Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

Currently, little is known about the evolution of epigenetic regulation in animal stem cells. Here we demonstrate, using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases that their function as tumor suppressors in mammalian stem cells is conserved over a long evolutionary distance. To investigate the potential conservation of a genome-wide epigenetic regulatory program in animal stem cells, we assess the effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumor suppressors among the affected genes that are likely candidates for mediating MLL3/4 tumor suppression function. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation

Organ specific gene expression in the regenerating tail of Macrostomum lignano

Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano

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

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

The challenges of detecting subtle population structure and its importance for the conservation of emperor penguins

Understanding the boundaries of breeding populations is of great importance for conservation efforts and estimates of extinction risk for threatened species. However, determining these boundaries can be difficult when population structure is subtle. Emperor penguins are highly reliant on sea ice, and some populations may be in jeopardy as climate change alters sea-ice extent and quality. An understanding of emperor penguin population structure is therefore urgently needed. Two previous studies have differed in their conclusions, particularly whether the Ross Sea, a major stronghold for the species, is isolated or not. We assessed emperor penguin population structure using 4,596 genome-wide single nucleotide polymorphisms (SNPs), characterized in 110 individuals (10–16 per colony) from eight colonies around Antarctica. In contrast to a previous conclusion that emperor penguins are panmictic around the entire continent, we find that emperor penguins comprise at least four metapopulations, and that the Ross Sea is clearly a distinct metapopulation. Using larger sample sizes and a thorough assessment of the limitations of different analytical methods, we have shown that population structure within emperor penguins does exist and argue that its recognition is vital for the effective conservation of the species. We discuss the many difficulties that molecular ecologists and managers face in the detection and interpretation of subtle population structure using large SNP data sets, and argue that subtle structure should be taken into account when determining management strategies for threatened species, until accurate estimates of demographic connectivity among populations can be made.</p

The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source. DOI: http://dx.doi.org/10.7554/eLife.20062.00

Aboobaker Lab Schmidtea mediterranea transcriptome dataset

<p>This fileset contains data associated with the consolidated Schmidtea mediterranea transcriptome. The publication associated with this fileset is currently under review. This transcriptome was consolidated using 5 independently assembled transcriptomes:</p> <p>-Blythe MJ, Kao D, Malla S, Rowsell J, Wilson R, et al. (2010) A Dual Platform Approach to Transcript Discovery for the Planarian Schmidtea Mediterranea to Establish RNAseq for Stem Cell and Regeneration Biology. PLoS ONE 5(12): e15617. doi:10.1371/journal.pone.0015617</p> <p>-Catherine Adamidi, Yongbo Wang, Dominic Gruen, et al. (2011) De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics. Genome Research 2011. doi:10.1101/gr.113779.110</p> <p>-Thomas Sandmann, Matthias C Vogg, Suthira Owlarn, et al. (2011) The head-regeneration transcriptome of the planarian Schmidtea mediterranea. Genome Biology 2011, 12:R76 doi:10.1186/gb-2011-12-8-r76</p> <p>-Resch AM, Palakodeti D, Lu Y-C, Horowitz M, Graveley BR (2012) Transcriptome Analysis Reveals Strain-Specific and Conserved Stemness Genes in Schmidtea mediterranea. PLoS ONE 7(4): e34447. doi:10.1371/journal.pone.0034447</p> <p>-Labbé, R. M., Irimia, M., Currie, K. W., Lin, A., Zhu, S. J., Brown, D. D.R., Ross, E. J., Voisin, V., Bader, G. D., Blencowe, B. J. and Pearson, B. J. (2012), A Comparative Transcriptomic Analysis Reveals Conserved Features of Stem Cell Pluripotency in Planarians and Mammals. STEM CELLS, 30: 1734–1745. doi: 10.1002/stem.1144</p> <p> </p> <p>The following files are included in this fileset:</p> <p>OX_smed_1.0.fa - transcriptome sequences in fasta format.</p> <p>OX_smed_1.0.blast - cleaned blast results for transcriptome. Hits to hypotheticals or unknowns are filtered out.</p> <p>OX_smed_1.0.go - Gene ontology results for transcriptome.</p> <p>OX_smed_1.0.pfam - PFAM HMM-A scan results for transcriptome.</p> <p>OX_smed_1.0.tf - Potential transcription factors found in the transcriptome using DBD transcription factor database.</p> <p>OX_smed_1.0.source - The source transcripts/EST that made up each consolidated transcript.</p> <p>OX_Gtig_0.1.fa - G. tigrina transcriptome assembled from Roche 454 reads.</p> <p>Supp01-08 - Supplemental files for publication in submission.</p

In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: The role of RGD and PHSRN domains

Polymorphonuclear leukocytes, monocytes/macrophages, foreign body giant cells (FBGC), and lymphocytes are central in directing the host foreign body and inflammatory/immune reactions that impact material biostability, biocompatibility, and device efficacy. We employed the functional architecture of fibronectin to probe the mechanisms of protein-cell interaction in modulating leukocyte behavior. We demonstrated previously that the RGD and PHSRN domain of fibronectin and the spatial orientation between the motifs were crucial in regulating macrophage function in vitro. The current study delineates the role of RGD and PHSRN in modulating the host inflammatory response and macrophage behavior in vivo. Oligopeptides containing RGD and/or PHSRN domains were grafted onto a polyethyleneglycol-based substrate and subcutaneously cage implanted into rats. Peptide identity played a minimal role in modulating the host inflammatory response and adherent macrophage density. The RGD motif, either alone or at the terminal position with the PHSRN-containing flanking sequence, elicited a rapid macrophage fusion to form FBGCs at the early stage of implantation. Both the RGD motif and the PHSRN motif were important in mediating FBGC formation at the later implantation time. However, the PHSRN motif, specifically in the configuration of G3RGDG6PHSRNG, evoked a larger extent of FBGC coverage. Our results indicate the importance of RGD and PHSRN in modulating macrophage function in a time and orientation dependent fashion in vivo. © 2001 Elsevier Science Ltd. All rights reserved.Link_to_subscribed_fulltex

The planarian regeneration transcriptome reveals a shared but temporally shifted regulatory program between opposing head and tail scenarios

BACKGROUND: Planarians can regenerate entire animals from a small fragment of the body. The regenerating fragment is able to create new tissues and remodel existing tissues to form a complete animal. Thus different fragments with very different starting components eventually converge on the same solution. In this study, we performed an extensive RNA-seq time-course on regenerating head and tail fragments to observe the differences and similarities of the transcriptional landscape between head and tail fragments during regeneration. RESULTS: We have consolidated existing transcriptomic data for S. mediterranea to generate a high confidence set of transcripts for use in genome wide expression studies. We performed a RNA-seq time-course on regenerating head and tail fragments from 0 hours to 3 days. We found that the transcriptome profiles of head and tail regeneration were very different at the start of regeneration; however, an unexpected convergence of transcriptional profiles occurred at 48 hours when head and tail fragments are still morphologically distinct. By comparing differentially expressed transcripts at various time-points, we revealed that this divergence/convergence pattern is caused by a shared regulatory program that runs early in heads and later in tails.Additionally, we also performed RNA-seq on smed-prep(RNAi) tail fragments which ultimately fail to regenerate anterior structures. We find the gene regulation program in response to smed-prep(RNAi) to display the opposite regulatory trend compared to the previously mentioned share regulatory program during regeneration. Using annotation data and comparative approaches, we also identified a set of approximately 4,800 triclad specific transcripts that were enriched amongst the genes displaying differential expression during the regeneration time-course. CONCLUSION: The regeneration transcriptome of head and tail regeneration provides us with a rich resource for investigating the global expression changes that occurs during regeneration. We show that very different regenerative scenarios utilize a shared core regenerative program. Furthermore, our consolidated transcriptome and annotations allowed us to identity triclad specific transcripts that are enriched within this core regulatory program. Our data support the hypothesis that both conserved aspects of animal developmental programs and recent evolutionarily innovations work in concert to control regeneration.S

Python script for filtering .SAM formatted mapping files aligned with BWA mem

The filter.py script works on sorted .SAM formatted mapping files from BWA mem alignment. For every pair of mapped forward and reverse reads, it parses out the CIGAR field (column 6 of the SAM file) and the MD tag to calculate the number of insertions, deletions, and mismatches. If a pair of reads have mismatches less than or equal to five and insertion/deletions less than or equal to two, then the pair is kept and printed to linux standard output. SAM header lines are ignored by the parser but also printed to standard output for compatible down-stream analysis