Decision letter: Modular, cascade-like transcriptional program of regeneration in Stentor (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor’s transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures. Editor's evaluation This ground-breaking study builds on recent genome annotation to report the gene expression pattern that drives morphogenesis of Stentor, a large and beautifully organized single-celled organism with a remarkable ability to regenerate after damage. The study has been greatly strengthened by the addition of two molecular perturbation experiments that provide important insights into the regeneration process. https://doi.org/10.7554/eLife.80778.sa0 Decision letter eLife's review process Introduction While much is known about the molecular composition of cells, the mechanism by which those components are arranged into complex patterns and structures is far less understood (Kirschner et al., 2000; Shulman and St Johnston, 1999; Harold, 2005; Marshall, 2020). How does a cell create and maintain pattern? Historically, study of regeneration has played a key role in revealing mechanisms of animal development, because regeneration allows specific developmental processes to be induced experimentally (Morgan, 1901a). Understanding how cells are able to rebuild cellular components and re-establish global patterning holds the promise of shedding new light on the largely unanswered fundamental question of how cells perform morphogenesis and pattern formation. A second reason to study regeneration in single cells is to identify the mechanisms by which cells repair and recover from wounds. Regeneration and wound healing are processes that are typically studied at the tissue level in multi-cellular organisms, but individual cells also must be able to repair wounds following mechanical disruption, and a cellular scale response to injury is a crucial feature of repair even in multi-cellular organisms. Injured cells must be able to not only patch over the site of injury to prevent leakage of cytoplasm, they also need to re-establish polarity, rebuild organelles, and reorganize the cytoskeleton (Tang and Marshall, 2017). Deficiencies in cell repair have become implicated in disease, for example in diseases of the heart, lung, and nervous system (Oeckler and Hubmayr, 2008; Wang et al., 2010; Angelo and Mao, 2015), including ARDS (acute respiratory distress syndrome) (Cong et al., 2017). Yet, there is still much to be learned about how an individual cell responds to a wound. Ciliates provide an excellent opportunity for investigating the mechanisms of cellular patterning and regeneration due to the fact that they have highly stereotyped cell shapes, with easily visible surface structures that serve as landmarks to assess the progress of regeneration (Aufderheide et al., 1980; Frankel, 1989). The giant ciliate Stentor coeruleus (Tartar, 1961; Marshall, 2021) is a single cell that can fully regenerate its complex subcellular structure after injury. In this classical system, virtually any portion of the cell, when excised, will give rise to a normally proportioned cell with intact subcellular organization (Morgan, 1901b; Tartar, 1961). Stentor provides a unique opportunity to study regeneration and patterning at the cell scale. Its large size (up to 1–2 mm in length), clear anterior/posterior axis, detailed cortical patterning, and remarkable ability to heal even large wounds in the cell membrane make it especially amenable to surgical manipulation and imaging approaches. But despite roughly a century of analysis by microscopy and microsurgery, the mechanism by which Stentor forms and regenerates its complex body pattern remains a mystery, because a lack of genetic and genomic tools in the organism prevented detailed molecular analysis of its processes. Now that the S. coeruleus genome has been determined (Slabodnick et al., 2017) and RNA interference methods developed (Slabodnick et al., 2014), molecular and genomic approaches enable new ways to explore this classic model system. In Stentor, principles of single-cell wound healing and regeneration can be studied without confounding effects of surrounding cells that may non-autonomously influence an intracellular injury response in the context of tissues. Transcriptome studies of various multi-cellular animal species, including zebrafish and planaria, all of which are capable of regeneration throughout life, have begun to reveal key regulators of multi-cellular regeneration. Many of these studies have delineated the molecular players in regeneration by identifying genes that are expressed when stem cells differentiate into various cell types required to rebuild lost tissue or organs. This transcriptomic approach has thus proven its utility in revealing cell-specific requirements for regeneration in the context of tissues. We have sought to take a similar approach to the problem of single-cell regeneration in Stentor, focusing on two regenerative processes: regeneration of the oral apparatus (OA) after its removal by sucrose shock, and regeneration of cells that have been bisected into two half-cells. One of the most dramatic and tractable regeneration paradigms in Stentor is the regeneration of the OA. The OA is a prominent structure on the anterior side of the cell that contains thousands of basal bodies and cilia (Paulin and Bussey, 1971) organized into a ciliated ring known as a membranellar band (MB). At one end of the ring is an invagination of the plasma membrane, where food particles are ingested. This invagination together with its associated cytoskeletal structures is known as the mouth. The OA can be induced to shed using sucrose shock (Tartar, 1957) after which a new OA regenerates over the course of 8 hr, progressing through a series of well-characterized morphological stages (Figure 1A; Tartar, 1961). Removal of the macronucleus, at any stage, causes OA regeneration to halt at the next stage, suggesting that several waves of gene expression may be required to drive different processes at different stages (Tartar, 1961). In addition to the macronucleus, each Stentor cell contains multiple micronuclei, which are generally not visible because they are closely adjacent to the macronucleus. The micronuclei are required for sexual reproduction but are dispensable for regeneration and for mitotic cell division (Schwartz, 1935; Tartar, 1961). Chemical inhibitor studies showed that regeneration of the OA requires transcription (Whitson, 1965; James, 1967; Burchill, 1968; Younger et al., 1972) and it is also known that overall levels of RNA synthesis increase several fold during the OA regeneration process (Ellwood and Cowden, 1966; Burchill, 1968; Younger et al., 1972). Figure 1 Download asset Open asset Transcriptional profiling of Stentor regeneration. (A) Morphological events in Stentor regeneration. At t=0,the membranellar band is shed during sucrose shock. The body cilia remain on the cell. After sucrose shock the frontal field protrudes, resulting in the anterior end of Stentor becoming rounded rather than cone-shaped. One hour after the start of regeneration, basal bodies begin to form at the locus of stripe contrast. After 3 hr, the first cilia of the new membranellar band are visible. These cilia show uncoordinated beating. After 5 hr, the new membranellar band elongates and extends along the anterior-posterior axis. A site for the new mouthparts is cleared at the posterior end of the membranellar band. During this stage, the cilia become oriented with respect to each other and their beating begins to become coordinated, forming multiple short metachronal waves. The nodes of the macronucleus begin to condense. By 6 hr, the mouthparts are completely formed and the macronucleus is fully condensed. At 7 hr the membranellar band and mouth migrate to the anterior end of the cell. The macronucleus extends into a sausage-like shape. By 8 hr after sucrose shock the Stentor is fully regenerated. The membranellar band completely wraps around the anterior of the cell, all of the oral cilia coordinate to form a single metachronal wave, the macronucleus is re-nodulated, and the cell resumes normal feeding activity. (B) Surgical bisection of Stentor. When a Stentor cell is cut in half perpendicular to the long axis, two cell fragments are produced, an anterior half-cell and a posterior half-cell. Immediately after cutting, both half-cells heal their wounded edges. The anterior half-cell then regenerates a new posterior body including the hold-fast, and the posterior half-cell regenerates a new anterior body including the oral apparatus (OA). Oral regeneration in the posterior half-cell has the same general morphological events and timing as oral regeneration in the sucrose-shocked cells. Both fragments are able to regenerate because the elongated macronucleus that is divided into both halves during surgery is highly polyploid, ensuring that each half-cell retains many copies of the genome. (C) Comparative transcriptional profiling. We performed RNA sequencing (RNAseq) on sucrose-shocked cells regenerating as in panel A, as well as on both the anterior and posterior half-cells regenerating after bisection as in panel B. These three datasets are represented by the three circles of the Venn diagram, with blue representing genes expressed during regeneration in bottom half-cells after bisection, green representing genes expressed during regeneration in top half-cells after bisection, and coral representing genes expressed during regeneration in cells following sucrose shock. Genes were grouped into modules according to correlated expression patterns shared between two or more fragments. For example, the ‘general regeneration’ module was defined based on genes showing differential expression in all three cases of regeneration (OA, anterior half, and posterior half). The second regeneration paradigm we consider is bisection (Figure 1B). When a Stentor cell is cut in half along its longitudinal axis, the posterior half regenerates a new OA, while the anterior half regenerates a new tail (Morgan, 1901b). Thus, the list of genes expressed during regeneration in posterior halves of bisected cells presumably includes genes involved in building a new OA, but is also expected to include genes involved in building other anterior structures such as the contractile vacuole, while the list of genes expressed in the anterior half would include genes involved in rebuilding lost posterior structures. Both halves are expected to express genes involved in recovering the normal metabolic state of the cell following closure of the large wound created in the plasma membrane during surgical bisection. Given that transcription is required for regeneration, there is likely to be a set of genes whose products drive the regenerative process. By learning the identity of these genes, we can determine the molecular pathways and building blocks involved in building new cellular structures. Proteomic analysis (Lin et al., 2022) has shown that the OA, anterior half-cell, and posterior half-cell, all have distinct protein compositions, hence we expect their regeneration to involve expression of different sets of genes. Transcriptional analysis provides a complementary method to proteomics for identifying new components of distinct cellular structures in Stentor while also allowing identification of genes whose products are needed to build the structure but that do not encode components of the final structure, and so would be missed by proteomics. This type of approach has previously been used successfully to identify genes involved in ciliogenesis, by identifying genes expressed in cells as they regenerate flagella (Schloss et al., 1984; Stolc et al., 2005; Albee et al., 2013). In addition to producing lists of candidate genes involved in assembly, the timing with which different genes are expressed will potentially reveal sequential steps in the assembly process itself. Furthermore, by revealing the timing of gene expression during regeneration, transcriptional analysis can provide clues about how the process is initiated. OA regeneration can be triggered by several different procedures, including removal of the OA but also rotation of the OA relative to the rest of the cortical pattern (Tartar, 1961) raising the question of how the cell recognizes these geometric perturbations. One approach is to determine which genes are expressed earliest in the pathway, and then move upstream to identify signals required to turn these early-expressed genes on. In terms of the timing and sequential logic of the expression program itself, we note that even if the ultimate goal is to understand how Stentor achieves complex spatial patterning, pattern formation is an inherently spatiotemporal process, such that both spatial and temporal control are important. It has been possible to use RNAseq to show that a portion of the genome becomes expressed during regeneration in Stentor (Sood et al., 2017; Onsbring et al., 2018; Wei et al., 2020). These studies focused on two different regeneration paradigms. In Sood et al., 2017, and Wei et al., 2020, genes were identified that were upregulated during OA regeneration in S. coeruleus, induced using sucrose shock or urea shock, respectively. The list of genes identified was presumably a mixture of those involved in building the new OA and genes involved in recovery from the stress of sucrose or urea shock that was used to remove the OA. In Onsbring et al., 2018, regeneration of bisected Stentor polymorphus cells were analyzed. Each of these three prior studies focused on a different regeneration paradigm in different species. However, we hypothesized that by comparing the transcriptional programs of regeneration in sucrose-shocked versus bisected cells, all within the same species, it would become possible to distinguish shared modules of gene expression, common to all regenerative processes, from structure-specific regeneration modules. Here, we report a comparative transcriptomic analysis in which RNAseq was used to analyze gene expression during OA regeneration following sucrose shock as well as regeneration in posterior and anterior cell fragments following surgical bisection. By comparing gene sets expressed in these different situations, we can identify genes specific to regeneration of the OA and the posterior tail, as well as additional sets of genes involved in the recovery from surgical wounding. The timing with which specific groups of genes are expressed correlates with the formation of specific cellular structures. These results suggest a modular organization of the regeneration program. By focusing on earlier stages of regeneration, we identified conserved transcriptional regulators as well as RNA-binding proteins that are differentially expressed during regeneration. Inhibition of protein translation allows the transcriptional program to be divided into an early set of genes whose expression does not require protein synthesis, and a later set of genes whose expression is apparently dependent on production of proteins encoded by the early genes, suggesting a cascade-like logic. This work opens a new window into the molecular details underlying the century-old question of regeneration in this extraordinary single-celled organism. Results Identifying gene expression modules by comparative transcriptomics S. coeruleus cells were subjected to sucrose shock to remove the OA (Figure 1A), or else surgically bisected to produce anterior and posterior half-cells (Figure 1B). These experiments produced three regenerating samples: intact cells from which the OA had been removed, which then regenerated the OA; posterior half-cells which regenerated a new anterior half, including the OA and other anterior structures; and anterior half-cells which retained the pre-existing OA and regenerated a new posterior half including contractile tail and holdfast. For each sample, cells were collected at regular intervals and analyzed by RNAseq (see Materials and methods). The process of building a new OA takes ~8 hr as detailed in Figure 1. RNA samples from ~20 cells were collected prior to sucrose shock, at 30 min and at 1, 1.5, 2, 3, 4, 5, 6, and 7 hr after sucrose shock. The number of time points was based on the time required to complete regeneration. At each stage, RNA was extracted, and RNAseq libraries were sequenced (see Materials and methods). We combined reads from all samples and replicates and used these to assemble a genome-guided de novo transcriptome (Grabherr et al., 2011) using TRINITY. To identify genes with dynamic expression patterns during regeneration, we first mapped reads from each sample to this transcriptome and then identified differentially expressed genes over the time course in each sample. To identify genes that were differentially expressed we compared two models – a generalized additive model where changes in expression over time are modeled by natural splines, and one in which there is no dependence on time. Overall, we identified 4323 genes that exhibited dynamic expression patterns through regeneration in sucrose-shocked cells, 1020 in posterior half-cells following bisection, and 229 in anterior half-cells following bisection. As indicated in Figure 1C, these three samples showed partially overlapping expression patterns, but each also expressed its own unique set of genes. The anterior half-cells showed by far the smallest number of differentially expressed genes, while the sucrose-shocked cells showed the largest. In total we detected 4811 differentially expressed genes, which constitutes roughly 10% of the Stentor genome. Based on the Venn diagram in Figure 1C, we defined six sets of differentially expressed genes: genes expressed in both sucrose shock and regenerating posterior halves, which we take to indicate genes required for OA regeneration; sucrose shock-specific, which we interpret as reflecting aspects of OA regeneration in sucrose-shocked but not bisected cells, possibly including osmotic response to the sucrose shock itself; genes expressed only in regenerating posterior halves, which we interpret as relating to regeneration of anterior structures other than the OA; genes expressed only in regenerating anterior halves, which we interpret as being involved in regenerating the tail of the cell; genes expressed in both regenerating half-cells but not sucrose shock, which we term bisection-specific; and, finally, genes expressed in all three samples, which we take to indicate general regeneration genes. Each of these sets of genes were clustered using clara (Kaufman and Rousseeuw, 1990). Differentially expressed genes specific for OA regeneration OA regeneration can be stimulated in a number of ways. If Stentor cells are treated with sucrose, the OA detaches via an autotomy process (Tartar, 1957). If Stentor cells are cut in half, the posterior half will regenerate a new OA. Analyzing genes expressed in either situation alone will reveal OA regeneration-specific genes but also genes that may be induced by the stresses of sucrose shocking or bisection, respectively. Thus, in order to obtain a list of highest confidence OA-specific genes, we compared sucrose-shocked cells and regenerating posterior half-cells in order to identify a set of genes that are differentially expressed in both samples and thus likely to be specific for OA regeneration. These genes fall into five clusters (Figure 2A). Cluster 1 consists of genes whose expression starts out high at t=0 (prior to removal of the OA) and decreases during the course of regeneration, possibly suggesting that these genes encode proteins whose function is dispensable for, or possibly even inhibitory of, the regeneration process. The other four clusters correspond to upregulated genes whose peak of expression takes place at successively later times during regeneration. The identity of OA-specific genes corresponding to gene models in the Stentor genome are in 1. of genes in the OA-specific module are in Figure includes genes proteins, kinases, and RNA-binding Figure Download asset Open asset Oral apparatus (OA) regeneration program. (A) OA-specific gene expression determined using combined sucrose shock and bisection Genes are clustered into five groups using indicated by the on the sucrose shock or bisection is on the 1 contains all genes whose expression decreases during regeneration compared to The peak expression of each of genes with developmental identified in Figure is and with of representing expression than the and blue than the (B) of gene types in each for a set of gene (C) of genes expressed in sucrose-shocked cells that are not in the OA-specific set of panel of gene types in each for sucrose genes. time course of a set of genes, showing their coordinate expression at a time in regeneration when bodies are forming at the start of OA time course of a set of highly conserved genes proteins known to be involved in structure and with panel that gene expression later than gene of gene expressed in regenerating posterior half-cells that are regenerating new anterior showing genes specific to the regenerating posterior and not in the OA-specific set of panel of gene types in each for regenerating posterior half-cells. showing of all centriole and cilia genes showing in the two paradigms shock and posterior that most upregulated genes are specific to sucrose shock. Among the genes in the OA-specific set many were that are to centriole in with the fact that a large number of new basal bodies form during OA regeneration in order to as basal bodies for the cilia of the of the OA. 1 lists the key genes to be upregulated in the OA-specific It is that most of these are expressed in clusters and 3, of genes whose expression at hr after sucrose shock, during the of time at which the first of an oral becomes visible in microscopy (Paulin and Bussey, and at which microscopy that thousands of new basal bodies are being formed de a and These basal bodies will into and become the basal bodies that the of the OA. Among the upregulated genes are is as one of the earliest known involved in the structure of the centriole et al., of the most conserved centriole genes, we that are expressed in either or This expression of the centriole gene set at the when basal bodies are forming thus provides a of One is expressed later than the in of this expression of a gene will be A of genes involved in assembly are expressed in later including several proteins, but no genes components of the were differentially expressed in any 1 of body genes in the oral apparatus module (Figure 2A). Cluster of peak genes In to the OA-specific which was defined by for genes shared in common between the expression program during OA regeneration in bisected cells and sucrose-shocked cells, the sucrose module groups genes differentially expressed only in sucrose-shocked cells. Figure genes showing differential expression in sucrose-shocked cells but not in regenerating posterior or anterior half-cells. These genes may be involved in stress response to the sucrose shock, or the of the OA after the shock. may also additional OA biogenesis that to be expressed during sucrose shock to the OA-specific module (Figure we five clusters of gene expression the sucrose genes (Figure The timing of these clusters that in the OA-specific The identity of sucrose genes corresponding to gene models in the Stentor genome are in Genes upregulated during sucrose shock response of the centriole genes. As with the OA-specific gene these genes were in of the sucrose shock for example and In many the sucrose shock module includes of these genes that were not expressed in the OA-specific with the of between bisection and sucrose OA formation. Cluster 3 contains genes relating to assembly such as the the the expressed of the key centriole biogenesis protein is not expressed highly in regeneration The expression timing of a set of genes is in Figure In to the lack of genes in the OA-specific gene set we that clusters 3 and of the sucrose gene set a large number of genes that encode proteins components of of genes components of the and which are clusters 2, 3, and In addition to the two other are required to coordinate and and the complex et al., 2017). Genes the complex are expressed during 3 1, 2, 3, 4, 5, and of genes components begins in 3 and is most in 4, where proteins are expressed 3, 4, and The with the and several proteins are upregulated during clusters The of cilia contains proteins that are at the between the A and These proteins are expressed in clusters 3 and We thus that clusters 3 and 4, in expression