1,721,027 research outputs found
Understanding the epigenetic regulation of stem cell fate in planarians using functional genomics
Full text linkPlanarians owe their remarkable regenerative capabilities to somatic pluripotent stem cells or neoblasts which, as the only dividing cells in the body, are able to differentiate into all cell types. Research into planarians so far has established the cellular and molecular basis of regeneration, stem cell pluripotency and differentiation. Recent studies using single-cell transcriptomics have improved our understanding of neoblast heterogeneity and the different cell types in planarians. Yet, the epigenetic mechanisms underlying neoblast pluripotency and the determination of cell fate remain understudied. This work aims to understand the epigenetic regulation of stem cell fate in planarians using an array of next-generation sequencing methods.
Planarians have a single methyl-CpG binding domain protein, MBD2/3, which is part of the NuRD complex that regulates transcription. Planarians lack endogenous cytosine methylation. Research into the role of MBD2/3 in an organism free of DNA methylation can provide important evolutionary insights into its DNA methylation-independent role. I show that knockdown of Smed-mbd2/3 affects both regeneration and tissue homeostasis. Using markers for stem cells and progeny, Smed-mbd2/3 was found to be essential for differentiation but not for stem cell maintenance. The different stem cell sub-populations were also unaffected by the knockdown of Smed-mbd2/3. To determine the targets of MBD2/3 involved in the lineage commitment of stem cells, I analysed the transcriptome of stem cells and stem cell progeny after knockdown of Smed-mbd2/3 at two time points. Genes misregulated after the inhibition of Smed-mbd2/3 were identified and will form an important resource to identify new markers of stem cell differentiation.
The identity of cis-regulatory elements remains elusive in planarians. Accessible regions of chromatin in the three planarian cell compartment were identified using ATAC-seq. ChIP-seq of planarian neoblasts was performed and correlated with gene expression and open regions of chromatin to identify putative enhancers. Information from the three types of sequencing datasets was used to create a catalogue of enhancers with different combinations of enhancer properties. This lays the groundwork for further studies to provide a better understanding of the heterogeneity of planarian stem cells and identify gene regulatory networks specific to the progression of different lineages
Radiation sensitivity and the DNA damage response in planarian stem cells
Full text linkIonizing radiation (IR) can inflict various types of DNA lesions which if not repaired, can induce genomic instability and subsequent oncogenic transformation. Potentially immortal and highly regenerative animals are hypothesized to have enhanced genome maintenance mechanisms to protect their stem cells. One such example, the freshwater planarian, Schmidtea mediterranea, contains a large population of collectively pluripotent adult stem cells called neoblasts that are completely ablated following exposure to lethal doses of IR (30 Gy). We identified a non-lethal dose of IR (15 Gy) that leads to a significant decrease in neoblasts but where full recovery of the stem cell number occurs over time. However, there is no evidence that DNA repair is required during regeneration and normal neoblast function. Here we show, exposure to 15 Gy of IR following knockdown of DNA repair gene is lethal, proof of principle that well-known DNA damage response (DDR) genes have a role in stem cell survival and repopulation post IR. We provide evidence that a new non-canonical role of DDR is to combat DNA damage during stem cell migration and that in the absence of a fully functioning DDR machinery, stem cells fail to migrate. Using an in-vivo shielded-irradiation assay, that allows cell migration to be tracked, we observed that neoblasts pre-exposed to IR migrate much slower, in a dose dependent manner, but eventually reach the wound. Migrating neoblasts were also more sensitive to IR than stationary cells suggesting that the mechanical stress due to changes in nuclear shape during migration represents a significant load on repair mechanisms. Our results provide an in vivo demonstration that a major novel role of DNA repair mechanisms may be to allow stem cell migration. Despite enormous efforts to treat cancer, radiotherapy is still the major treatment to kill cancerous cells. There is growing evidence that tumour-initiating cancer stem cells survive and adapt to repeated rounds of IR eventually leading to cancer-recurrence. This radio-tolerance is dependent on an efficient DDR signalling. However, the molecular basis of variations in IR resistance is not well understood. The extraordinary capacity of neoblasts to tolerate high doses of IR offer an opportunity to get novel mechanistic insights into radiation resistance. Using RNA-sequencing we delineate the transcriptional response to IR in planarian stem cells. We identified genes that were differentially expressed in response to IR and characterized the role of transcription factors (FHL-1) and a tetraspanin family of genes in stem cell repopulation post IR. We further extended our investigation by comparing the transcriptome of irradiated planarian stem cells with a human fibrosarcoma cell line, HT1080. We identified conserved transcriptional responses to IR providing a rich resource to identify radiation responsive genes. Given the conservation between pASCs and mammalian stem cells these conserved genes may include novel druggable targets for combining with radiotherapy
Genome-wide study of DNA methylation in the early development of the amphipod crustacean Parhyale hawaiensis
No full textDNA methylation is a crucial epigenetic modification associated with the regulation of gene expression in vertebrates and has dynamic patterns associated with the reprogramming of parental methylomes during early embryogenesis. However, the dynamics and underlying mechanisms of DNA methylation during early invertebrate development remain inadequately understood. Maternal gene products initially control early animal development until the maternal genome hands over control to the zygotic genome, known as maternal to zygotic transition (MZT). In vertebrates, DNA methylation plays a key role in ensuring the successful transfer of developmental control across generations during MZT. It remains unclear whether and how DNA methylation is involved in the MZT of invertebrates. The amphipod crustacean Parhyale hawaiensis (Parhyale) genome encodes DNA methyltransferases (DNMT1 and DNMT3) and exhibits higher DNA methylation levels than many other invertebrates. Therefore, in this thesis, I used Parhyale as an animal model to investigate the potential function of DNA methylation in relation to the control of gene expression and the control of transposon activity, both of which are essential factors in early development.
First, I confirmed the presence of DNA methylation in the Parhyale genome by analysing the distribution of DNA methylation using Whole-Genome Bisulfite Sequencing (WGBS) and PacBio single molecule real-time (SMRT) data from distinct Parhyale tissues and mixed-stage embryos. Next, I applied Enzymatic Methyl-seq to three early developmental stages surrounding the onset of MZT during Parhyale embryogenesis. This revealed that 8% of CpGs in Parhyale were methylated, with a particular focus on repetitive elements within non-coding regions. These methylation data were then integrated with RNA-seq data. A positive correlation between gene body methylation and gene expression was discovered, particularly at the highly expressed genes, which were found to have high repetitive content and elevated intronic repeat methylation. The functional significance of DNA methylation was further investigated using the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AzaD), which was administered to pre-MZT embryos. The study found that 5AzaD treatment caused developmental delays and was associated with impaired transcription of genes with higher levels of methylation in early development.
Our findings indicate that DNA methylation may play a role in the early development of Parhyale, potentially acting as an epigenetic regulator of precise temporal dynamics of gene expression. DNA methylation deposition is necessary for the successful transfer of developmental control from maternally supplied gene products to those synthesized from the zygotic genome. These results contribute to our understanding of the evolutionary conservation of DNA methylation patterns and offer valuable insights into the epigenetic regulation of developmental processes in invertebrates
Regenerative responses following DNA damage – β-catenin mediates head regrowth in the planarian Schmidtea mediterranea
Full text linkPluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS
An investigation of cellular and molecular mechanisms of stem cell regulation in Schmidtea mediterranea
Full text linkThe planarian Schmidtea mediterranea discovered by Benazzi, Baguñà, Ballester, Puccinelli & Del Papa, 1975 is a classic model organism for the investigation of regenerative processes. The source of new tissues is a population of proliferative cells called 'neoblasts'. The level of heterogeneity among this population of cells is unknown. However at least a proportion of neoblasts are pluripotent stem cells, and these are sometimes referred to as clonogenic neoblasts (cNeoblasts). Although the Schmidtea mediterranea genome has been sequenced, and RNAi techniques are well established, our knowledge of the molecular regulators of neoblast behaviours such as migration in-vivo remain limited. This thesis presents an assay for tracking spatio-temporal processes such as stem cell migration and division. Data presented herein supports the assays potential as an adjunct method for functional testing of molecular regulators of stem cell biology. Moreover this thesis demonstrates the effect of various gene knockdowns on cell migration in vivo. The matrix metalloprotease MTMMPA has been shown herein to inhibit stem cell and progeny migration. Conversely the serine threonine kinase and tumor suppressor SMG-1 has been shown to positively effect cell migration and regeneration time frames. This 'over activity' in SMG RNAi has also been demonstrated to ultimately result in the formation of ectopic growths, analogous with tumor masses seen in cancer. The characterisation of the Methyl-CpG-binding domain protein MBD 2/3 has been expanded upon to include a migration effect. MBD 2/3 RNAi animals exposed to shielded irradiation fail to regenerate as previously published by Jaber 20141 and through the use of Fluorescent Insitu Hybridisation (FISH) visualisation we can confirm that this phenotype is in part attributable to cell migration failure. This body of work also demonstrates the ability of the developed assay to uncover otherwise undetectable phenotypes. Knockdown of the well-known cancer implicated zinc finger protein SNAIL has previously failed to give rise to regeneration defects in planarians. However in the shielded irradiation paradigm SNAIL RNAi does result in a lethal regenerate defect. SNAIL RNAi animals are able to maintain their stem cell and progeny populations, suggesting SNAIL does not have a role in cell maintenance and differentiation. However, investigations using a Fluorescent In situ Hybridisation technique (FISH) show that the cells of SNAIL RNAi animals fail to migrate, supporting the broadly proposed role for SNAIL in the promotion of cell migration. Four additional genes selected using the Oncomine web-based microarray database have been identified as having a role in planarian biological processes. Knockdown of gene UDP-Nacteylglucosamine pyrophosphorylase 1 (UAP1) caused homeostatic animals to regress their heads. Further investigations using FISH to visualise underlying cell behaviours is required, however head regression is associated with stem cell defect in the planarian model2. Knockdown of ribosomal biogenesis protein (WDR12), thyroid hormone receptor interactor 13 (TRIP13) and Bystin (BYSL) resulted in regeneration defects. To the best of our knowledge these genes have never before been investigated in planarians. The phenotypes, all characterised by a failure of animals to regrow a head, were only observable in shielded irradiation experiments. Detailed characterisation of the underlying cellular and molecular mechanisms of these defects is required. However the observations presented herein are adequate to propose that the assay developed has significant potential as a novel technique for the planarian community to investigate important cell behaviours, particularly cell migration, which has a key role in disease, specifically cancer metastasis
Towards understanding the regulation of pluripotency of planarian stem cells
No full textThe planarian Schmidtea mediterranea serves as a model organism for studying regeneration due to its robust stem cell system, driven by neoblasts. While significant progress has been made in understanding neoblast morphology, injury responses, and heterogeneity, the regulatory mechanisms underlying neoblast pluripotency remain poorly understood. In the current study, we investigated neoblast pluripotency through both molecular ("micro") and cellular ("macro") perspectives.
At the molecular level, we examined the role of mbd2/3, a gene encoding a NuRD complex component essential for stem cell pluripotency. RNA interference of mbd2/3 revealed a phenotype of defective differentiation, despite intact proliferation. Single-cell RNA sequencing analysis highlighted specific lineages impacted by mbd2/3 loss. Further, transcriptomic analysis identified zfp-9 as a key regulatory factor upregulated following mbd2/3 RNAi. Functional assays demonstrated that co-inhibition of mbd2/3 and zfp-9 restored differentiation capacity. Our findings suggest that the MBD2/3-NuRD complex maintains pluripotency by suppressing zfp-9, which represses fate-specific transcription factors critical for differentiation.
At the cellular level, we refined the neoblast pluripotency model by proposing a previously overlooked "Pre-G1" phase during the transition from mitosis to G1. This phase, characterized by transcriptional quiescence and lack of fate specification, facilitates the reset of lineage information and the restoration of pluripotency. Notably, cells enriched in this phase were observed following sub-lethal irradiation, suggesting a role in radiation-induced neoblast recovery. This discovery further demonstrates that the pluripotency of neoblasts is integrated within the cell cycle rather than being restricted to a specific sub-population.
Together, our findings advance the understanding of neoblast pluripotency, linking molecular regulators to cellular transitions, and offering a revised model for pluripotency regulation in planarian stem cell
An epigenetic analysis of planarian stem cells
Full text linkPlanarian flatworms possess somatic pluripotent stem cells, called neoblasts (NBs), which are able to differentiate into all cell types that constitute the adult body plan. Consequently, planarians possess remarkable regenerative capacities, and have become an invertebrate model system to study stem cell responses during regeneration. Transcriptomic studies have revealed the genes needed to both maintain NB pluripotency and ensure correct lineage specification during differentiation. However, these studies have not elucidated how this regulation of expression is controlled at the epigenetic level, and in particular by diverse histone modifications. In this thesis, we present a case for elevating planarians as a model system for studying the epigenetic regulation of stem cell pluripotency and differentiation.
Firstly, we describe an expression-based annotation of the asexual Schmidtea mediterranea genome. For each annotated locus, we allocate proportional values for a geneâs expression in either S/G2/M NBs (X1), G1 NBs + post-mitotic progeny (X2), or differentiated cells (Xins) â the three broadly-defined cellular compartments that can be isolated from planarians using Fluorescence Activated Cell Sorting (FACS). The production of a well-annotated genome incorporating transcriptomic information serves as the basis for correlating the presence of particular histone modifications with underlying gene expression. We have developed an optimized ChIP-seq protocol for planarian NBs and show that the active histone marks H3K4me3 and H3K36me3 and suppressive H3K4me1 and H3K27me3 marks correlate with the transcriptional output of genes. We also show that genes with little transcriptional activity in NBs, but which switch on in post-mitotic progeny during differentiation are bivalent, being marked by both H3K4me3 and H3K27me3 at the promoter-proximal region. Bivalent histone modifications in mammalian embryonic and germline stem cells enable transcriptional poising of genes, and consistent with this we find that bivalent genes in planarian NBs are marked by paused RNA Pol II at the promoter-proximal region.
In addition to histone modifications, enhancers and their associated transcription factors can also directly influence gene expression. Consequently, we elucidate the potential TF repertoire of planarians, and identify those enriched in NBs. We also present preliminary evidence to suggest that ATAC-seq on planarian cells can identify both transcriptionally permissive gene promoters, at least in differentiated cells, as well as putative enhancers that correlate with expression of neighbouring genes. In the future, we hope to be able to build gene regulatory networks by identifying TF-binding sites in open chromatin regions and elucidating enhancer targets in a range of planarian cell types. </p
Development of a model system for studying crustacean immunity
No full textOur current understanding of crustacean immunity lacks sufficient scope because it mainly comes from research on insects and decapods. Little is known specifically about the immunity of amphipods, which constitutes a large proportion of crustacean species. The goal of my thesis is to develop a model system for studying crustacean immunity using the amphipod Parhyale hawaiensis, which is an emerging animal model in many research areas. P. hawaiensis was challenged with Vibrio bacteria to elicit an immune response. P. hawaiensis was resistant to Vibrio bacteria under normal conditions but injury significantly increased mortality rate due to increased bacteria entry. Whole-body transcriptomic response after 24h bacterial challenge yielded hundreds of differentially expressed genes. Additional in vivo bacteria, qPCR, and RNA-seq measurements across the infection time course provided a comprehensive overview the immune system after bacterial challenge. The expression pattern of Relish closely matched that of in vivo bacteria. Key effector genes (i.e., TEP, ALFs, and crustin) upregulation was critical in determining survival. At 4h, upregulation involved pathogen recognition receptors and Toll/IMD pathway components. At 8h, immune regulation and effector activities peaked with the aid of JAK-STAT pathway components. At 24h, peak bacterial load pushed the immune system to the limit. Immune system returned to near normal at 168h. Based on in vivo bacterial measurements and stronger transcriptomic response, females seemed to exhibit higher immunocompetence. Only some immune related genes (e.g., IMD and JAK-STAT pathway components) were differentially expressed in both sexes. Toll pathway components were only differentially expressed in females. Hemocytes imaged using imaging flow cytometry were divided into five subpopulations based on size and granularity. The size of larger hemocytes increased by 9.3% after bacterial challenge and these cells may be involved in antioxidant defense and phagocytosis bacterial clearance based on orthologs. The hemocytic transcriptome was very distinct from whole animal and hemocytic response to bacteria was much stronger with 2077 upregulated and 1599 downregulated genes. Key immune genes (e.g., scavenger B, GNBPs, and Tollip) with hemocytic pathogen recognition functions in other taxa were identified. Findings from my thesis better our understanding of the understudied amphipod immunity and provide additional insight into the immune system of the phylogenetically close decapods
Radiation response and tolerance in planarian stem cells
Full text linkIonizing radiation threatens cellular survival, a property strategically exploited in radiotherapy to eradicate cancer cells. However, the efficacy of radiotherapy is impeded by two co-existing challenges: the presence of radiotolerant cancer stem cells capable of withstanding treatment and subsequently driving cancer recurrence; coupled with collateral damage to normal stem cells, which are typically radiosensitive. Despite the pressing demand for radiotherapy, the underlying mechanisms governing radiosensitivity in normal stem cells and radiotolerance in cancer stem cells remain enigmatic, particularly within an in vivo setting. Here, we leverage the planarian Schmidtea mediterranea, which harbours abundant populations of experimentally tractable adult stem cells, as an in vivo model system to investigate stem cell radiation response and recovery. Employing a functional genomics approach, we aim to assess the transcriptional response of planarian stem cells to gamma radiation at both bulk and single-cell levels, as well as to uncover novel genes crucial for planarian radiotolerance.
We generated SPLiT-Seq libraries from irradiated planarians to explore their response to acute gamma radiation at the single-cell level. Our single-cell atlas delineates distinct stem cell clusters exhibiting varying sensitivity to gamma radiation. RNA velocity analyses further reveal dose and time-dependent changes in cell fate trajectories following irradiation. By integrating marker patterns and cell fate trajectories, we proposed a model linking radiosensitivity to cell cycle phase, with stem cells in S/G2/M phases exhibiting heightened sensitivity to gamma radiation.
Guided by insights from our single-cell and previous bulk RNA-sequencing data, we conducted an RNAi screen to identify novel regulators of planarian radiotolerance. We silenced the expression of 105 candidate genes and exposed animals to a sub-lethal dose of gamma radiation. Our screen unveiled six genes, namely Slmap, Kin-17, Dkc, Rab32, Rasl-12, and DMXL-1, as essential regulators of planarian radiotolerance in vivo. An additional 20 single-gene knockdowns were observed to significantly delay the post-irradiation stem cell recovery process, although these knockdowns alone were insufficient to cause animal mortality. Multiple members of the FHL gene family were amongst the single knockdowns causing this delay phenotype. To account for potential redundancies, we knocked down the FHL family members in all possible combinatorial pairs, revealing distinct RNAi pairs that rendered planarians sensitive to gamma radiation.
We then examined the in vivo functions of Slmap, Kin-17, Dkc, Rab32, Rasl-12, and DMXL-1 in the regulation of planarian radiotolerance. We determined that following sub-lethal irradiation, the residual surviving stem cells in Slmap, Kin-17, and Dkc RNAi-treated animals were proliferation incompetent. In contrast, RNAi of Rab32, Rasl-12, and DMXL-1 led to an abnormally prolonged state of cell-cycle arrest in the remaining stem cells. Whether it was complete abolition of stem cell recovery in the former set of gene knockdowns or partial recovery in the latter, the knockdown-induced impairments prevented animal survival following exposure to sub-lethal irradiation. Among these six genes, we identified Dkc as a key player for DNA strand break repair in planarians.
In the wider framework, the genes demonstrated in this study to be vital for planarian radiotolerance could be tested in other model organisms, potentially uncovering novel and conserved mechanisms employed by adult stem cells to counteract the deleterious effects of ionizing radiation. The potential conservation of these mechanisms in mammals may also hold biomedical relevance, as targeting these genes could be explored for imparting radioprotection to normal stem cells, or to sensitize cancer stem cells to radiotherapy
Investigating the regulation of early development of the amphipod crustacean Parhyale hawaiensis
Full text linkThe amphipod crustacean Parhyale hawaiensis is an ideal model organism for studying development, evolution, and regeneration. Its ease of rearing in the lab, accessibility of embryos at all developmental stages, large broods produced year-round, and the variety of functional experiments that can be conducted on animals and embryos make it a valuable resource for investigating many biological questions. As an out group to insects, Parhyale offers a platform for studying biological diversity through comparative studies. Recent research has identified a full repertoire of single-copy genes encoding the machinery associated with DNA methylation in the Parhyale genome. This discovery provides an opportunity to explore the role of DNA methylation in early embryogenesis using an invertebrate model system. In this thesis, we present Parhyale hawaiensis as a tractable model to study DNA methylation dynamics during embryonic development. Our aim was to investigate the regulation during early embryo development, describe the maternal-to-zygotic transition (MZT) and zygotic-genome-activation (ZGA) in Parhyale, and explore the potential function of DNA methylation during embryogenesis while examining its regulatory role in gene expression.
Parhyale possesses a large genome size of approximately 3.6 Gb, which was initially sequenced by Kao et al. in 2016. An update using Dovetail technology enhanced the assembly by generating large scaffolds but left significant gaps. Therefore, our objective was to further improve the existing assembly by integrating PacBio data to close the gaps and correct assembly errors. This effort proved successful, as over 70% of the gaps in the assembly were closed, suggesting further coverage would give further improvement. Subsequently, we performed an expression-driven annotation using a wide range of RNA-seq data from various embryonic stages and different adult conditions. The well-annotated genome served as the foundation for analyzing transcriptomic data from early embryonic stages to detect de novo zygotic transcription using intronic RNA signals.
We described the transcriptome of early Parhyale embryos and proposed a model for the maternal-tozygotic-transition (MZT) and zygotic-genome-activation (ZGA) timelines. Our findings demonstrated that zygotic transcription begins as early as 11 hours post-fertilization and occurs in two waves. The minor wave of ZGA in Parhyale commences at the 32-cell stage, while the major wave takes place at the start of the blastodisc formation stage. We discovered that the earliest transcribed genes in Parhyale are typically short, intron-less or intron-poor, and newly evolved. Furthermore, we validated the presence of DNA methylation mediator genes, with a focus on DNMT1, DNMT3, and MBD2/3. We analyzed the expression pattern of DNA methylation machinery genes during the early stages of Parhyale embryogenesis and found that they are provided maternally. To explore the relationship between DNA methylation and gene expression, we correlated our gene expression datasets with methylseq datasets. Our results revealed a positive correlation between gene-body methylation and gene expression levels.
Lastly, we conducted functional experiments targeting DNMT1, DNMT3, and MBD2/3 to understand their roles during embryonic development. We utilized CRISPR/Cas9 to generate knockout animals for each of the three genes, revealing that the loss of any of these genes is lethal to embryos. Additionally, we performed RNA interference (RNAi) knockdown on MBD2/3, which also resulted in an early embryonic lethality, confirming its essential role in embryogenesis. Profiling the transcriptome of knockdown embryos revealed that the knockdown of MBD2/3 altered the expression of many genes, including developmental transcription factors with low levels of gene-body methylation.
The work presented in this thesis offers a comprehensive understanding of the early embryonic development of Parhyale and emphasizes the crucial role of DNA methylation during embryogenesis. In future, we aim to investigate whether MBD2/3 regulates gene expression through its association with the NuRD complex, and if this occurs in a DNA methylation-dependent, independent, or both manners. Furthermore, the improved assembly and annotation presented in this thesis will greatly facilitate more precise analyses, enabling us to address intriguing questions regarding the promising model organism Parhyale hawaiensis
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