Satellite Cell Expansion Is Mediated by P-Eif2α-Dependent

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STEM CELLS AND REGENERATION RESEARCH ARTICLE Satellite cell expansion is mediated by P-eIF2α-dependent Tacc3 translation Ryo Fujita1,2,*, Solenè Jamet2, Graham Lean1,2, Harry Chun Man Cheng1,2, Steven Hébert2, Claudia L. Kleinman1,2 and Colin Crist1,2,‡

ABSTRACT cell self-renewal (Wang et al., 2019). How satellite cells achieve this Translational control of gene expression is an important regulator of proliferative capacity, while maintaining the fidelity of cell division, adult stem cell quiescence, activation and self-renewal. In skeletal remains unclear. muscle, quiescent satellite cells maintain low levels of protein Quiescent satellite cells have few activated mitochondria and synthesis, mediated in part through the phosphorylation of eIF2α generate low levels of ATP (Rocheteau et al., 2012; Rodgers et al., (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with 2014). To manage available energy resources, quiescent satellite the small molecule sal003 maintains P-eIF2α and permits the cells likely maintain low rates of protein synthesis, mediated in part α expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also by the phosphorylation of translation initiation factor eIF2 α increases the translation of specific mRNAs, which is mediated by (Zismanov et al., 2016). Phosphorylation of the eIF2 subunit of P-eIF2α-dependent read-through of inhibitory upstream open reading the eIF2 complex turns eIF2 into a competitive inhibitor of the frames (uORFs). Here, we ask whether P-eIF2α-dependent mRNA guanine nucleotide exchange factor eIF2B to prevent the recycling translation enables expansion of satellite cells. Using transcriptomic of the eIF2-GTP-initiator methionyl-tRNA ternary complex (eIF2- Met and proteomic analyses, we show a number of genes associated with GTP-tRNA ) needed to deliver the first amino acid to the the assembly of the spindle pole to be upregulated at the level of ribosome (Krishnamoorthy et al., 2001). Genetic inactivation of α α protein, without corresponding change in mRNA levels, in satellite eIF2 phosphorylation (P-eIF2 ) in satellite cells leads to increased cells expanded in the presence of sal003. We show that uORFs in the global protein synthesis, activation of the myogenic program and α 5′ UTR of mRNA for the mitotic spindle stability gene Tacc3 direct failure to self-renew. Pharmacological inhibition of eIF2 P-eIF2α-dependent translation. Satellite cells deficient for TACC3 dephosphorylation with the small molecule sal003 (Boyce et al., exhibit defects in expansion, self-renewal and regeneration of skeletal 2005; Costa-Mattioli et al., 2007) maintains low levels of protein muscle. synthesis and enables ex vivo expansion of cultured satellite cells that retain their regenerative potential (Lean et al., 2019; Zismanov KEY WORDS: Muscle stem cell, Satellite cell, TACC3, Skeletal et al., 2016). muscle regeneration Although maintaining low levels of protein synthesis, quiescent satellite cells also repress the translation of specific transcripts that INTRODUCTION maintain satellite cells ‘primed’ to activate the myogenic program Skeletal muscle regeneration relies on a population of resident adult and the cell cycle. Myf5 transcripts are repressed by miR-31 and stem cells, named ‘satellite cells’ for their position around the accumulate in cytoplasmic RNA granules (Crist et al., 2012), which outside of myofibers and underneath the basal lamina (Mauro, require P-eIF2α for their assembly and maintenance (Zismanov 1961). Satellite cells express members of the paired homeodomain et al., 2016). MyoD (Myod1) transcripts are repressed by the activity family of transcription factors PAX7 and, in a subset of muscle, of RNA-binding proteins TTP (ZFP36; Hausburg et al., 2015) and PAX7 and PAX3 (Relaix et al., 2006). Satellite cells are mitotically STAUFEN 1 (STAU1; de Morrée et al., 2017), and transcripts quiescent and activate the myogenic program and the cell cycle in required to activate the cell cycle, such as Dek, are repressed by response to muscle injury. Activated satellite cells exhibit miR-489 (Cheung et al., 2012). remarkable proliferative capacity to rapidly expand the population Although the role of post-transcriptional mechanisms repressing of myogenic progenitors required to efficiently regenerate gene expression in satellite cells is well documented, a more muscle. Moreover, this proliferative phase is marked by complete picture requires that we also understand how specific symmetric cell divisions, which drive expansion of the satellite mRNAs escape repression and are translated efficiently. When cells cell pool, and asymmetric cell divisions, mediated in part through an are under stress, P-eIF2α-dependent mRNA translation is illustrated EGFR-AuroraA kinase signaling pathway, which ensure satellite by selective translation of mRNAs for Atf4 and Chop (Ddit3), which are required to initiate an integrated stress response (ISR) (Palam et al., 2011; Vattem and Wek, 2004). These P-eIF2α-dependent 1Department of Human Genetics, McGill University, 3640 University St., Montréal H3A 0C7, Canada. 2Lady Davis Institute for Medical Research, Jewish General mRNAs have inhibitory upstream open reading frames (uORFs) Hospital, 3755 chemin de la Côte Ste. Catherine, Montréal H3T 1E2, Canada. that prevent the translation of the main ORF under normal *Present address: Division of Regenerative Medicine, Transborder Medical conditions, whereas stress-induced P-eIF2α permits the read- Research Center, University of Tsukuba, Ibaraki, 305-8577 Japan. through of the uORFs to enhance the translation of the main ORF ‡Author for correspondence ([email protected]) required for the ISR. We propose a model by which P-eIF2α-dependent mRNA R.F., 0000-0002-8392-0122; C.C., 0000-0002-0258-1158 translation regulates satellite cell quiescence, self-renewal and Handling Editor: Benoit Bruneau expansion. In this study, we determined how satellite cells modify

Received 25 June 2020; Accepted 4 December 2020 their transcriptome and proteome while expanded with sal003. We DEVELOPMENT

1 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480 show that sal003 pushes satellite cell gene expression towards a abdominal muscle of adult Pax3GFP/+ mice (Montarras et al., 2005) progenitor cell phenotype, while preventing differentiation. By after 4-day culture in the presence of 10 µM sal003 (Table S1; focusing on genes upregulated at the level of protein, without a Zismanov et al., 2016). Principal component analysis (PCA) reveals corresponding increase in mRNA, we demonstrate that satellite cell a strong, global effect on the transcriptome, with sal003 treatment expansion is mediated in part through P-eIF2α-dependent translation explaining 90% of the variance in the data (Fig. 1A). Consistent with of mRNA for Tacc3 (transforming acidic coiled coil protein 3), an the effect of sal003 to reduce global protein synthesis (Zismanov AuroraA kinase substrate that is essential for microtubule assembly, et al., 2016), we observed a general trend towards downregulation of maintenance of the spindle pole and ensuring the fidelity of cell genes that produced an asymmetrical volcano plot (Fig. 1B), as division (Burgess et al., 2015; Kinoshita et al., 2005). well as a larger number of downregulated genes (958 versus 383, P-value<0.00001). We generated a z-score heatmap to visualize RESULTS selected genes that are upregulated or downregulated at least 2-fold in Pharmacological inhibition of eIF2α dephosphorylation the presence of 10 µM sal003 (Fig. 1C). leads to expansion of satellite cells with distinct To understand the mechanisms underlying changes in gene transcriptional profiles expression when satellite cells are cultured in the presence of sal003, To begin asking how sal003/P-eIF2α enables satellite cell we used Enrichr (Chen et al., 2013) to perform gene ontology (GO) expansion, we first used RNA-seq to determine global changes in analyses (GO Biological Process 2018), as well as to identify gene expression in satellite cells isolated from diaphragm and enrichment of consensus target genes for transcription factors

Fig. 1. Transcriptome analysis of satellite cells cultured in the presence of 10 µM sal003. (A) A PCA plot derived from RNA-seq transcriptome profiling of satellite cells cultured under normal conditions (DMSO, blue) and 10 µM sal003 (red). (B) A volcano plot depicting differentially expressed genes (mRNA) between satellite cells cultured in DMSO (control) and 10 µM sal003. The threshold of P-value 0.05 is indicated by green and gray data points. The top three significantly downregulated and upregulated genes are indicated. (C) A z-score heatmap of selected differentially expressed genes in satellite cells after 4-day culture in 10 µM sal003. (D-I) Analysis of upregulated (D,F,H) and downregulated (E,G,I) genes was performed by gene set enrichment analysis for GO Biological Process (D,E), ENCODE and ChEA Consensus Transcription Factors (F,G) and ENCODE Histone Modifications (H,I). Intensity and length of bars indicate significance (−log10 P-value). The top 10 enriched gene sets are shown in each category. DEVELOPMENT

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(ENCODE and ChEA Consensus TFs from ChIP-X) and histone and moreover, downregulated genes are enriched for chromatin marks modifications (ENCODE Histone Modifications 2015). Amongst common to repressed gene bodies (H3K27me3) in non-skeletal the 383 genes upregulated greater than 2-fold (log2>1), there is muscle tissue (Fig. 1I). strong enrichment for GO Biological Process gene sets related to DNA replication, mitosis and cell cycle regulation (Fig. 1D). When Pharmacological inhibition of eIF2α dephosphorylation satellite cells are cultured in the presence of sal003, we observe modifies the satellite cell proteome enrichment for target genes of known transcriptional regulators of As sal003 inhibition of P-eIF2α dephosphorylation lowers rates of myogenic progenitor survival and proliferation (E2f1, Sin3a, E2f6, protein synthesis (Zismanov et al., 2016) and is expected to impact E2f4, Foxm1) (Wang et al., 1996) (Fig. 1F). Upregulated genes also rates of mRNA translation independent of changes in transcription, show enrichment for chromatin marks associated with actively we simultaneously asked how sal003 modifies the satellite cell transcribed genes in myogenic progenitors (H3K4me3, H3K9ac, proteome. We cultured satellite cells under normal conditions or in H3K6me3) (Fig. 1H). the presence of sal003, then labeled cell lysates with tandem mass Amongst the 958 genes downregulated greater than 2-fold tag (TMT) reagents to identify and quantify changes in protein (log2<−1) there is strong enrichment for GO Biological Process expression by high resolution mass spectrometry (Fig. 2A; gene sets related to differentiated skeletal muscle, including muscle Table S2). contraction, muscle filament sliding, myofibril assembly and Of the 180 genes upregulated 2-fold or greater (log2>1) sarcomere organization (Fig. 1E). Amongst these genes, consensus quantified by mass spectrometry, there is strong enrichment for target genes for transcription factor MYOD is predominant (Fig. 1G) genes expressed in mouse progenitor and stem cell lines (BioGPS,

Fig. 2. Proteome analysis of satellite cells cultured in the presence of 10 µM sal003. (A) Schematic of satellite cell proteome analysis by TMT labeling and mass spectrometry (MS). (B-G) Analysis of upregulated (B,D,F) and downregulated (C,E,G) genes performed by gene enrichment analysis for tissue expression in the Mouse Gene Atlas (B,C), GO Biological Process (D,E) and GO Cellular Component (F,G). Intensity and length of bars indicate significance

(−log10 P-value). Gray bars, P<0.05. The top 10 enriched gene sets are shown in each category. DEVELOPMENT

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Fig. 3. Satellite cells upregulate protein levels independent of mRNA when cultured in sal003: P-eIF2α dependent Tacc3 mRNA translation. (A) Venn diagram showing the relationship of genes upregulated at the level of mRNA (red) and protein (green) in satellite cells cultured in 10 µM sal003: 140 genes (green shading) are upregulated at the level of protein (mass spectrometry, log2>1), without a corresponding increase reported by mRNA (RNA-seq, log 2<1; blue). (B) Scatterplot of gene expression reported by transcriptome (x-axis) and proteome (y-axis) analyses. Pax7 and Tacc3 are highlighted (green) within the subset of genes upregulated at the level of protein (mass spectrometry, log2>1), without a corresponding increase reported by mRNA (RNA-seq, log 2<1; green dotted line). (C) Immunoblotting against TACC3 and β-actin from cell lysates of Pax3GFP/+ satellite cells cultured in the presence of DMSO (D, control) and sal003 (S). Relative levels of TACC3 normalized to β-actin are indicated, with representative immunoblots shown. (D) Relative Pax7, myogenin (MyoG) and Tacc3 levels, determined by RT-qPCR, after 4-day culture in the presence of DMSO (control) or 10 µM sal003. Levels are reported normalized to Actb and relative to DMSO conditions. (E) Immunoblotting against TACC3 and β-actin from cell lysates of Pax3GFP/+ satellite cells cultured in the presence of DMSO, sal003 or sal003 with ISRIB (200 nM). Relative levels of TACC3 normalized to β-actin are indicated, with representative immunoblots shown. (F) EdU incorporation (green), combined with immunolabeling with antibodies against PAX7 (red) and TACC3 (white) after 4-day culture of satellite cells in the absence (DMSO) or presence of 10 µM sal003. Merged images with DAPI are shown (right panels). (G) Quantification of numbers of PAX7(+), TACC3(+), EdU(+) cells in F. (H) Relative Firefly:Renilla luciferase activity in 293 cells transfected with pGL3-Atf4-luc, pGL3-Tacc3-luc (5′ UTR uORFs) or pGL3-Atf4mut-luc, pGL3-Tacc3mut-luc (5′ UTR ΔuORFs) (firefly), and pRL-TK (Renilla) after overnight culture in the presence of 1 µM TG. Atf4 (red) and Tacc3 (orange) 5′ UTRs are indicated to scale, with positions of deleted start codons at uORFs (ΔuORFs) indicated. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed Student’s t-test). n.s., not significant. Scale bar: 50 µm.

spindle assembly (Tacc3, Cdc20, Tpx2, Nedd1, Racgap1, Espl1, Spag5, Incenp, Vps4b, Kif2c, Ska3) (Fig. 3A,B; Table S3).

P-eIF2α-dependent translation of Tacc3 mRNA We further focused our attention on Tacc3, which was the most significantly upregulated gene reported by mass spectrometry and is a representative gene of the most significantly enriched gene ontology (spindle pole). Tacc3 mRNA contains five uORFs in its 5′ untranslated region (UTR), potentially enabling selective translation of the main ORF for Tacc3 by P-eIF2α. TACC3 is a substrate of AuroraA kinase and functions at the centrosome to regulate Fig. 2B). Upregulated genes are enriched in chromatid and microtubule nucleation, promote stability of the spindle apparatus chromosome separation (GO Biological Process, Fig. 2D), and are and the fidelity of cell division (Burgess et al., 2015; Kinoshita associated with the mitotic spindle, centromere and microtubules et al., 2005). Inhibition of TACC3 by knockout, knockdown and (GO Cellular Component, Fig. 2F). In contrast, the presence of pharmacological strategies reveal a role for TACC3 in maintaining 10 µM sal003 in satellite cell culture conditions downregulates 185 or expanding adult and cancer stem cell populations, although it is genes (2-fold, log2<−1) quantified by mass spectrometry. These dispensable for stem cell differentiation (Piekorz et al., 2002; genes are enriched in mouse skeletal muscle tissue (BioGPS, Wurdak et al., 2010; Yao et al., 2016; Zhou et al., 2015). Fig. 2C), are associated with muscle contraction and muscle First, we confirmed that 4-day culture of satellite cells in the filament sliding (GO Biological Processes, Fig. 2E), and are presence of 10 µM sal003 increases TACC3 protein levels (Fig. 3C), components of striated muscle thin filament, actin cytoskeleton, without a corresponding change in Tacc3 mRNA (Fig. 3D), focal adhesions and actomyosin (GO Cellular Component, Fig. 2G). validating the identification of Tacc3 in our global gene expression To reveal transcripts potentially translated in a P-eIF2α- profiles (Fig. 3A,B). Next, we cultured satellite cells in the presence dependent manner, we identified 140 genes that are upregulated at of sal003 to induce P-eIF2α levels and in the presence of integrated the level of protein (mass spectrometry, log2>1), without a stress response inhibitor (ISRIB), a small molecule inhibitor of corresponding increase reported by mRNA (RNA-seq, log 2<1) P-eIF2α first identified using a high throughput cell-based screen (Fig. 3A,B). Upregulated genes include inhibitors of myogenic for inhibitors of uORFs present in Atf4 mRNA. Mechanistically, differentiation (Pax7, Id3, Cabin1) and chromatin modifiers ISRIB activates eIF2B to recycle the eIF2-GTP-tRNAMet ternary (Kdm5c, Kmt2a, Setd1a, Setd2) that may account for, in part, complex, bypassing the effect of P-eIF2α (Sidrauski et al., 2015). transcriptional changes in gene expression observed (Fig. 1). The Enhanced eIF2B activity leads to translation initiation at the most significantly represented class of genes are those involved in inhibitory uORFs. We showed that additional treatment of satellite DEVELOPMENT

4 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480 cells with ISRIB lowers TACC3 protein levels to normal culture activated satellite cells, we first used magnetic cell sorting (MACS) conditions, further linking Tacc3 mRNA translation to the effects of to isolate satellite cells from uninjured and injured tibialis anterior P-eIF2α (Fig. 3E). Finally, we used immunofluorescence with muscle (TA) muscle. TACC3 is strongly upregulated in activated antibodies against PAX7 and TACC3, combined with EdU labeling, satellite cells in vivo, 3 days after cardiotoxin (ctx) injury (Fig. 4B,C). to reveal increased levels of TACC3 protein in proliferating In activated satellite cells that remain associated with cultured single PAX7(+) cells after 4-day culture in the presence of sal003 extensor digitorum longus (EDL) myofibers, strong immunolabeling (Fig. 3F,G). of TACC3 is observed in an average of 56% of PAX7(+) satellite cells To ask whether uORFs present in the 5′ UTR of Tacc3 mRNA after 24 h in culture, and in 96% of satellite cells after 48 h in culture, in mediate P-eIF2α-dependent translation in a manner similar to Atf4, which it appears to be localized to perinuclear areas and to spindle we cloned the 5′ UTRs of these two mRNAs upstream of a poles (Fig. 4D,E). luciferase reporter. We generated additional control reporters that During ex vivo culture of satellite cells, P-eIF2α is detectable in eliminate uORFs by mutation of the corresponding ATG start codon PAX7-expressing cells, but not in MYOD-expressing cells that have (Fig. 3H). We transfected 293 cells with Atf4 and Tacc3 P-eIF2α activated the myogenic program (Zismanov et al., 2016). We reporters, and further cultured 293 cells under normal conditions or therefore asked whether TACC3 accumulates preferentially in PAX7- in the presence of thapsigargin (TG) to induce high P-eIF2α levels expressing cells. After 4-day culture of isolated satellite cells, TACC3 (Vattem and Wek, 2004). P-eIF2α-dependent translation of the remains expressed in >80% of PAX7-expressing myogenic luciferase reporter occurs in the presence of TG when uORFs in Atf4 progenitors, in 65% of MYOD-expressing progenitors and in <20% or Tacc3 are present, but not when these uORFs are eliminated by of myogenin-expressing cells undergoing differentiation (Fig. 4F,G). mutation of the start codon (Fig. 3H). Tacc3 is required for expansion of self-renewing satellite TACC3 is abundant in PAX7-expressing satellite cells, and cells ex vivo downregulated upon differentiation As sal003 upregulates TACC3 and facilitates ex vivo expansion of Next, we asked when TACC3 is expressed during the myogenic self-renewing satellite cells, we next asked whether Tacc3 is program. Low-level TACC3 expression is observed by required for satellite cell expansion and self-renewal. We examined immunoblotting cell lysates of fresh isolated satellite cells, compared satellite cells from Pax7CreERT2/+; Tacc3fl/fl mice (Murphy et al., with differentiating satellite cells that have been cultured for 4 days 2011; Yao et al., 2007, 2016), such that tamoxifen (tmx) treatment (Fig. 4A). To compare TACC3 expression in quiescent versus would result in loss of TACC3 from PAX7-expressing satellite cells.

Fig. 4. TACC3 is expressed in activated PAX7(+) satellite cells and is downregulated in differentiating myoblasts. (A) Immunoblotting lysates of fresh isolated satellite cells (D0) and after 4-day culture (D4), with antibodies against TACC3 and β-actin. Quantification of TACC3 levels, relative to β-actin levels, by densitometry of n=3 independent experiments, with representative immunoblots shown. (B) Immunofluorescence analysis with antibodies against PAX7 (green) and TACC3 (red) on fresh isolated satellite cells isolated from uninjured TA and 3-days after ctx injury (CTX 3 day). (C) Quantification of TACC3 total cell fluorescence of fresh isolated satellite cells from TA muscle, uninjured (green) and 3 days after ctx (red), in B. Background immunofluorescence revealed without anti-TACC3 antibody (−Ab) control (black). (D) Representative immunofluorescence analysis of a single EDL myofiber after 48 h culture, with antibodies against PAX7 (green) and TACC3 (red). (E) Fraction of PAX7(+) cells that are TACC3(+), on single EDL myofibers (isolated from n=3 mice), after 24 h and 48 h culture. (F) Immunofluorescence analysis with antibodies against PAX7 (upper panels), MYOD (middle panels) and myogenin (lower panels), combined with TACC3 after 4-day culture of isolated satellite cells (n=3 plates). Merged images with DAPI are shown (right panels). White asterisks indicate the presence of MYOD(+) or myogenin(+), TACC3(−) cells. (G) Fraction of TACC3(+) cells that are PAX7(+), MYOD(+) or myogenin(+) cells in F. Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed Student’s t-test). Scale bars: 20 µm in B; 10 µm in D; 20 µm in F. DEVELOPMENT

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First, satellite cells were isolated from tmx-treated Pax7CreERT2/+; cross sections of regenerating TA muscle with EdU labeling Tacc3fl/fl mice and further cultured in the presence of 4- combined with immunofluorescence with antibodies against PAX7 hydroxytamoxifen (4-OHT) to eliminate Tacc3 expression in and myogenin. TA muscle of Pax7CreERT2/+; Tacc3fl/fl mice had cultured satellite cells (Fig. 5A-C). In the absence of TACC3, numerous EdU(+) proliferating cells and differentiating PAX7 is present diffusely within the nucleus and cytoplasm myogenin(+) myogenic progenitors, but a marked decrease in (Fig. 5B,D), and low PAX7 levels are confirmed by western blotting PAX7(+) nuclei (Fig. S4). To examine specifically the satellite cell (Fig. 5E) and RT-qPCR (Fig. 5F). Satellite cells defective for Tacc3 compartment of regenerating muscle, we used MACS to isolate have a pronounced defect to expand into large myocolonies ex vivo myogenic progenitors from regenerating muscle. Although we did (Fig. 5G,H). Further analysis of myocolonies present after 4-day not observe significant differences in EdU incorporation amongst culture indicate precocious differentiation, as revealed by an the total population of isolated myogenic progenitors (Fig. 6G,H), increase in PAX7(−), MYOD(+) nuclei, and a decrease in self- the PAX7-expressing compartment of EdU(+) cells is specifically renewal, as indicated by the near absence of PAX7(+), MYOD(−) reduced in Pax7CreERT2/+; Tacc3fl/fl mice (Fig. 6G,I). Similar results nuclei representing the ‘reserve cell’ population (Fig. 5G,H). We were revealed by analysis of TA sections with antibodies against used EdU incorporation assays to compare rates of proliferation PAX7 and Ki67 (Fig. 6J,K). between wild-type Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl The inability of Tacc3-deficient satellite cells to expand in vivo satellite cells over the course of 4-day culture (Fig. 5I-K). We should lead to a reduction in the pool of myogenic progenitors observe a marked decrease in EdU incorporation in the progeny of required for efficient muscle regeneration. Although Tacc3-deficient Pax7CreERT2/+; Tacc3fl/fl satellite cells at later time points in culture satellite cells differentiate to express myogenin (Fig. 6L,M; Fig. S4), (days 3-4) (Fig. 5J), and moreover, the PAX7-expressing we observe defective regeneration 7 days after injury, illustrated by compartment of EdU(+) proliferating progeny is specifically reduced weak, disorganized expression of embryonic myosin heavy chain in Tacc3-deficient satellite cells (Fig. 5K). Next, we rescued TACC3 (embMHC), and by large nuclei-dense areas devoid of regenerating expression in satellite cells isolated from tmx-treated Pax7CreERT2/+; myofibers (Fig. 6L,M; Fig. S4). The delay in regeneration mediated Tacc3fl/fl mice with lentiviral vectors driving Tacc3 expression under by Tacc3-deficient satellite cells continues 21 days after ctx the phosphoglycerate kinase 1 (PGK) promoter (Fig. 5L). Increased injury, revealed by the presence of disorganized, smaller myofibers TACC3 levels led to more robust PAX7 expression and localization of (Fig. 6N,O). PAX7 to the nucleus (Fig. 5L-N). We further asked whether TACC3 was important for the effect of DISCUSSION sal003 in expanding satellite cells ex vivo. We isolated satellite cells P-eIF2α-dependent Tacc3 translation permits satellite cell from diaphragm and abdominal muscle from tmx-administered expansion ex vivo Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice and cultured them In this work, we set out to understand how sal003-mediated for 4 days under normal conditions or in the presence of 10 µM inhibition of eIF2α dephosphorylation enables the expansion of sal003. In the presence of sal003, wild-type Pax7CreERT2/+ satellite satellite cells and further identify important regulators of satellite cells gave rise to >80% PAX7-expressing progeny after 4-day cell behavior during the expansion phase. Genetic manipulations culture (Fig. S1A,C), as we have reported previously (Zismanov that eliminate eIF2α phosphorylation cause satellite cells to activate, et al., 2016; Lean et al., 2019). In contrast, Pax7CreERT2/+; Tacc3fl/fl enter the cell cycle and contribute to differentiation, while being satellite cells failed to expand and instead gave rise to small colonies defective for self-renewal. The presence of sal003 in culture composed of fewer PAX7-expressing cells (Fig. S1B,C). Similar conditions leads to decreased rates of proliferation (Zismanov et al., results were obtained for satellite cells isolated from hindlimb 2016), and we propose that expansion occurs because slowly muscle (Fig. S1D-F). proliferating satellite cells avoid differentiating into post-mitotic The importance of TACC3 to promote satellite cell expansion, myoblasts that undergo fusion to form the myofiber. Our data rates of proliferation and satellite cell self-renewal, while preventing supports this model because genes required for differentiated precocious differentiation, is also confirmed by siRNA knockdown skeletal muscle tissue are the most significantly downregulated in of Tacc3 (Fig. S2) and by TACC3 inhibition with the small the presence of 10 µM sal003 (Figs 1,2). compound KHS101 (Fig. S3; Wurdak et al., 2010). Here, we asked whether sal003 enables satellite cell expansion by P-eIF2α-dependent translation of specific mRNAs. Culture of Tacc3 deficient satellite cells expand poorly, leading to satellite cells in the presence of sal003 broadly leads to maintenance defects in muscle regeneration of gene expression associated with stem and progenitor cells To reveal a role for TACC3 in satellite cells in vivo, we inactivated (Figs 1,2). Amongst our candidates for P-eIF2α-dependent Tacc3 by tmx administration to Pax7CreERT2/+; Tacc3fl/fl mice. In translation, enriched genes were most commonly associated with uninjured muscle, numbers of PAX7(+) nuclei remain unchanged 5 the spindle pole, centrosome and microtubule assembly (Fig. 3; and 28 days after tmx administration, suggesting that TACC3 does Table S1), suggesting that sal003 permits the translation of mRNAs not play a role in maintaining the quiescent satellite cell pool required to maintain the fidelity of cell division. Amongst these (Fig. 6A,B). genes, we focused on Tacc3 because its 5′ UTR includes five We therefore asked whether TACC3 is required for expansion of uORFs and we demonstrate read-through of these inhibitory uORFs activated satellite cells in vivo. We used ctx to injure TA muscle of in a P-eIF2α-dependent manner. Moreover, Tacc3 depletion leads to tmx-treated Pax7CreERT2/+; Tacc3fl/fl mice (Fig. 6C). Seven days a loss of PAX7 expression in satellite cells and precocious after acute injury, we confirmed decreased numbers of TACC3(+) differentiation, whereas Tacc3 overexpression leads to increased and PAX7(+) satellite cells (Fig. 6C-E) and the decrease in PAX7 expression of PAX7 in satellite cells. It remains unclear whether the expression is confirmed by western blotting of lysates of isolated changes in PAX7 expression that we observe in relation to perturbed satellite cells (Fig. 6F). To examine defects in proliferation in TACC3 levels is indirectly associated with altering the balance of Pax7CreERT2/+; Tacc3fl/fl mice in vivo, we administered EdU to mice self-renewing versus differentiating satellite cells or mediated via after ctx injury to TA muscle. Seven days after injury, we analyzed direct mechanisms, for example interactions between PAX7 and DEVELOPMENT

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Fig. 5. TACC3 is required for satellite cell expansion and self-renewal ex vivo. (A,B) Isolation of satellite cells from tmx-treated Pax7CreERT2/+; Tacc3fl/fl mice, followed by 4-day culture in the presence of 4-OHT leads to the absence of TACC3 reported by immunoblotting of lysates with antibodies against TACC3 and β-actin (A) and immunofluorescence of satellite cell cultures with antibodies against PAX7 (green) and TACC3 (red) (B). White arrows indicate presence of TACC3(+) satellite cells that are strongly immunolabeled for PAX7. (C) Quantification of TACC3-positive nuclei in B, after 4-day culture of satellite cells isolated from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (D) Numbers of PAX7(+) nuclei in B. (E) Immunoblotting of satellite cell lysates with antibodies against PAX7 and β-actin after 4-day culture of satellite cells isolated from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (F) RT-qPCR analysis of Tacc3 and Pax7 expression after 4-day culture of satellite cells isolated from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (G) Immunofluorescence analysis with antibodies against PAX7 (green) and MYOD (red) after 4-day culture of satellite cells isolated from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. White arrows indicate PAX7(+), MYOD(−) nuclei. (H) Quantification of satellite cells undergoing self-renewal [reserve cell population, PAX7(+), MYOD(−)], activation [PAX7(+), MYOD(+)] and differentiation [PAX7(−), MYOD(+)] in G. (I) EdU incorporation (green) combined with immunofluorescence analyses of PAX7-expressing cells (red) after 3-day culture of satellite cells isolated from Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (J) Numbers of EdU(+) cells after 2, 3 and 4-day culture of satellite cells isolated from Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (K) Fraction of EdU(+) cells that express PAX7 after 2, 3 and 4-day culture of satellite cells isolated from Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (L) Immunofluorescence analysis with antibodies against PAX7 (green) and TACC3 (red) after 4-day culture of satellite cells isolated from tmx-treated Pax7CreERT2/+; Tacc3fl/fl mice and transduced with lentiviral vectors overexpressing Tacc3 ( pLenti-Tacc3). (M) Numbers of PAX7(+), TACC3(+) cells in L. (N) Numbers of PAX7(+) nuclei in L. Data are mean±s.e.m. (n=3 plates). *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed Student’s t-test). ns, not significant. Scale bars: 50 µm.

TACC3. TACC3 has been reported to regulate the activity of Satellite cell expansion and self-renewal ex vivo and in vivo transcription factors, for example TACC3 regulates subcellular requires TACC3 localization and physical association of GATA1 and FOG1 The efficiency of skeletal muscle regeneration is dependent on a

(ZFPM1) to control hematopoiesis (Garriga-Canut and Orkin, 2004). balance between satellite cells undergoing self-renewal, DEVELOPMENT

7 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480

Fig. 6. TACC3 is required for satellite cell expansion and skeletal muscle regeneration in vivo. (A) Schematic of in vivo analyses. (B) Numbers of PAX7(+) satellite cells immediately (D0) and 28 days after (D28) after tmx administration to Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice. (C) Immunofluorescence analysis with antibodies against PAX7 (green) and TACC3 (red) on transverse sections of TA muscle from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice, 7 days after ctx injury. White arrows indicate the position of PAX7(+) nuclei. (D) Quantification of numbers of TACC3(+), PAX7(+) satellite cells in C. (E) Quantification of PAX7(+) nuclei per field of view in C. (F) Immunoblotting of lysates obtained from MACS-sorted satellite cells, 7 days after ctx injury, with antibodies against PAX7 and β-actin. Quantification of PAX7 levels, relative to β-actin levels, by densitometry of n=3 independent experiments, with a representative immunoblot shown. (G) EdU incorporation (green) and PAX7 (red) immunolabeling of MACS-isolated satellite cells from TA muscle of tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice, 7 days after ctx injury. (H) Average fraction of MACS-isolated myogenic progenitors that are EdU(+) in G from n=3 mice. (I) Average fraction of EdU(+) cells that are PAX7(+) in G from n=3 mice. (J) Average number of Ki67(+) cells per field of view (FOV) on transverse sections of TA muscle of tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice (n=4), 7 days after ctx injury. (K) Average number of PAX7(+), Ki67(+) cells per field of view on transverse sections of TA muscle of tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice (n=4), 7 days after ctx injury. (L) Immunofluorescence analysis, with antibodies against embMHC (green) and myogenin (red), on transverse sections of TA muscle of tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice, 7 days after ctx injury. Merged images with DAPI (blue) are shown (right panels). White dotted line indicates nuclei-dense regions devoid of embMHC(+) myofibers. (M) Average numbers of myogenin(+) nuclei in L, from n=6 mice. (N) Immunofluorescence analysis with antibodies against laminin (red) counterstained with DAPI (blue) on transverse sections of TA muscle from tmx-treated Pax7CreERT2/+ and Pax7CreERT2/+; Tacc3fl/fl mice, 21 days after ctx injury. (O) Quantification of myofiber cross section area (CSA) in N from n=4 mice. Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (unpaired two-tailed Student’s DEVELOPMENT t-test). ns, not significant. Scale bars: 50 µm in C; 25 µm in G; 50 µm in L; 100 µm in N.

8 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480 proliferation and differentiation. Our results point to a role for isofluorane (CDMV) inhalation and 50 µl of 10 µM ctx (Sigma-Aldrich) TACC3 in permitting the expansion of PAX7(+) myogenic was injected into the TA muscle. For 5-ethynyl-2′-deoxyuridine (EdU) progenitors ex vivo and in vivo. Interestingly, the progeny of labeling (Life Technologies), mice received 200 µg EdU in 100 µl PBS by activated Tacc3-deficient satellite cells remain capable of limited intraperitoneal injections five times at 12-h intervals before analysis at day 7 proliferation, can activate the myogenic program and differentiate. after ctx injury. At indicated time points, muscle was harvested for analysis by immunofluorescence. However, the proliferating pool of PAX7-expressing myogenic progenitors is specifically depleted ex vivo and in vivo. The resulting Cell and single-fiber isolation and culture balance between self-renewal and differentiation is perturbed, Satellite cells were isolated from abdominal and diaphragm muscle of leading to defective muscle regeneration at early (7 days) and late 8-week-old Pax3GFP/+, Pax7CreERT2/+, R26tdtomato/+; Pax7CreERT2/+, (21 days) stages after acute injury. R26tdtomato; Tacc3fl/fl mice by flow cytometry (GFP or tdTomato) as Recently the regulation of stem cell polarity has emerged as an previously described (Zismanov et al., 2016) using a FACSAriaII cell sorter important mechanism facilitating stem cell expansion and (BD Biosciences), or alternatively from Pax7CreERT2/+ or Pax7CreERT2/+, differentiation. In satellite cells, an EGFR-AuroraA kinase Tacc3fl/fl mice by MACS Satellite Cell Isolation Kit, together with anti- signaling pathway orients the mitotic spindle apicobasally to Integrin α-7 MicroBeads (Miltenyi). Single myofibers were isolated by facilitate asymmetric cell divisions that are required to expand trituration of 0.5% collagenase D (Sigma-Aldrich)-treated EDL muscle of myogenic progeny needed for efficient skeletal muscle regeneration 8-week-old adult mice. Cells and single EDL myofibers were cultured in 39% DMEM, 39% F12, 20% fetal calf serum (FCS) (Life Technologies), (Wang et al., 2019). TACC3 has been identified as an EGFR 2% UltroserG (Pall Life Sciences). When indicated, culture conditions were binding partner, promoting EGFR stability at the cell surface to supplemented with 0.1% dimethylsulfoxide (DMSO control, Sigma- increase EGFR-dependent signaling pathways (Pettschnigg et al., Aldrich), 10 µM sal003 (Sigma-Aldrich), 200 nM ISRIB (Cayman), 2017). Moreover, TACC3 is amongst several substrates of AuroraA 10 µM 4-OHT (Cayman) or 2.5 µM KHS101 (Sigma-Aldrich). For kinase that influence mitotic spindle assembly (Burgess et al., siRNA experiments, satellite cells were transfected after 48 h culture with 2015). Whether TACC3 stabilizes EGFR at the membrane of siRNAs against Tacc3 (20 nM) or Mission® siRNA Universal Negative satellite cells and whether TACC3 regulates the orientation of the control (Sigma-Aldrich) with jetPRIME® transfection reagent (Illkirch- mitotic spindle required for satellite cell polarity are important Graffenstaden, France). Transfected satellite cells were cultured for an questions that require further investigation. additional 2 days. For EdU incorporation assays (Life Technologies), 10 µM In addition to the balance between satellite cell self-renewal, EdU was included in culture for 2 h or 6 h, when indicated. We cultured 293 cells in 90% DMEM, 10% FCS and supplemented when indicated with proliferation and differentiation, rates of apoptosis would impact 1 µM TG (Sigma-Aldrich). satellite cell expansion ex vivo and in vivo. Germline deletion of Tacc3 has been reported to result in high levels of apoptosis in Tacc3 overexpression hematopoietic progenitors present in the thymus by embryonic day The mouse Tacc3 sequence was amplified by PrimeSTAR® Max DNA 18.5 (Piekorz et al., 2002). In our study, we did not detect increased polymerase (TAKARA) using forward 5′-CTCCCCAGGGGGATCATG- rates of apoptosis in satellite cells in vivo 7 days after ctx injury, nor AGTCTGCATGTCTTAAAT-3′ and reverse 5′-GAGGTTGATTGTCGA- ex vivo after siRNA knockdown of Tacc3 (data not shown). These TCAGATCTTCTCCATCTTAG-3′ primers. The purified fragment was discrepancies may reveal a cell- or temporal-dependent context role cloned into BamHI-SalI site of pLenti-PGK-GFP (Crist et al., 2009) using for Tacc3 in apoptosis. Whether TACC3 protects against apoptosis In-Fusion cloning HD kit (TAKARA). HEK293T cells were transfected in conditions of increased or extended proliferative stress, for with the pLenti-PGK-Tacc3 or pLKO.1 TRC (control, gift from David Root, example during embryonic development of muscle, chronic muscle Broad Institute of MIT and Harvard, USA, Addgene plasmid #10879), degeneration or in aging satellite cells that are more prone to pMD2.G (gift from Didier Trono, École polytechnique fédérale de Lausanne, France, Addgene plasmid #12259) and psPAX2 (gift from apoptosis, requires further investigation. Didier Trono, Addgene plasmid #12260) with jetPRIME® transfection Increasing evidence points to translational control of gene reagent (Illkirch-Graffenstaden). Six hours later, the medium was changed expression as an important regulator of adult stem cell quiescence and 42 h later, supernatant was collected, filtrated through a 0.45 µm filter and self-renewal, with multiple mechanisms mediated by microRNA and concentrated using a Lenti-XTM Concentrator (TAKARA). Titers and RBPs repressing the translation of specific mRNAs. Here, we (∼1×108 infectious units/ml) were calculated by GFP analysis of transduced show increases in protein production, independent of mRNA levels, 293T cells. Satellite cells isolated by either MACS or FACS described above for a number of genes expressed in satellite cells cultured in the (1×104 cells/35 mm dish or a well of a 6-well plate) were transduced with presence of sal003. We demonstrate P-eIF2α-dependent translation 4 µl of lentivirus solution with polybrene (5 µg/ml). Twenty-four hours after of mRNA for Tacc3 and show a requirement for Tacc3 expression in transduction, the lentivirus-containing medium was carefully removed and expanding satellite cells ex vivo and in vivo. Our findings suggest an replaced with fresh satellite cell medium. Transduced satellite cells were cultured for an additional 3 days. additional role for P-eIF2α-dependent translation of mRNA in the maintenance of adult stem cell populations and outside the context of Luciferase assay the ISR. The Atf4- and Tacc3-luciferase constructs were made by cloning gBlock gene fragments (Integrated DNA Technologies) corresponding to 5′ UTRs MATERIALS AND METHODS of Mus musculus Atf4 and Tacc3, fused to the 5′ end of the firefly luciferase Mice ( fluc) gene up to the NarI restriction enzyme site. Mutant versions of Atf4 Animal care practices were in accordance with the federal Canadian Council and Tacc3 5′ UTRs were designed with each ATG start codon of uORFs on Animal Care, as practiced by McGill University. All mice were deleted. These 5′ UTR gene fragments were cloned into HindIII, NarI sites maintained on a C57/Bl6 background. Tacc3fl/fl mice were kindly provided upstream of the fluc gene in pGL3-promoter plasmids, thereby maintaining by R. Yao (Yao et al., 2016). Pax7CreERT2/+ (Murphy et al., 2011) and the final overlapping uORF with the main ORF for fluc (Promega). HEK293 Rosa26tdTomato (Madisen et al., 2009) mice were obtained from Jackson cells were plated in 24-well plates at a density of 25,000 cells/well and Laboratories. Tmx (Cayman Chemical) was administered in corn oil, 30% incubated overnight. Fluc reporter plasmids and the pRL-TK Renilla ethanol by intraperitoneal injections (2.5 mg/day) for 5 days and, when luciferase (Rluc) transfection control plasmid (Promega) were co-transfected indicated, mice were maintained on a tmx diet (80 mg/kg body weight/day, into these cells using jetPRIME® transfection reagent (Illkirch-

Envigo). For muscle regeneration, 8-week-old mice were anesthetized by Graffenstaden). Cells were incubated overnight in the presence or absence DEVELOPMENT

9 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480 of 1 µM TG (Sigma-Aldrich). Cells were then lysed and the Fluc/Rluc ratio consensus transcription factor binding sites, ENCODE histone was determined using the Promega Dual-Luciferase Reporter kit. modifications, mouse tissue expression, GO Biological Process and GO Cellular Component. Venny2.1.0 was used to generate a list of genes Immunodetection upregulated at the protein level (log2>1), without a corresponding increase Cultured satellite cells were fixed in 4% paraformaldehyde (PFA), in mRNA expression (log2<1). permeabilized with 0.2% Triton, 50 mM NH4Cl and blocked in 5% horse serum (HS). Single EDL myofibers were fixed with 4% PFA, permeabilized RNA-seq analysis with 0.1% Triton in PBS and blocked in 5% HS with 0.1% Triton in PBS. Trimmomatic v0.32 (Bolger et al., 2014) was used to trim sequencing reads, TA muscles were fixed for 2 h in 0.5% PFA at 4°C and equilibrated including adaptors and other Illumina-specific sequences, the first four overnight in 20% sucrose at 4°C. Tissues were mounted in Frozen Section bases from the start of each read and low quality bases identified using a 4 bp Compound (VWR) and flash frozen in a liquid nitrogen-cooled isopentane sliding window where quality fell below 30 (phred33<30). Finally, reads bath. Transverse sections (10 µm) were permeabilized with 0.1% Triton, shorter than 30 base pairs were removed. Cleaned reads were aligned to the 0.1 M Glycine in PBS, and blocked in M.O.M. reagent (Vector mouse reference genome build mm10 using STAR v2.3.0e (Dobin et al., Laboratories). For immunoblotting, cell lysates were obtained in RIPA 2013) with default settings. Reads mapping to more than 10 locations in the buffer (Thermo Fisher Scientific) supplemented with Complete protease genome (MAPQ<1) were discarded. Gene expression levels were estimated inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Sigma- by quantifying uniquely mapped reads to exonic regions (the maximal Aldrich). genomic locus of each gene and its known isoforms) using featureCounts Primary antibodies were against PAX7 [Developmental Studies (v1.4.4) (Liao et al., 2014) and the Ensembl gene annotation set. Hybridoma Bank (DSHB) 1:100], MYOD (Santa Cruz Biotechnology, Normalization (mean of ratios) and variance-stabilized transformation of sc-304 and sc-377460, 1:300), TACC3 (Abcam, 134154, 1:200), Ki67 (BD the data were performed using DESeq2 (v1.14.1) (Love et al., 2014). Biosciences, B56, 1:300), laminin (Sigma-Aldrich, L9393, 1:500), Multiple control metrics were obtained using FASTQC (v0.11.2), samtools myogenin (Abcam, 124800, 1:200), myogenin (Santa Cruz (v0.1.20) (Li et al., 2009) BEDtools (v2.17.0) (Quinlan and Hall, 2010) and Biotechnology, sc-12732, 1:200), embMHC (DSHB, F1.652, 1:100) and custom scripts. For visualization, normalized Bigwig tracks were generated β-actin (Sigma-Aldrich, A5441, 1:2000). Alexa Fluor-488, Alexa Fluor-594 using BEDtools and UCSC tools. PCA was carried out using the 1000 most and Alexa Fluor-647 conjugated secondary anti-mouse IgG1, anti-mouse variant genes. IgG and IgG2b or anti-rabbit antibodies (Life Technologies, A21121, A21207, A21145 and A31573, 1:500) were used for immunofluorescence. Statistical analysis Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit Graphs are presented as mean±s.e.m., as indicated in figure legends. Unless secondary antibodies (Jackson ImmunoResearch, 115-035-003 and 111- otherwise indicated, three independent replicates of each experiment were 035-003, 1:2000) were used with the ECL Prime Western Blotting performed. Significance was calculated by unpaired Student’s t-tests with Detection reagents (GE Healthcare). Densitometry of immunoblots and two-tailed P values: *P<0.05, **P<0.01, ***P<0.001. total cell fluorescence analyses were performed using ImageJ. Acknowledgements RNA analysis We thank C. Young for assistance with flow cytometry, C. Borchers and D. Smith for RNA was isolated from cells with TRIzol reagent (Life Technologies) and assistance with mass spectrometry. R. Yao generously provided Tacc3fl/fl mice. Data treated with DNase (Roche) before reverse transcription with iScript reverse analyses were enabled by computer and storage resources provided by Compute transcription supermix (Bio-Rad). RT-PCR primers were: Pax7 forward 5′- Canada and Calcul Québec. AGGCCTTCGAGAGGACCCAC-3′, reverse 5′-CTGAACCAGACCTG- GACGCG-3′; myogenin forward 5′-CAACCAGGAGGAGCGCGATCT- Competing interests CCG-3′, reverse 5′-AGGCGCTGTGGGAGTTGCATTCACT-3′; Tacc3 The authors declare no competing or financial interests. forward 5′-GAGCTTCAGAGACCCATCAGA-3′, reverse 5′-AGTTGG- AGAGATGGGACGAG-3′; Actb forward 5′-AAACATCCCCCAAAGT- Author contributions TCTAC-3′, reverse 5′-GAGGGACTTCCTGTAACCACT-3′. Levels of Conceptualization: C.C.; Methodology: R.F., S.J., G.L., C.L.K., C.C.; Software: S.H., mRNA were measured using SYBR Green on a 7500 Fast Real Time C.L.K.; Validation: C.C.; Formal analysis: R.F., S.J., G.L., S.H., C.L.K.; Investigation: PCR System (Applied Biosystems). R.F., S.J., G.L., H.C.M.C., S.H., C.L.K.; Data curation: S.H., C.L.K.; Writing - original draft: C.C.; Writing - review & editing: R.F., S.J., G.L., H.C.M.C., C.C.; Visualization: R.F., S.J., G.L., H.C.M.C., C.C.; Supervision: C.L.K., C.C.; Project administration: RNA-seq, mass spectrometry and bioinformatic analysis C.C.; Funding acquisition: C.C. For both RNA and protein analysis, satellite cells were isolated from GFP/+ Pax3 adult mice and seeded at 7500 cells per 35 mm plate and Funding subsequently cultured in 10 µM sal003 (n=4) or DMSO (n=4) for 4 days. C.C. and coworkers are funded by the Canadian Institutes of Health Research For RNA analysis, total RNA was isolated from satellite cell cultures using (CIHR; 399258), the Stem Cell Network, the Fonds de Recherche du Québec – the RNeasy® Micro Kit (Qiagen). The RiboGone™-mammalian (Takara Santé(FRQS) and the Richard and Edith Strauss Foundation. R.F. is funded by a Bio) kit was subsequently used to eliminate ribosomal and mitochondrial Japan Society for the Promotion of Science Overseas Research Fellowship, the RNA. cDNA libraries for RNA-seq were then prepared using the Uehara Memorial Foundation, the Mochida Memorial Foundation for Medical and SMARTer® Stranded RNA-Seq kit (Takara Bio). With all kits, the Pharmaceutical Research, and a Ministry of Education, Culture, Sports, Science and manufacturer’s protocols were followed. The resulting cDNA libraries Technology Leading Initiative for Excellent Young Researchers grant. C.L.K. is supported by the CIHR (156086) and the FRQS. were sequenced with the Illumina MiSeq system (Genome Quebec). For protein analysis, the TMTsixplex™ Isobaric Tag Kit (Thermo Fisher Data availability Scientific) was used to isolate and label peptides from satellite cells cultured RNA-sequencing data have been deposited in GEO under accession number in 10 µM sal003 (n=12 plates) or DMSO (n=12 plates), per the GSE164774. manufacturer’s protocol. After labeling peptides with unique isobaric tags, the peptide solutions were pooled and analyzed via high pH reversed Supplementary information phase fractionation and liquid chromatography-mass spectrometry. All data Supplementary information available online at were acquired with Thermo Orbitrap Fusion™ Tribrid™ 2.1 software https://dev.biologists.org/lookup/doi/10.1242/dev.194480.supplemental (Thermo Fisher Scientific), analyzed using Proteome Discoverer 1.4 (Thermo Fisher Scientific) and MASCOT v2.4 software (Matrix Science). Peer review history Raw data files were searched against a Uniprot Mouse database. Gene lists The peer review history is available online at were examined using Enrichr (Chen et al., 2013) for ENCODE and ChEA https://dev.biologists.org/lookup/doi/10.1242/dev.194480.reviewer-comments.pdf DEVELOPMENT

10 STEM CELLS AND REGENERATION Development (2021) 148, dev194480. doi:10.1242/dev.194480

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