The planarian TCF/LEF factor Smed-tcf1 is required for the regeneration of dorsal-lateral neuronal subtypes
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ABSTRACT: The adult brain of the planarian Schmidtea mediterranea (a freshwater flatworm) is a dynamic structure with constant cell turnover as well as the ability to completely regenerate de novo. Despite this, function and pattern is achieved in a reproducible manner from individual to individual in terms of the correct spatial and temporal production of specific neuronal subtypes. Although several signalling molecules have been found to be key to scaling and cell turnover, the mechanisms by which specific neural subtypes are specified remain largely unknown. Here we performed a 6 day RNAseq time course on planarians that were regenerating either 0, 1, or 2 heads in order to identify novel regulators of brain regeneration. Focusing on transcription factors, we identified a TCF/LEF factor, Smed-tcf1, which was required to correctly pattern the dorsal-lateral cell types of the regenerating brain. The most severely affected neurons in Smed-tcf1(RNAi) animals were the dorsal GABAergic neurons, which failed to regenerate, leading to an inability of the animals to phototax away from light. Together, Smed-tcf1 is a critical regulator, required to pattern the dorsal-lateral region of the regenerating planarian brain.
Project description:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete.
Project description:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete. In the RNAcompete assay, a purified GST-tagged protein is incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.
Project description:The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians, but proteomic studies are still in the early stages. Here, a global proteomics analysis of regenerating head fragments in planarians at 0h, 2h and 6h after amputation was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics coupled with LC-MS/MS strategy. Then the significantly changed proteins were identified. The correlation betwwen protein expression profiles and signaling pathways/biological processes was analyzed by bioinformatics and systems biology.
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.
Project description:Epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to the external environment. Interestingly, the epidermes of different species show broad morphological and functional diversity yet it is unclear whether this diversity came from modification of an existing gene regulatory network or de novo innovation of new genes. Here we identify the transcriptional regulators underlying the differentiation program of planarian epidermal lineage. We classify Smed-p53 as the most upstream molecule in this transcriptional cascade, suggesting a potentially conserved role for this gene in epidermal differentiation similar to TP63 in vertebrates. Moreover, we find that homologs of Sox and Pax family transcription factors, Smed-soxP-3 and Smed-pax-5, act cooperatively to activate the expression of epidermal markers. Together, these data show that planarian epidermal differentiation is regulated by a combination of conserved elements (p53/p63), recruitment of a non-conventional transcription module (soxP-3/pax-5), and novel genes (prog); they also suggest that specialized adpatations, such as epidermal mucus-secretion, arise from complex changes to gene networks.
Project description:The planarian epidermis provides an excellent model to explore adult stem cell (ASC) lineage development due to well-characterized and distinct spatiotemporal phases during lineage progression. Using flow cytometry-isolated cells enriched in epidermal progenitors, we performed transcriptional profiling and RNAi screening to uncover regulators of epidermal differentiation. We identified a MYB-type transcription factor (Smed-myb-1) required for specification of the first temporal phase of post-mitotic maturation. Knockdown of myb-1 abolished the early progenitor phase of differentiation without ceasing production of subsequent epidermal progenitors or homeostatic turnover and regeneration of the epidermis. Further examination revealed accelerated maturation of ASC descendants, with premature entry into subsequent progeny phases and ultimately the epidermis. These results demonstrate that a spatiotemporal shift in lineage progression occurs in the absence of the early progenitor state after myb-1 RNAi, and identify myb-1 as a critical regulator of the early temporal window in the step-wise specification during planarian epidermal differentiation.
Project description:Adult stem cells are tissue-specific cells with the capacity to self-renew and differentiate to continually replace cells lost to normal physiological turnover or injury. Neoblasts, the planarian stem cells, are widely distributed throughout the body mesenchyme, driving constitutive renewal of tissues during homeostasis and endowing planarians with the remarkable capacity to regenerate wholly from tiny tissue fragments. Neoblasts are the only dividing cells in planarians and are believed to be collectively comprised of both a heterogeneous population of pluripotent cells with broad differentiation potential and also lineage-committed progenitor cells that give rise to specific tissues. Recent technology has allowed one to isolate stem cells so we used a well-established method to isolate planarian stem cells by Hoechst blue staining and flow cytometry. To understand the molecular mechanisms underlying neoblast differentiation, we performed an RNA-Seq analysis of X1 and Xins cells looking at the differentially expressed genes between the two populations. Examine gene expression profiles of adult flatwormâ??s X1 and Xins cell types. The experiment was performed in quadruplicate yielding a total of 8 samples.
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:Epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to the external environment. Interestingly, the epidermes of different species show broad morphological and functional diversity yet it is unclear whether this diversity came from modification of an existing gene regulatory network or de novo innovation of new genes. Here we identify the transcriptional regulators underlying the differentiation program of planarian epidermal lineage. We classify Smed-p53 as the most upstream molecule in this transcriptional cascade, suggesting a potentially conserved role for this gene in epidermal differentiation similar to TP63 in vertebrates. Moreover, we find that homologs of Sox and Pax family transcription factors, Smed-soxP-3 and Smed-pax-5, act cooperatively to activate the expression of epidermal markers. Together, these data show that planarian epidermal differentiation is regulated by a combination of conserved elements (p53/p63), recruitment of a non-conventional transcription module (soxP-3/pax-5), and novel genes (prog); they also suggest that specialized adpatations, such as epidermal mucus-secretion, arise from complex changes to gene networks. This experiment aims to identify regulators involved in epidermal differentiation at the neoblast stage by using RNA Seq to examine transcriptional changes in neoblasts (X1 population) isolated from animals treated with zfp-1 or p53 dsRNA.