Project description:Among the Chordates tunicates demonstrate the highest capacity for regeneration, ranging from full-body regeneration in colonial ascidians to appendage regeneration in solitary ascidians. Here we present a parallel study of mRNA and microRNA expression at three stages of oral siphon regeneration in the solitary ascidian Ciona robusta (a.k.a., Ciona intestinalis), and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed (DE) across the course of regeneration, we also identified 57 novel microRNAs, several of which are among the most highly DE. Analysis functional categories identified transcripts related to stress responses and apoptosis enriched at the wound healing stage, various signaling pathways including Wnt and TGF-β enriched during early regrowth, and negative regulation of extracellular remodeling proteases as enriched in late stage regeneration. Additionally, comprehensive 3’-UTR binding site prediction and probability of conserved targeting for all C. robusta microRNAs (including those identified here) was calculated using TargetScanS. A microRNA-target network was subsequently constructed by calculating Pearson correlation coefficients for all predicted microRNA-mRNA target pairs expressed during regeneration. Network based clustering associated one or more microRNAs and their targets into 22 non-overlapping groups. Functional analysis of mRNA targets in each network cluster showed that enrichment of stress response, signaling pathway and extracellular remodeling categories associated with specific stages could also be associated with specific microRNAs. Finally, predicted targets of the miR-9 network cluster suggest a role in regulating differentiation and proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle. This work represents a significant advance in the prediction of microRNA effects on appendage regeneration and provides a foundation for investigating evolutionary conservation of microRNAs during regeneration in chordates.

Project description:Variability of regenerative potential among animals has long perplexed biologists. Based on their amazing regenerative abilities, planarians have become important models for understanding the molecular basis of regeneration; however, planarian species with limited regenerative abilities are also found. Despite the importance of understanding the differences between closely related, regenerating and non-regenerating organisms, few studies have focused on the evolutionary loss of regeneration, and the molecular mechanisms leading to such regenerative loss remain obscure. Here we examine Procotyla fluviatilis, a planarian with restricted ability to replace missing tissues, utilizing next-generation sequencing to define the gene expression programs active in regeneration-permissive and regeneration-deficient tissues. We found that Wnt signaling is aberrantly activated in regeneration-deficient tissues. Remarkably, down-regulation of canonical Wnt signaling in regeneration-deficient regions restores regenerative abilities: blastemas form and new heads regenerate in tissues that normally never regenerate. This work reveals that manipulating a single signaling pathway can reverse the evolutionary loss of regenerative potential. RNA-seq experiments to identify gene expression changes following amputation in body regions with variable regenerative potential. Adult Procotyla fluviatilis were amputated at sites either anterior or posterior to the pharynx. After 24 hours post-amputation, tissues near the amputation site were excised and RNA was extracted. Similar tissues were excised from uncut control animals. Samples were processed for RNA-seq using Illumina procedures. We generated a de novo P. fluviatilis transcriptome and used RNA sequencing (RNA-seq) to characterize transcripts from excised tissue fragments in Reg+ and Reg- body regions 24 hours post-amputation. We performed parallel analyses on tissues excised from intact animals at identical body regions to account for regional differences in transcripts, thereby identifying changes resulting from amputation. Samples A1-A3 = Regeneration-proficient (Reg+) tissue excision 24 hours after amputation. Samples B1-B3 = Tissue excision from regeneration-proficient (Reg+) region but not amputated. Samples C1-C3 = Tissue excision from regeneration-deficient (Reg-) tissues 24 hours after amputation. Samples D1, D3-D4 = Tissue excision from regeneration-deficient (Reg-) region that was not amputated.

Project description:Fusarium graminearum and F. verticillioides are devastating cereal pathogens with very different life history and ecological characteristics. F. graminearum is homothallic, and sexual spores are an important component of its life cycle, responsible for disease initiation. F. verticilloides is heterothallic, and produces only modest numbers of fruiting bodies, which are not a significant source of inoculum. To identify corresponding differences in the transcriptional program underlying fruiting body development in the two species, comparative expression was performed, analyzing six developmental stages. To accompany the transcriptional analysis, detailed morphological characterization of F. verticillioides development was performed and compared to a previous morphological analysis of F. graminearum. Morphological development was similar between the two species, except for the observation of possible trichogynes in F. verticillioides ascogonia, which have not been previously reported for any Fusarium species. Expression of over 9000 orthologous genes were measured for the two species. Functional assignments of highly expressed orthologous genes at each time-point revealed the majority of highly expressed genes fell into the M-bM-^@M-^XM-bM-^@M-^Xunclassified proteinsM-bM-^@M-^YM-bM-^@M-^Y category, reflecting the lack of characterization of genes for sexual development in both species. Simultaneous examination of morphological development and stage-specific gene expression suggests that degeneration of the paraphyses during sexual development is an apoptotic process. Expression of mating type genes in the two species differed, possibly reflecting the divergent roles they play in sexual development. Overall, the differences in gene expression reflect the greater role of fruiting bodies in the life cycle and ecology of F. graminearum versus F. verticillioides. mRNA were sampled and compared from six time points across sexual reproduction in two Fusarium species

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The onset of whole-body regeneration in Botryllus schlosseri: morphological and molecular characterization

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