Methods for cell isolation and analysis of the highly regenerative tunicate Polycarpa mytiligera

Background:Polycarpa mytiligera is the only molecularly characterized solitary ascidian capable of regenerating all organs and tissue types. The cellular basis for regeneration in P. mytiligera is largely unknown, and methods for isolating live cells from this species for functional analyses are unavailable.Results: Here, we developed a method for isolating live cells from P. mytiligera, overcoming major experimental challenges, including the dissociation of its thick body wall and native cellular autofluorescence. We demonstrated the applicability of our approach for tissue dissociation and cell analysis using three flow cytometry platforms, and by using broadly used non-species-specific cell labeling reagents. In addition to live cell isolation, proof-of-concept experiments showed that this approach was compatible with gene expression analysis of RNA extracted from the isolated cells, and with ex vivo analysis of phagocytosis.Conclusion: We presented efficient methods for cell purification from a highly regenerative ascidian, which could be transferable to diversity of non-model marine organisms. The ability to purify live cells will promote future studies of cell function in P. mytiligera regeneration

Planarian Epidermal Stem Cells Respond to Positional Cues to Promote Cell-Type Diversity

Successful regeneration requires that progenitors of different lineages form the appropriate missing cell types. However, simply generating lineages is not enough. Cells produced by a particular lineage often have distinct functions depending on their position within the organism. How this occurs in regeneration is largely unexplored. In planarian regeneration, new cells arise from a proliferative cell population (neoblasts). We used the planarian epidermal lineage to study how the location of adult progenitor cells results in their acquisition of distinct functional identities. Single-cell RNA sequencing of epidermal progenitors revealed the emergence of distinct spatial identities as early in the lineage as the epidermal neoblasts, with further pre-patterning occurring in their post-mitotic migratory progeny. Establishment of dorsal-ventral epidermal identities and functions, in response to BMP signaling, required neoblasts. Our work identified positional signals that activate regionalized transcriptional programs in the stem cell population and subsequently promote cell-type diversity in the epidermis.National Institutes of Health (U.S.) (Grant R01GM080639

A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians

Regeneration starts with injury. Yet how injuries affect gene expression in different cell types and how distinct injuries differ in gene expression remain unclear. We defined the transcriptomes of major cell types of planarians—flatworms that regenerate from nearly any injury—and identified 1,214 tissue-specific markers across 13 cell types. RNA sequencing on 619 single cells revealed that wound-induced genes were expressed either in nearly all cell types or specifically in one of three cell types (stem cells, muscle, or epidermis). Time course experiments following different injuries indicated that a generic wound response is activated with any injury regardless of the regenerative outcome. Only one gene, notum, was differentially expressed early between anterior- and posterior-facing wounds. Injury-specific transcriptional responses emerged 30 hr after injury, involving context-dependent patterning and stem-cell-specialization genes. The regenerative requirement of every injury is different; however, our work demonstrates that all injuries start with a common transcriptional response.Broad Institute of MIT and Harvard. Klarman Cell ObservatoryHoward Hughes Medical Institute (Investigator)National Institutes of Health (U.S.) (NIH grant R01GM080639)European Molecular Biology Organization (Fellowship)National Institutes of Health (U.S.) (NIH grant F32 HD075541

The Single-Nucleotide Resolution Transcriptome of Pseudomonas aeruginosa Grown in Body Temperature

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen

Comparative transcriptomics of pathogenic and non-pathogenic Listeria species

Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs

Comparative transcriptomics across the prokaryotic tree of life

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to ‘transcriptional noise’. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5′UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome

Mutation Detection with Next-Generation Resequencing through a Mediator Genome

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes

RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs

The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5′- and 3′-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and ‘RNA walk’ to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite’s life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes

Table1_Methods for cell isolation and analysis of the highly regenerative tunicate Polycarpa mytiligera.XLSX

Background:Polycarpa mytiligera is the only molecularly characterized solitary ascidian capable of regenerating all organs and tissue types. The cellular basis for regeneration in P. mytiligera is largely unknown, and methods for isolating live cells from this species for functional analyses are unavailable.Results: Here, we developed a method for isolating live cells from P. mytiligera, overcoming major experimental challenges, including the dissociation of its thick body wall and native cellular autofluorescence. We demonstrated the applicability of our approach for tissue dissociation and cell analysis using three flow cytometry platforms, and by using broadly used non-species-specific cell labeling reagents. In addition to live cell isolation, proof-of-concept experiments showed that this approach was compatible with gene expression analysis of RNA extracted from the isolated cells, and with ex vivo analysis of phagocytosis.Conclusion: We presented efficient methods for cell purification from a highly regenerative ascidian, which could be transferable to diversity of non-model marine organisms. The ability to purify live cells will promote future studies of cell function in P. mytiligera regeneration.</p