anterior or posterior wound. The mechanisms that differentiate anterior from posterior and direct the replacement of the appropriate missing body parts are unknown. We found that in the planarian Schmidtea mediterranea, RNA interference (RNAi) of β-catenin or dishevelled causes the inappropriate regeneration of a head instead of a tail at posterior amputations...

Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris

Boran Altincicek and Andreas Vilcinskas

"...We identified numerous immune-inducible genes in Hydra and Schmidtea that show a similar distribution corresponding to their physiological roles, although lineages of both animals split from their common ancestor for more than five hundred millions of years. The present study is the first analysis of immune-inducible genes of these two phylogenetically distant model organisms of regeneration and provide numerous candidate genes that we can use as a starting point for comparative examination of interrelationships between immunity and homeostasis."