Foxm1 regulates neural progenitor fate during spinal cord regeneration
2021; Springer Nature; Volume: 22; Issue: 9 Linguagem: Inglês
10.15252/embr.202050932
ISSN1469-3178
AutoresDiane Pelzer, Lauren S. Phipps, Raphaël Thuret, Carlos J. Gallardo-Dodd, Syed Murtuza Baker, Karel Dorey,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoArticle24 August 2021Open Access Transparent process Foxm1 regulates neural progenitor fate during spinal cord regeneration Diane Pelzer Diane Pelzer orcid.org/0000-0001-6906-2451 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UKThese authors contributed equally to this work Search for more papers by this author Lauren S Phipps Lauren S Phipps orcid.org/0000-0001-9324-5469 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UKThese authors contributed equally to this work Search for more papers by this author Raphael Thuret Raphael Thuret Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Carlos J Gallardo-Dodd Carlos J Gallardo-Dodd orcid.org/0000-0002-6086-0550 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Syed Murtuza Baker Syed Murtuza Baker orcid.org/0000-0002-6633-333X Division of Informatics, Imaging & Data Sciences, Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Karel Dorey Corresponding Author Karel Dorey [email protected] orcid.org/0000-0003-0846-5286 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Diane Pelzer Diane Pelzer orcid.org/0000-0001-6906-2451 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UKThese authors contributed equally to this work Search for more papers by this author Lauren S Phipps Lauren S Phipps orcid.org/0000-0001-9324-5469 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UKThese authors contributed equally to this work Search for more papers by this author Raphael Thuret Raphael Thuret Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Carlos J Gallardo-Dodd Carlos J Gallardo-Dodd orcid.org/0000-0002-6086-0550 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Syed Murtuza Baker Syed Murtuza Baker orcid.org/0000-0002-6633-333X Division of Informatics, Imaging & Data Sciences, Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Karel Dorey Corresponding Author Karel Dorey [email protected] orcid.org/0000-0003-0846-5286 Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK Search for more papers by this author Author Information Diane Pelzer1,†, Lauren S Phipps1, Raphael Thuret1, Carlos J Gallardo-Dodd1,†, Syed Murtuza Baker2 and Karel Dorey *,1 1Division of Developmental Biology and Medicine, Faculty of Biology, Medicine and Health, School of Medical Sciences, University of Manchester, Manchester, UK 2Division of Informatics, Imaging & Data Sciences, Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, UK †Present address: Institut Curie, Sorbonne Université, CNRS UMR3215, INSERM U934, PSL Research University, Paris, France †Present address: Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, Sweden *Corresponding author. Tel: +44 161 275 5319; E-mail: [email protected] EMBO Reports (2021)22:e50932https://doi.org/10.15252/embr.202050932 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue-specific response to injury remains poorly understood. Using a combination of bulk and single-cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1−/− tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord-specific response to injury and reveal a new role for neuronal differentiation during regeneration. Synopsis Differentiation of neural progenitor cells (NPCs) is a hallmark of successful spinal cord regeneration. This study shows that Foxm1 controls the switch from proliferation to differentiation of NPCs during spinal cord regeneration in Xenopus tropicalis. A regeneration-specific population of cells, characterized by the expression of foxm1, was identified during spinal cord regeneration using single cell RNA sequencing. Foxm1−/− tadpoles do not fully regenerate their spinal cord and tail after amputation. Foxm1 promotes neuronal differentiation during regeneration, without affecting proliferation or the overall length of the cell cycle in NPCs. Introduction Mammals have limited tissue regeneration capabilities, particularly in the case of the central nervous system. Spinal cord injuries (SCIs) are often irreversible and lead to the loss of motor and sensory function below the site of the damage (McDonald & Sadowsky, 2002). In contrast, amphibians such as Xenopus (X.) tadpoles have far greater regenerative abilities as they can regenerate a fully functional tail following amputation, including their spinal cord (Deuchar, 1975; Love et al, 2011; Kakebeen et al, 2020). The injured spinal cord is sealed within a day by the formation of the neural ampulla, and lineage tracing has revealed that the spinal cord regenerates from its original stump (Gargioli & Slack, 2004; Slack et al, 2008). A hallmark of spinal cord regeneration is the re-activation of Sox2/3+ neural progenitor cells (NPCs) to induce both regrowth of the spinal cord and the generation of new neurons (Muñoz et al, 2015). In axolotl, the spinal cord regrows from extensive proliferation of NPCs located in the "source zone" adjacent to the site of amputation (Mchedlishvili et al, 2007; Rost et al, 2016). This increase in proliferation is tightly regulated as progenitors switch from a neurogenic to a proliferative division. A key factor driving that switch is the planar cell polarity (PCP) pathway which is re-activated in the spinal cord following amputation (Rodrigo Albors et al, 2015). However, how the balance between self-renewal proliferation and differentiation is controlled during regeneration is currently not well understood. During development, the switch from a proliferative to a neurogenic division depends at least in part on changes in the relative length of the different phases of the cell cycle (Cheffer et al, 2013; Hardwick & Philpott, 2014). We therefore took an unbiased approach to identify cell cycle regulators expressed specifically during X. tropicalis spinal cord regeneration by RNA-seq. This led to the identification of Foxm1, a transcription factor known to promote G2/M transition, as a potential key transcription factor for spinal cord regeneration. Foxm1−/− X. tropicalis tadpoles develop normally but their ability to regenerate their spinal cords is impaired. Using single-cell (sc)RNA-seq and immunolabelling experiments, we show that foxm1+ cells in the regenerating spinal cord undergo a transient but dramatic change in the relative proportions of cells in different phases of the cell cycle, suggesting a change in their ability to differentiate. Surprisingly, Foxm1 does not regulate the rate of progenitor proliferation or the length of the cell cycle but is required for neuronal differentiation leading to successful spinal cord regeneration. Results Foxm1 is specifically expressed in the regenerating spinal cord We compared the transcriptome of isolated spinal cords at 1 day post-amputation (1 dpa) and 3 dpa to spinal cords from intact tails (0 dpa, Fig 1A). Principal component plot, dendrogram of sample-to-sample distances and MA plot of the log2 fold change (FC) of expression in relation to the average count confirmed the quality of the data (Fig EV1-EV5). Between 0 dpa and 1 dpa, 2447 differentially expressed (DE) transcripts (|log2(FC)|> 1 and FDR < 0.01) were identified (1,125 down- and 1,322 upregulated). Between 0 dpa and 3 dpa, 5,383 genes are differentially expressed (2,746 down- and 2,637 upregulated, Fig EV1E, Dataset EV1). Figure 1. Analysis of differentially expressed genes during spinal cord regeneration Twenty spinal cords of NF50 tadpoles were isolated at 0, 1 and 3 days post-amputation (dpa) and pooled for RNA sequencing. Genes with a |log2(FC)|> 1 and P-adj< 0.01 were used for hierarchical clustering. For each cluster, the gene list was uploaded on Fidea (http://circe.med.uniroma1.it/fidea/) (D'Andrea et al, 2013). The five most significant enrichment of GO (biological processes) terms are shown, and the −log10(P_value) with Bonferroni correction is shown. The dataset was uploaded on the Ingenuity Pathway Analysis software (Qiagen). Genes with a |log2(FC)|> 1 and P_adj < 0.01 were considered. The software identified upstream regulators based on the changes in expression levels of known downstream targets. Each upstream regulator is attributed a z-score, corresponding to the negative log of the P-value derived from the Fisher's exact test. Changes in the expression of foxm1 and known downstream targets in the whole tail comparing day 0 and day 3 (WT_d0d3) and in the spinal cord comparing day 0 and day 1 (SC_d0d1) and day 0 and day 3 (SC_d0d3). The whole tail dataset was obtained from (Chang et al, 2017). Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole-mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate (r) at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord. Total RNA was isolated from regenerating tails at indicated timepoints post-amputation, reverse-transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean ± SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparison test was used. *P = 0.0149. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Metadata from the RNA-seq experiment of the time course of isolated spinal cord regeneration A. Principal component analysis to assess overall similarities between all samples. The biological replicates of day 0 (0 dpa, green square), day 1 (1 dpa, red circle) and day 3 post-amputation (3 dpa, blue triangle) cluster together whilst showing wide variation in the two dimensions shown on the graph. B. Hierarchical clustering of the nine datasets. C, D. MA plots depicting the log2 fold change against the mean of normalised counts. DE genes (P_adj< 0.05) are coloured in red when comparing day 0 versus day 1 (C) and day 0 versus day 3 (D). E. Total number of differentially up- and downregulated (|Log2(FC)|> 1, P_adj< 0.01) transcripts in 0 dpa versus 1 dpa and 0 dpa versus 3 dpa samples. F. Schematic of the experiment designed to identify the signals upstream of foxm1 expression. After amputation, the tails were left to heal for 36h before inhibitor treatments were started. The tails were collected at 72hpa, and foxm1 expression was determined by RT–qPCR. G–I. Effects of treating tadpoles with 4 µM DPI (a NOX inhibitor, G), 20 µM SU5402 (an FGFR inhibitor, H) and 2.5 µM cyclopamine (a Hedgehog signalling inhibitor, I) on foxm1 expression. DMSO was used as a control for G and H and ethanol for I. Data presentation: The graphs in G–I represent the mean with standard deviation of four independent experiments with at least 15 tails per experiments, ef1α was used to normalise expression, and significance was assessed with an unpaired t-test, *P < 0.05. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Establishment of a foxm1 knockout line A. The CRISPR/Cas9 system was used to generate foxm1 knockdown and knockout animals, and gRNA was designed to target the foxm1 gene. The target region contains the restriction site for NcoI and was used to test efficiency by RFLP. B. Embryos were either uninjected (UI) or coinjected with gRNA and Cas9mRNA, 0.6 ng Cas9 protein or 1.5 ng Cas9 protein. Genomic DNA was extracted and a region amplified around the gRNA target site by PCR. Half of the PCR product was digested with NcoI. By comparing the ratio of the digested product with an intact restriction site (lower band) to the non-digested product containing a mutated restriction site (upper band) after the addition of NcoI (+) gives an indication of the efficiency of the induction of mutations. C. Frogs injected with the CRISPR/Cas9 system and raised to adulthood. The F1 embryos were sequenced for mutations in foxm1. Four frameshift mutations were identified. D. Genotypes used in this study. E. Tadpoles from a foxm1+/− cross were raised to NF50, amputated and the tails collected at 3dpa for RNA expression and the heads for genotyping. Foxm1 expression was analysed by qPCR, using ef1α as a reference (n = 3 with at least 3 embryos per sample). The data are expressed as the mean ± SD. F. A third of the tails of foxm1 knockdown (Crispr mosaic F0) and wt tadpoles at NF50 were amputated and the tadpoles left to regenerate for 9 days. The images show representative tails at 9dpa. G, H. To quantify the rate of regeneration, the ratio of the length of the regenerate to the length that has originally be amputated was compared for the spinal cord (G) and the whole tail (H). The graph represents the mean ± SD of three independent experiments with at least five tadpoles in each experiment. Data presentation: For testing statistical significance, an unpaired t-test was used in E and a two-way ANOVA followed by a Sidak multiple comparison test in G and H. *P < 0.05 and ****P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Characterisation of the Xenopus spinal cord by single-cell RNA sequencing Metadata of the scRNA-seq experiment. t-SNE representation of the dataset from 0 dpa with the different cell types identified using a dynamic tree cut algorithm. Bubble plot representing the proportion of cells (size of the dot) and level of expression (colour of the dot) for the genes used to identify the cell types in (B). Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Characterisation of the foxm1 positive cells UMAP representation of the scRNA-seq dataset before (left panel) and after (right panel) batch correction using Seurat. Unbiased acceptance rate at the indicated subsampling percentile in the raw data (Counts) and after batch correction using Harmony or Seurat algorithm. Unsupervised pseudo-time of the whole scRNA-seq dataset. The distribution of the different clusters along the pseudo-time is indicated with the colours and numbers as described in Fig 3F. Pseudo-time of the whole scRNA-seq dataset with cells from 0 dpa in orange and from 3 dpa in green. Pseudo-time representation showing the cells expressing foxm1 (red dots). Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Effect of impairing foxm1 expression on the organisation of the regenerating spinal cord A. Tails from tadpoles foxm1 knockdown (mosaic Crispr F0, kd) and control (wt) NF50 tadpoles were amputated and left to regrow for 3 days. RNA was isolated from the regenerates and expression levels of sox2 and ntubulin analysed by qPCR using ef1α as a reference. sox2: n = 4, ntubulin: n = 6, with at least 20 tails per sample, B. Effect of DPI treatment on the expression of known transcriptional targets of Foxm. Embryos were treated with DPI as described in Fig EV1F, and the expression of ccnb3 (a Foxm1 target gene), ntub (a marker of differentiated neurons) and ami (a gene expressed in endothelial cells) was analysed by RT–qPCR using ef1α as control. C, D. Rose plot histograms showing the percentage frequency distribution of the angles of DAPI+ nuclei (C) or Sox3+ nuclei (D) in wt (blue) or foxm1−/− spinal cords (red). Angles are distributed into 12 bins from 0 to 180 degrees using a MATLAB script. Dorsal = 0 degrees, lateral = 90 degrees and ventral = 180 degrees. The inner, middle and outer circle corresponds to 5, 10 and 15%, respectively. Ten sections from n = 4 animals were analysed per genotype. Total cell counts were as follows: wt anterior (DAPI+ 2166; Sox3+, 701), foxm1−/− anterior (DAPI+, 2246; Sox3+, 707), wt regenerate (DAPI+, 1927 nuclei; Sox3+, 826) and foxm1−/− regenerate (DAPI+, 2393; Sox3+, 1125). P < 0.0001 for regenerate spinal cords and P > 0.05 for anterior spinal cords as analysed by Kolmogorov–Smirnov tests. E, F. Quantification of the absolute number of cells per section expressing Sox3 (E) and nuclei (DAPI, F) in the regenerate of wild type (wt) and foxm1−/− knockout tadpoles at 5 dpa. The number is derived from the same sections analysed in Fig 5C, and the quantification is derived from the analysis of 8 tadpoles with an average of 15 sections per tail. G. The tails of control and foxm1KD animals, fixed at 5 days post-amputation, were sectioned and labelled with the Sox3 or Myt1 antibody followed by DAPI staining. The ratio of Sox3 and Myt1 per number of DAPI stained nuclei in the spinal cord was quantified and compared between control and foxm1KD tadpoles. Sox3 wt n = 6 with 41 sections, CRISPR/Cas9 n = 5 with 50 sections, Myt1 wt = 8 with 67 sections and foxm1KD n = 7 with 53 sections. Data information: In A and B, the graph represents the mean ± SD of three independent experiments normalised to wt. In E and F, the central line represents the median, the box the 25th/75th percentile and the whiskers the min and max values. In G, the graph shows the mean ± SD. For A, E, F and G, the significance was tested with an unpaired t-test, and for B, a one-way ANOVA with a Tukey post hoc test was used. ns: non-significant, *P < 0.05, ***P < 0.001 and ****P < 0.0001. Download figure Download PowerPoint To identify the most enriched biological processes by gene ontology (GO), a non-biased hierarchical clustering for all DE genes was performed (Fig 1B). We observed three phases: first an increase in expression of genes involved in metabolic processes (cluster I), then a strong upregulation of genes associated with cell cycle regulation (clusters II and III) and finally, a downregulation of expression of genes involved in nervous system development (clusters IV and V, Fig 1B). Using Ingenuity Pathway Analysis (IPA), we identified potential upstream regulators that could explain changes in expression of downstream target genes, with Foxm1 showing the highest significance at 3 dpa (Fig 1C). Using published RNA-seq of tail regeneration in X. tropicalis (Chang et al, 2017), we compared changes in expression of known Foxm1 target genes between 0 and 3 dpa in whole tail (WT_d0d3), 0 and 1 dpa (SC_d0d1) and 0 and 3 dpa (SC_d0d3) in spinal cord. Foxm1 and its transcriptional targets are significantly upregulated only in the spinal cord at 3 dpa, but not in the whole tail (Fig 1D). We wanted to confirm the expression of foxm1 during regeneration by in situ hybridisation (ISH) and RT–qPCR. ISH shows that foxm1 is not expressed in the spinal cord at 0 and 1 dpa but is restricted to the regenerating spinal cord at 3 dpa (Fig 1E). The whole-mount ISH was then sectioned to confirm that foxm1 is expressed specifically in the regenerating spinal cord (Fig 1E). We then performed RT–qPCR for foxm1 over a period of 7 days, and its expression peaks at 3 dpa and decreases back to baseline levels at 7 dpa (Fig 1F). We next wanted to identify the upstream signal(s) regulating its expression. As foxm1 expression starts at 3 dpa, it is not a direct response to the injury. We tested whether signalling pathways required for tail regeneration promote foxm1 expression at 3 dpa. A sustained increase of reactive oxygen species (ROS) in the tail is required for its regeneration (Love et al, 2013). ROS levels were decreased following amputation using DPI, an inhibitor of the NADPH oxidases (NOX). In NF50 tadpoles treated with DPI from 36hpa until 72hpa, foxm1 expression decreases by 69% (P = 0.032) compared to DMSO controls (Fig EV1F and G). ROS are upstream of different signalling pathways, including FGF (Lin & Slack, 2008; Love et al, 2013). Furthermore, Sonic hedgehog (Shh) signalling is also required for tail regeneration (Beck et al, 2003; Hamilton et al, 2021) and induces Foxm1 expression in the developing cerebellar granule neuron precursors (Schüller et al, 2007). However, foxm1 expression is not affected by treating amputated tails treated with an FGF receptor kinase inhibitor (SU5402, Fig EV1H) or a Shh signalling inhibitor (cyclopamine, Fig EV1I). Foxm1 is required for spinal cord regeneration To test the role of Foxm1 during regeneration, we designed a guide RNA (gRNA) targeted at bases 129–152 downstream of the ATG to knock down and knock out foxm1 expression using CRISPR/Cas9 (Fig EV2A). The efficacy of the gRNA was assessed by restriction fragment length polymorphism analysis (RFLP). Co-injection of the gRNA with cas9 mRNA did not lead to the destruction of the NcoI site but co-injection with 0.6 and 1.5 ng of Cas9 protein leads to NcoI-resistant PCR products in a dose-dependent fashion (Fig EV2B). Sequencing of individual clones revealed that indels occur in 50–90% of the foxm1 locus. We have identified four frameshift mutations that were germline transmitted and F0 frogs with these mutations were backcrossed with wild types to establish foxm1+/− lines used to generate foxm1−/− tadpoles (Fig EV2C and D). We then confirmed these mutations lead to a decrease in foxm1 expression by RT–qPCR, with homozygotes mutants displaying an 80% reduction in foxm1 expression compared to wild type (P < 0.0001, Fig EV2E). We then analysed the ability of NF40 tadpoles to regenerate their spinal cord depending on their genotype (Fig 2A and B). About 40% of the tails were removed and the ratio of regeneration was determined between 0 to 3 and 0 to 7 dpa by dividing the length of the regenerate by the length of the amputated tail. No differences were observed for 0 to 3 dpa, but for 0 to 7 dpa the rate of regeneration was on average 35% lower (P < 0.0001) compared to controls (Fig 2B). Interestingly, foxm1+/− tadpoles have an intermediate phenotype (reduction of 17%, P = 0.0055), suggesting a dose-dependent effect of foxm1 expression on appendage regeneration. The same effect was observed in F0 mosaic tadpoles at NF50, suggesting that the impairment in spinal cord regeneration is not stage or mutation specific (Fig EV2-EV5). Figure 2. Foxm1 is required for spinal cord regeneration but does not regulate the length of the cell cycle NF40 tadpoles with the following genotypes foxm1−/− (mut), foxm1+/− (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site. Quantification of the rate of regeneration. The ratio of the length of the tail regenerate at 3 and 7 dpa was compared to the length of the tail originally amputated at 0 dpa. The graph represents the mean ± SD of five independent experiments from three different clutches with at least five tadpoles in each experiment. Experimental setup for EdU labelling, foxm1 knockout and wt tadpoles were amputated and left to regenerate for 3 days. Tadpoles were then injected with EdU and 2 days later the tails were fixed, sectioned and stained for EdU and DAPI. Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. The graph represents the mean ± SD of EdU+ cells over the total number of cells in the spinal cord of 5–12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal. Experimental setup for Dual-Pulse S-phase Labelling: NF50 tadpoles at 3 dpa were injected with EdU, and 3 h later, the same tadpoles were injected with BrdU. Six hours after the first injection, the tails were fixed, sectioned and labelled for Sox3, Edu, BrdU and DAPI. Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. Quantification of images in (G). The graph represents the mean ± SD of 6 tadpoles with an average of 13 sections per tadpole analysed. Data information: Two-way ANOVA with Tukey's post hoc tests was used for B and E and an unpaired t-test for H. ns: non-significant, **P < 0.01 and ****P < 0.001. Scale bar is 500 µm in A and 25 µm in D and G. Download figure Download PowerPoint Could the impaired regeneration be caused by defective proliferation? To determine the rate of proliferation in the regenerating spinal cord, wt and foxm1−/− tadpoles were injected with EdU at 3 dpa, followed by a 2-day chase (Fig 2C–E). As expected, we observed a higher proportion of EdU+ cells in the regenerate than in the non-regenerating spinal cord (˜45 and ˜20%, respectively). However, no difference in the proportion of EdU+ cells between wt and foxm1−/− was observed, suggesting that Foxm1 does not affect the overall length of cell cycle. To confirm these data, we measured the absolute length of the cell cycle by Dual-Pulse S-phase Labelling (DPSL; Thuret et al, 2015). We first established the growth fraction by quantifying the proportion of cycling progenitors in the regenerating spinal cord (PCNA+ Sox3+) in wt and mutant tadpoles. In both cases, around 95% of Sox3+ cells are also PCNA+ (Appendix Fig S1A and B). To establish the absolute length of the cell cycle at 3 dpa, the tadpoles were injected first with EdU, then 3 h later with BrdU and fixed 3 h later (Fig 2F). The total length of the cell cycle (Tc) was about 50 h both in wild type and in foxm1−/− tadpoles, confirming that Foxm1 does not regulate the overall length of the cell cycle during spinal cord regeneration (Fig 2G and H). As it has been shown that knocking down foxm1 expression decreases the proliferation of neuronal progenitors during primary neurogenesis in X. laevis (Ueno et al, 2008), we tested whether this was also the case in X. tropicalis (Appendix Fig S2). Embryos at 1-cell stage were injected either with morpholino control (MOC) or with morpholino targeted to the exon3–intron3 (MOe3i3) and intron3–exon 4 (MOi3e4) splice sites of the foxm1 mRNA (MOF1). In parallel, we also analysed embryos injected with Cas9/gRNA against foxm1 (Crispr F1) or Cas9 alone (Crispr C). We first analysed the ability of the morpholino and gRNA/Cas9 to reduce foxm1 expression by RT–qPCR when injected at the one-cell stage (solid bars) or in one cell of a 2-cell stage embryo (hatch bars Appendix Fig S2A). Injection of MOF1 at 1-cell stage leads to a reduction of ˜70% of foxm1 expression (˜40% when injected in 1 cell at 2-cell stage), whilst injection of Crispr F1 causes a reduction of about 55% (and ˜30% when injected in 1 cell at 2-cell stage, Appendix Fig S2A). We then injected 1 cell at the 2-cell stage with MOC, MOF1, Crispr F1 or Crispr C together with a rhodamine-DEXTRAN as a tracer (Appendix Fig S2B). Embryos were fixed at NF13 and stained for phospho-Histone H3 (pH3). Quantification of pH3 cells in the neural plate shows no difference between injected and non-injected sides in all conditions, suggesting that Foxm1 does not promote proliferation during primary neurogenesis in Xenopus tropicalis (Appendix Fig S2C). Organisation of the Xenopus spinal cord at stage NF50 To understand the role of Foxm1 during spinal cord regeneration, we wanted to characterise this cell population at the molecular level using single-cell RNA sequencing (scRNA-seq). As the cellular organisation of the Xenopus spinal cord is not well described, we first used the 10X Genomics platform to sequence 2908 cells from uninjured spinal cord (Fig EV3-EV5). Twelve clusters were identified comprising the different cell types expected in the spinal cord: roof and fl
Referência(s)