Neoblast-enriched zinc finger protein FIR1 triggers local proliferation during planarian regeneration
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Regeneration, relying mainly on resident adult stem cells, is widespread. However, the mechanism by which stem cells initiate proliferation during this process in vivo is unclear. Using planarian as a model, we screened 46 transcripts showing potential function in the regulation of local stem cell proliferation following 48 h regeneration. By analyzing the regeneration defects and the mitotic activity of animals under administration of RNA interference (RNAi), we identified factor for initiating regeneration 1 (Fir1) required for local proliferation. Our findings reveal that Fir1, enriched in neoblasts, promotes planarian regeneration in any tissue-missing context. Further, we demonstrate that DIS3 like 3′-5′ exoribonuclease 2 (Dis3l2) is required for Fir1 phenotype. Besides, RNAi knockdown of Fir1 causes a decrease of neoblast wound response genes following amputation. These findings suggest that Fir1 recognizes regenerative signals and promotes DIS3L2 proteins to trigger neoblast proliferation following amputation and provide a mechanism critical for stem cell response to injury.
KEYWORDSlocal proliferation adult stem cells Dis3l2 wound recognition planarians Schmidtea mediterranea
Regeneration is a common phenomenon throughout the animal kingdom. For example, in mammals, hair follicle and epidermis can regenerate following injury (Fuchs and Segre, 2000; Seifert et al., 2012), and some invertebrates such as Hydra are capable of whole-animal regeneration from tissue pieces (Govindasamy et al., 2014; Sanchez Alvarado, 2000). There are two general regeneration groups: epimorphosis, which comprises all cases of regeneration that involve proliferation to form new tissue, and morphallaxis, in which regeneration can occur in the absence of cell proliferation (Morgan, 1901). The source of proliferative cells varies among the organisms exhibiting epimorphic regeneration. Adult stem cells (ASCs), residing in adult tissues, are undifferentiated cells and divide to replenish senescent cells and regenerate wounded tissues (Beachy et al., 2004; Clarke et al., 2000). The proliferation of ASCs is essential to initiate regeneration. It is reported that many signaling pathways are involved in the regulation of adult stem cell proliferation. For example, the transforming growth factor-β signaling is implicated in the control of muscle stem cell proliferation during adult skeletal muscle regeneration (Carlson et al., 2008), while canonical Wnt signaling promotes the proliferation of peripheral olfactory stem cells during the peripheral olfactory regeneration (Wang et al., 2011). However, these signals come from extrinsic molecules, the intrinsic regulators that govern adult stem cell proliferation in vivo remain largely elusive.
Planarians are a classical model for studying regeneration, as they can regenerate their whole bodies after amputation even from little pieces (Morgan, 1898; Reddien and Sanchez Alvarado, 2004). This amazing regenerative capacity relies on a population of adult stem cells named neoblasts (Reddien and Sanchez Alvarado, 2004), which are constantly dividing to replenish all cell types in intact animals (Newmark and Sanchez Alvarado, 2000; Pellettieri and Sanchez Alvarado, 2007). Neoblasts proliferate following wounding and are the source of new cells for regeneration (Best et al., 1968). Upon amputation, neoblasts display two waves of proliferating response: one commencing 6–8 h following wounding, whereby proliferation increases throughout the body, followed by another occurred 40 h later, in which proliferation is restricted to the wounds (Wenemoser and Reddien, 2010). The first wave is triggered following all injury types, while the second wave is specific to ‘missing-tissue’ response (Wenemoser and Reddien, 2010; Wurtzel et al., 2015). Many genes were mainly expressed in neoblasts and could regulate neoblast proliferation during regeneration. For example, Smed-hp1-1 triggers neoblast proliferation by inducing the expression of Mcm5 (Zeng et al., 2013). However, Smed-hp1-1 is required for all neoblasts proliferation, not specifically for proliferation near the wounds. The intrinsic regulatory mechanisms of neoblasts that promote local proliferation responding to wound are poorly understood. Following amputation, a class of wound-induced genes was activated directly within neoblasts (e.g., runt-1 and cdc25-1) (Wenemoser et al., 2012). Moreover, a recent study has revealed that all kinds of injury activate a common wound-response transcriptional progress, and neoblasts express most wound-induced genes (Wurtzel et al., 2015). These findings suggest that neoblasts play critical roles in response to early injury. Nevertheless, the neoblast intrinsic genes controlling neoblast wound response are needed to be identified.
In the present study, we identified that Fir1 was required for local proliferation response for regeneration. Fir1 is mainly expressed in neoblasts and promotes regeneration following amputation. Further, we found that Dis3l2 is required for Fir1 phenotype. Moreover, the expression of neoblast wound response genes is reduced in Fir1(RNAi) animals following amputation. These results suggest that Fir1 senses regenerative signals and promotes DIS3L2 proteins to trigger neoblast proliferation following amputation and provide a mechanism critical for neoblast response to injury.
Identification of Fir1 required for local proliferation by screening
If these 23 genes mentioned above mainly play roles in maintaining the local proliferation 48 h post-amputation, they should not affect the neoblast population in homeostasis (Gavino et al., 2013; Wenemoser and Reddien, 2010). Therefore, we examined ‘X1’ population (referred to as neoblasts in FACS) after inhibiting each of the representative 23 genes respectively, and found that the percentage of ‘X1’ population was comparative to the control after inhibiting 13 genes, indicating that at this time point these genes might have no effects on neoblast maintenance in homeostasis (Fig. 1D). Strikingly, Fir1 was included in these 13 genes, indicating that Fir1(RNAi) does not affect neoblast maintenance before amputation. Furthermore, we investigated neoblast population and mitotic activity in intact Fir1(RNAi) animals at different time points, and we found that the neoblast population and the mitotic density appeared indistinguishable compared to control after Fir1 RNAi (Fig. S1F and S1G). We also found that Fir1(RNAi) animals displayed homeostasis defects following long-term RNAi (Fig. S1H). The number of neoblast early (prog-1+) and late (agat-1+) progenies decreased following Fir1 RNAi (Fig. S1I). These results suggest that Fir1 is required for neoblast differentiation in homeostasis, which was not mainly discussed in this paper.
To exclude the possibility that Fir1(RNAi) affects neoblast generic response 6–8 h after injury, we examined the mitotic activity 0 h and 7 h post-amputation in Fir1(RNAi) animals (Fig. 1E). After quantification of the mitotic density, Fir1(RNAi) animals displayed neoblast generic response (Fig. 1F). Taken together, these results suggest that Fir1 specifically controls local proliferation in early regeneration.
Fir1 is required for missing-tissue regeneration
To specify the regeneration phenotype after inhibition of Fir1, we inflicted some other injury types to planarians, including a large incision, excision of lateral tissue wedge, removal of head tips, and removal of eyes. We observed that Fir1(RNAi) animals could repair the large incision, while Fir1(RNAi) animals were not able to regrow in other injury conditions, indicating that Fir1 was required for missing-tissue regeneration (Fig. 2C).
Fir1 mRNA is predominantly expressed in neoblasts
Recently, it is reported that planarian neoblasts comprise two major and functionally distinct cellular compartments, denoted as zeta and sigma (van Wolfswinkel et al., 2014). By using dFISH, we detected Fir1 mRNA with zeta class marker zfp-1 and sigma class marker soxP-2 and found that 23.07% of Fir1+ cells expressed soxP-2 mRNA and 52.17% of Fir1+ cells expressed zfp-1 mRNA, suggesting that Fir1 may play a role in both cell types during regeneration (Fig. 3D and 3E). Furthermore, by analyzing macerated animals subjected to 4 h BrdU pulse and labeled with Fir1 and smedwi-1 FISH probes and BrdU IF, we found that 56% of BrdU+ cells were Fir1 mRNA positive, in which most cells were also smedwi-1 positive, indicating that dividing cells expressed Fir1 mRNA in homeostasis (Fig. 3F).
In regeneration, qRT-PCR results showed that like smedwi-1, Fir1 mRNA expression level increased significantly and reached the maximum at Day 3 post-amputation (Fig. S3E). To understand the change in the Fir1 mRNA expression pattern during regeneration, we performed colorimetric WISH on 10 regeneration time points and found that like smedwi-2, Fir1 mRNA expression dramatically increased 48 h post-amputation especially near the wounds, suggesting that Fir1 may play important roles in this region (Figs. 3G and S3F). Further, we examined Fir1 mRNA, smedwi-1 mRNA and mitosis marker H3P in tail pieces 48 h post-amputation (Fig. 3H). We observed that all H3P+ cells expressed Fir1 mRNA as well as smedwi-1 mRNA, suggesting that Fir1 may regulate local proliferation during regeneration.
Analysis of Fir1 downstream genes by screening
In other species, the homologous of FIR1 protein is POGZ (pogo transposable element-derived protein with zinc finger domain), which is poorly understood in stem cell research (Fig. S4A). FIR1 protein was predicted to contain 6 ZnF_C2H2 domains from SMART, suggesting that FIR1 may function as a transcription factor (Fig. S4B). We sought to define the molecular mechanism of FIR1 by expression-profiling experiments. We designed custom oligonucleotide microarrays representing 61,657 predicted S. mediterranea transcripts and isoforms from various sources (Kao et al., 2013; Labbe et al., 2012; Onal et al., 2012; Rouhana et al., 2012; Wenemoser et al., 2012). Based on our observations that mitoses decreased dramatically at the wound sites (part 1) while increasing significantly in the remaining Fir1(RNAi) tail pieces (part 2) 48 h post-amputation, we isolated ‘part 1’ and ‘part 2’ tissues for microarrays (Fig. 4B). Prior to microarray analysis, we first examined the Fir1 knockdown efficiency using qRT-PCR assay and found that Fir1 mRNA expression decreased dramatically in both part 1 and part 2 (Fig. 4C). To display an overview of the microarray results, we clustered the transcripts according to fold change using R language, which revealed that the expression level of most transcripts did not change in either part 1 or part 2 (Fig. 4D). In addition, we checked the fold change of several known neoblast regulators and found that 25/34 of these genes showed the similar fold change as our qRT-PCR results (Figs. S4C and 4A, data not shown). According to the criterion for assignment to Cluster three above, we identified 46 genes the expression of which decreased more than 50% in part 1 and had no change in part 2 in our microarrays (Fig. 4E, Table S3). Further, considering the possible function of gene orthologs, we finally chose 26 genes for RNAi analysis, and to find more genes promoting regeneration, we increased the dsRNA feeding times to 4. Finally, we found that only Dis3l2(RNAi) and Mrpl21(RNAi) animals exhibited regeneration defects, suggesting that these two genes might function as Fir1 downstream genes (Fig. 4F).
Fir1 functions through regulation of Dis3l2
To establish that either Dis3l2 or Mrpl21 functions as a Fir1 downstream gene, we detected the mRNA expression of Dis3l2 and Mrpl21 by qRT-PCR in animals irradiated for 24 h and 48 h. The expression of Mrpl21 was not reduced in animals irradiated for 48 h, indicating that Mrpl21 was not expressed in neoblasts. In the previous study, Fir1 was predominantly expressed in neoblasts and could control local neoblast proliferation. So, we considered that Mrpl21 was not a direct downstream gene of Fir1. Interestingly, we found that Dis3l2 mRNA expression reduced dramatically, with 50% reduction noted in samples irradiated for 48 h, indicating that Dis3l2 was expressed in neoblasts (Fig. 4G). Planarian DIS3L2 was similar to Drosophila DIS3L2 in protein sequences and, like DIS3L2 in other organisms, it may be involved in proliferation regulation (Fig. S4D). Further, we examined Dis3l2 mRNA expression in tail pieces regenerated for 48 h following Fir1(RNAi). We found that the Dis3l2 mRNA expression decreased at the wound sites, suggesting that Fir1 may promote Dis3l2 expression during regeneration (Fig. 4H).
To further validate this assertion, we examined the expression pattern of Dis3l2 mRNA by colorimetric WISH in intact animals and regenerating tails. We observed that the expression of Dis3l2 mRNA at the wound sites increased from 18 h to 2 days post-amputation with quite a low expression in intact worms, indicating that Dis3l2 was induced by tissue-missing injury (Fig. 5E). Taken together, these data suggest that Fir1 drive local proliferation, at least partially, by promoting Dis3l2 expression.
Fir1 is required for neoblast wound recognition during regeneration initiation
Moreover, we explored genes belonging to W1, W2 and W3 in Fir1(RNAi) regenerating pieces. The mRNA expression of W1 genes like jun-1 and egrl-1 decreased 1–3 h post-amputation in Fir1(RNAi) animals (Fig. S6B and S6C). Notably, amputated Fir1(RNAi) animals displayed a higher level of expression of some W2 genes (wnt1, notum and fst) than did controls 18 h after amputation, suggesting that the regenerative signals were blocked (Fig. 6C–E). The expression of some W3 genes, such as delta-1, also increased 24 h post-amputation in Fir1(RNAi) animals (Fig. 6F). To further explore whether the regeneration was initiated after Fir1(RNAi), we detected the expression pattern of wnt1 and notum 48 h post-amputation in Fir1(RNAi) animals. We observed that Fir1(RNAi) animals did not establish anterior-posterior polarity, suggesting that regenerative procedure could not be activated following Fir1(RNAi) (Fig. S6D).
The proliferation of adult stem cell in regeneration
In mammals, the proliferation of adult stem cells is essential for tissue/organ regeneration. For example, cardiomyocyte proliferation is critical for heart regeneration in neonatal mice (Porrello et al., 2011), and intestinal stem cell proliferation, regulated by Hippo pathway, plays an important role during Drosophila adult midgut regeneration (Shaw et al., 2010). Inhibition of cell proliferation blocks the regeneration of oral structures in the anthozoan cnidarian (Passamaneck and Martindale, 2012). In planarians, regeneration is mediated by neoblasts, the only dividing cells, which are responsible for tissue regeneration (Wagner et al., 2011). Planarian can regenerate any part of the body rapidly, which makes them an ideal model for studying stem cell function in vivo (Gentile et al., 2011; Sanchez Alvarado, 2003). After amputation, two phases of proliferation occur: the first is initiated after all kinds of injury, while the second is triggered only when regeneration is required (Wenemoser and Reddien, 2010). The second phase of proliferation is restricted to the wounds (also known as local proliferation), which is equivalent to the proliferation of adult stem cells for tissue/organ regeneration in mammals.
Neoblast-enriched Fir1 triggers local neoblast proliferation
To find neoblast intrinsic regulators required for local neoblast proliferation, we analyzed the regeneration phenotypes, the mitotic activity and the percent of neoblasts after inhibiting each of the 46 genes that were selected according to the severity of RNAi phenotypes and neoblast-enriched features reported in pertinent literature, as well as found in our proprietary database (Figs. S1B and 1B–D, Table S1). Despite the small size of our screening, we found that a novel gene Fir1 meets our requirements exactly. First, Fir1(RNAi) trunk pieces cannot regenerate blastemas (Figs. S1B and 2A). Second, Fir1(RNAi) tail pieces display reduced mitotic activity near the wounds 48 h post-amputation (Fig. 1B and 1C). Third, the percent of neoblasts was indistinguishable from that found in the control animals (Fig. 1D). Moreover, almost all Fir1+ cells express smedwi-1 (Fig. 3B). In our screening, we chose regenerating tail pieces, in which nearly all Fir1+ cells express smedwi-1, as the main model for screening genes required for local proliferation (Fig. 3H, data not shown). Therefore, these results suggest that Fir1 may function as a neoblast intrinsic regulator controlling local proliferation. To date, many genes are reported to be required for neoblast proliferation during regeneration, such as smedwi-2 (Reddien et al., 2005b), Smed-hp1-1 (Zeng et al., 2013), Smed-argonaute-2 (Li et al., 2011) and Smed-p53 (Pearson and Sanchez Alvarado, 2010). However, most of these genes regulate proliferation of all the neoblast throughout animal body, and it is presently unknown if there exist neoblast intrinsic regulators specifically required for local proliferation responsible for a missing-tissue injury. Here, for the first time, we identified neoblast-enriched gene Fir1 required for local proliferation, which provided in vivo mechanisms for adult stem cell proliferation during regeneration.
Dis3l2 is a potential target of Fir1
FIR1 protein is predicted to contain 6 ZnF_C2H2 domains, which suggests that Fir1 may function as a transcription factor. To find genes downstream of Fir1 in regulating local proliferation, we mainly analyzed the downstream transcripts adjacent to the wounds after RNAi knockdown of Fir1 by microarray. Indeed, the best way is isolating the neoblasts near the wounds to perform expression-profiling experiments. But we could not get enough neoblasts for microarray, and that is why we chose tissues near the wounds for microarray analysis instead. Although the expression of some reported neoblast regulators decreased dramatically, these genes did not affect the local proliferation required for regeneration as we expected, indicating that Fir1 is one of the few genes involved in this process. Importantly, we identified a novel gene Dis3l2 as a potential target of Fir1. First, Dis3l2 is downregulated after RNAi knockdown of Fir1 and promotes planarian regeneration (Fig. 4F and 4H). Second, Dis3l2 is expressed in neoblasts and colocalizes with Fir1 in the vicinity of wound 48 h post-amputation (Figs. 4G and 5A). Intriguingly, colorimetric WISH reveals that rare Dis3l2 mRNA is detected in intact animals, while Dis3l2 mRNA is induced adjacent to the wounds from 18 h post-amputation with obvious accumulation following 48 h of regeneration (Fig. 5E). Moreover, Dis3l2 mRNA disappears in this region as regeneration progresses, which suggests that Dis3l2 mRNA dynamics are consistent with its function during regeneration (Fig. 5E). Most importantly, Dis3l2 is required for local proliferation, suggesting that Dis3l2 may be a functional downstream gene of Fir1 (Fig. 5B and 5C). Dis3l2, mutations in which cause the Perlman syndrome, is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3′-5′ direction (Astuti et al., 2012). Dis3l2 plays a critical role in RNA metabolism and is essential for the regulation of cell growth and division. For example, Dis3l2 functions in the Lin28-mediated repression and degradation of let-7 microRNAs (miRNAs) in mouse embryonic stem cells (mESCs) (Chang et al., 2013; Ustianenko et al., 2013). Knockdown of Dis3l2 enhances the growth of human cancer cell lines (Astuti et al., 2012). However, in planarian, inhibition of Dis3l2 suppresses the local proliferation required for regeneration, suggesting that Dis3l2 may degrade the mRNA transcribed by genes inhibiting neoblast proliferation adjacent to the wounds during regeneration.
Neoblast response to injury requires Fir1 function
In planarians, three major classes of wound-induced genes that are expressed in differentiated tissues (W1, W2 and W3 genes) and a class of genes induced in neoblasts (W4 genes) have been identified, which constitute a molecular wound response program to elicit regeneration (Wenemoser et al., 2012). We examined the expression pattern of representative genes in four categories following Fir1 RNAi and found that the response of these genes to amputation displayed abnormally. The induction of W4 genes (runt-1 and cdc25-1) and W1 genes (jun-1 and egrl-1) was reduced in Fir1(RNAi) animals, while the induction of W2/3 genes like wnt1, notum, fst, and delta-1 increased significantly (Figs. 6A, 6C–F and S6A–C), suggesting that Fir1 is required for wound response during regeneration. It is reported that fst, a wound-induced gene expressed in differentiated tissues, is required for local mitosis by inhibiting Activin signaling (Gavino et al., 2013). The increase in the number of fst+ cells near wounds can be explained as the interruption of regenerative signals before neoblasts sensing them. Moreover, a recent study has revealed that neoblasts express most wound-induced genes, suggesting that neoblasts may play an important role in wound response (Wurtzel et al., 2015). Here, we for the first time identified Fir1 as a potential neoblast intrinsic regulator controlling neoblast response to missing-tissue injury.
Materials and methods
Planarian culture and irradiation
In this study, sexual Schmidtea mediterranea CIW4 strain was used in all experiments. These planarians were maintained as described elsewhere (Newmark and Sanchez Alvarado, 2000; Sanchez Alvarado et al., 2002; Wang et al., 2016). Briefly, planarians were cultured in 1× Montjuic salts at 21°C in the dark, fed homogenized beef liver paste two times per week, and amputated for expansion. Planarians were starved for 1–2 weeks before experiments. 4–6 mm-long animals were used for RNAi and 1–2 mm-long animals were used for in situ hybridization. For irradiation, planarians were exposed to 6,000 rads on a GammaCell 3000 irradiator (Table S4). The animals were kindly provided by P. Newmark (University of Illinois at Urbana-Champaign/Howard Hughes Medical Institute, Urbana, IL), P. Reddien (Massachusetts Institute of Technology/Howard Hughes Medical Institute, Cambridge, MA), and N. Oviedo (University of California, Merced, Merced, CA).
Gene cloning and RNAi
All planarian transcripts used in this study were cloned into the pMD18-T vector (Takara) from complementary DNA (cDNA) and verified by Sanger sequencing. RNA interference (RNAi) was mainly performed as described elsewhere (Rouhana et al., 2013). The template for producing dsRNA was generated by polymerase chain reaction (PCR) using primers with T7 promoters flanking on the 5′-ends. The sense and antisense RNA molecules were transcribed by T7 RNA polymerase (Promega) and annealed. The quality of dsRNA was assessed by non-denaturing agarose gel electrophoresis. 4 µg dsRNA with 20 μL beef liver was sufficient for inducing 15 animals RNAi. dsRNA for GFP was used as negative control. Generally, animals were fed dsRNA food every 3 days, and the animals were amputated 8 days after initial RNAi. We made a cartoon depicting experiment design for each figure, which described the number of RNAi treatments, time of amputations after initial RNAi, amputation position (as indicated with dotted red lines) and time of detection. For RNAi of Dis3l2 and Mrpl21 in Fig. 4F, animals got 4× dsRNA feedings. Unless otherwise noted, animals were fed 2× dsRNA food (Table S2).
Whole-mount immunofluorescence was performed as described elsewhere (Newmark and Sanchez Alvarado, 2000; Wenemoser and Reddien, 2010). Briefly, animals were sacrificed and fixed in Carnoy’s on ice for 1.5 h, following bleaching in 6% hydrogen peroxide/methanol solution, animals were blocked and incubated with rabbit anti-H3P antibody (1:100, Millipore), and the mitotic activity was developed using anti-rabbit Alexa 488 (1:600, Invitrogen).
Immunofluorescence on paraffin section
Tail pieces following 2 days regeneration were fixed in 4% formaldehyde for 2 h at 4°C. They were subsequently embedded in paraffin and sectioned adjacent to the wounds at 5 μm thickness. After deparaffination, antigen retrieval was performed in 0.01 mol/L citrate buffer, pH 6.0, for 20 min. Then the sections were blocked with 4% BSA and incubated in HP1-1 (1:100 dilution) and SMEDWI-1 (1:200 dilution) antibody solution (Zeng et al., 2013). HP1-1 and SMEDWI-1 were developed using rhodamine tyramide (1:2,000 dilution) and fluorescein tyramide (1:1,000 dilution), respectively. Horseradish peroxidase enzyme was inactivated for 20 min between labelings by 1% Hydrogen peroxide, in PBS containing 0.1% Triton-X100 (PBSTx). The sections were counterstained with DAPI (sigma, 1 μg/mL).
Whole-mount in situ hybridization
Whole-mount in situ hybridizations were performed as described elsewhere (Pearson et al., 2009). Hybridized RNA probes were labeled with DIG-11-UTP (Sigma), Fluorescein-12-UTP (Sigma) or DNP-11-UTP (Perkin Elmer) and purified as described (Lapan and Reddien, 2011). Tyramide was generated by conjugation of succinimidyl esters of rhodamine, FITC, and AMCA with tyramide-HCL (Sigma) (Hopman et al., 1998). For horseradish peroxidase enzyme inactivation, animals were incubated in 154 mmol/L sodium azide for 2 h (King and Newmark, 2013; van Wolfswinkel et al., 2014). Animals were counterstained with DAPI (Sigma, 3 μg/mL in PBSTx) for 1 h and mounted for imaging.
Fluorescence-activated cell sorting
The procedures of fluorescence-activated cell sorting were mainly performed as described elsewhere (van Wolfswinkel et al., 2014). Planarians were diced with a razor blade on ice-cold dishes, and the tissue mash was collected in CMFB (400 mg/L NaH2PO, 800 mg/L NaCl, 1,200 mg/L KCl, 800 mg/L NaHCO3, 240 mg/L glucose, 1% BSA, 15 mmol/L HEPES pH7.3) supplemented with 1 mg/mL collagenase (Sigma) (Reddien et al., 2005a). After digestion for 45 min under agitation at room temperature, cell suspensions were passed through a 35 μm cell-strainer cap (BD Biosciences), and pelleted. Then the cells were stained with Hoechst 33342 (Invitrogen) and propidium iodide and filtered again. Cells were sorted on a MoFlo (Beckman-Coulter), and Hoechst blue versus red plots were used to identify the ‘X1’ fraction that is high in DNA content (Hayashi et al., 2006).
BrdU labeling was performed as described elsewhere (Cowles et al., 2012). Briefly, animals were treated with 0.0625% N-acetyl cysteine (NAC) for 1 min, washed 3 times quickly, and incubated for 1 h in 1× Montjuıïc salts containing 25 mg/mL BrdU (Sigma) and 3% dimethyl sulfoxide in the dark. Animals were washed 3 times and inculcated for 4 h in the dark.
Single-cell FISH and immunofluorescence on cells
Cells from macerated animals or fluorescence-activated cell sorting were adhered to coverslips. Single-cell FISH was performed as described elsewhere (Scimone et al., 2014). For BrdU immunofluorescence after FISH, the procedures were similar to that in whole mount animals, with slight modifications: all washes were limited to 5 min, and the BrdU signal was developed using rhodamine tyramide (Newmark and Sanchez Alvarado, 2000).
qRT-PCR was performed as previously described (Li et al., 2011; Wang et al., 2016; Zeng et al., 2013). Briefly, total RNA of the regenerating pieces was isolated using TRIZOL (Invitrogen). M-MLV Reverse Transcriptase (Promega) was used to synthesize cDNA from 1 μg of total RNA. Gene-specific primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) (Table S2). qPCRs were performed with SYBR Green quantitative PCR master mix (Toyobo Co.) on a quantitative PCR system (7900HT Fast Real-Time PCR System, Applied Biosystems). Three biological replicates were performed for each group. The relative mRNA expression was plotted with GraphPad Prism.
We designed custom oligonucleotide microarrays representing 61,657 predicted S. mediterranea transcripts and isoforms from various sources (Kao et al., 2013; Labbe et al., 2012; Onal et al., 2012; Rouhana et al., 2012; Wenemoser et al., 2012) at the eArray website (Agilent Technologies). ‘part 1’ and ‘part 2’ RNA was harvested with Trizol (Invitrogen) from control and Fir1(RNAi) tail pieces 48 h post-amputation. Two biological replicates were used. RNA was amplified and labeled with Cy3-CTP using a low RNA input fluorescent linear amplification kit (Agilent Technologies). Custom oligonucleotide expression arrays (Agilent) were hybridized, scanned and analyzed as previously described (Zeng et al., 2013). To find the downstream genes of Fir1, genes were considered if they met a corrected P-value threshold of 0.05 and were downregulated in ‘part 1’ and showed invariable in ‘part 2’ in duplicate samples. Hierarchical clustering and heat map generation were performed using R.
Protein sequence for SMED-FIR1 and SMED-DIS3L2 was aligned with its homologous proteins in other organisms using ClustalW with the default setting (Thompson et al., 1994). The result of ClustalW was imported to MEGA 4.0, in which neighbor-joining tree was generated using default settings and 1,000 bootstrap replicates.
Animals were fixed and stained for TUNEL using a method described elsewhere (Pellettieri et al., 2010) with modifications: animals were bleached in formamide-bleaching solution (5% non-deionized formamide, 0.5× SSC, and 1.2% H2O2) (King and Newmark, 2013) for 4 h under bright light, after TdT reaction animals were washed 2 × 30 min at 65°C in 1 mmol/L EDTA, and the TUNEL signal was developed using FITC-tyramide solution (FITC-tyramide 1:1,000 and 0.006% H2O2 in PBS containing 0.01% Tween-20) for 20 min.
Image acquisition, processing and quantification
Live animals and whole-mount in situ hybridization samples were photographed using a microscope (SteREO Discovery.V20; Carl Zeiss) equipped with a Plan Apochromat 1.0× objective and a digital microscope camera (AxioCam HRc; Carl Zeiss) automated by AxioVision Rel.4.8 software (Carl Zeiss). Confocal images were captured on a Leica SP5 confocal microscope with a 20×, 40×, or 63× objective. All the quantifications were using the Measurement program of Volocity (Perkin Elmer) and normalized by the quantified animal area. For H3P quantifications, all the mitotic events were determined by counting nuclei labeled with the anti-H3P antibody. For quantification of notum+, wnt1+ or fst+ cells, all the probe signals surrounding the nucleus in the vicinity of the wounds were calculated. For cells under apoptosis quantifications, we obtained all the TUNEL signal using confocal microscope (about fifty 1 μm stacks), then we used Volocity software (PerkinElmer) to build 3D image for these stacks and quantify TUNEL+ nuclei in our region of interest (ROI), and finally the number of TUNEL+ nuclei was normalized by the area of the upper surface of ROI.
Results are presented as means ± SEM, and statistical analyses were performed in GraphPad Prism using the Student’s t test for two groups. P < 0.05 was considered significant.
We thank P. Newmark, P. Reddien, and N. Oviedo for kindly providing worms. We thank S. Lapan, D. Wenemoser, and K. Kravarik for FISH advice, J. Wolfswinkel for FACS assistance. We thank X. Qiu and J. Chen for critically reading the manuscript. We thank all members of Jing lab for comments and discussion. We thank the staff in the core facility (Institute of Health Sciences) for technical assistance. This work was supported in part by the National Key Research and Development Program of China (2017YFA0103700), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020903), and the National Natural Science Foundation of China (91739301, 91339205, and 31229002).
ASCs, adult stem cells; dFISH, double fluorescent in situ hybridization; FACS, fluorescence-activated cell sorting; H3P, phosphorylated histone H3 at serine 10; RNAi, RNA interference; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WISH, whole-mount in situ hybridization.
COMPLIANCE WITH ETHICS GUIDELINES
Xiao-Shuai Han, Chen Wang, Fang-hao Guo, Shuang Huang, Yong-Wen Qin, Xian-Xian Zhao and Qing Jing declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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