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Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse

Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse Background: Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. Results: Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the ntdTom endogenous Myogenin locus. Expression of tdTOMATO in Myog mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN myoblasts. Conclusions: We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts. Keywords: Myogenin, Knock-in mouse, tdTOMATO, Intravital imaging, Skeletal muscle Background single MRF knockout mice, only Myog-null homozygous Embryonic and postnatal myogenesis and adult muscle animals die at birth due to severe skeletal muscle defects regeneration are regulated by a family of basic helix- [1–3]. Thus, unlike the other myogenic bHLH factors, loop-helix myogenic regulatory factors (MRFs) compris- Myog has no redundant or compensatory mechanisms to ing Myf5, Mrf4, Myod and Myogenin (Myog). Following replace its function during development. Myoblasts lack- myogenic specification in the embryo, the MRFs are ing this gene accumulate in the muscle-forming areas expressed in a sequential manner to ensure commit- throughout the body and fail to form normal myofibers ment, proliferation, differentiation and fusion to give rise in vivo, pointing to its critical role in terminal differenti- to multinucleated skeletal myofibres. Single and com- ation of myoblasts [2–4]. While Myog-null embryos have binatorial mouse knockout models of the MRFs have some disorganised residual primary fibres, major differ- established a genetic hierarchy where Myf5, Mrf4 and ences between mutant and wild-type embryos become Myod control lineage commitment and proliferation of apparent during the initiation of secondary myofibre for- myogenic progenitors, and Myod, Mrf4 and Myog regu- mation [2, 4]. Unexpectedly, conditional ablation of late in terminal differentiation [1]. Notably, amongst the Myog during the perinatal and postnatal period does not result in noticeable defects in muscle morphology or −/− histology, suggesting that Myog myoblasts can still * Correspondence: shahragim.tajbakhsh@pasteur.fr Stem Cells & Development Unit, Institut Pasteur, 25 rue du Dr. Roux, 75015 contribute to muscle growth [5, 6]. Additionally, condi- Paris, France tional ablation of Myog in a Duchenne muscular dys- UMR CNRS 3738, Institut Pasteur, Paris, France trophy mouse model (mdx [7]) did not result in an Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 2 of 13 adverse phenotype, confirming that Myog is dispensable Generation of the Myog-ntdTomato construct for CRISPR- for adult muscle regeneration in this disease [8]. Never- Cas9-mediated homologous recombination theless, although Myog-null muscle stem cells (MuSCs) A fragment of 1000 bp from the last exon of Myog was proliferate and differentiate in culture as efficiently as amplified by PCR from murine gDNA (primers 1 and 2, wild-type cells, the muscle gene expression program is Supplementary Table 1), introducing SalI and NotI re- profoundly altered in the absence of Myog [6]. striction sites. This fragment was subcloned into the Adult muscle regeneration depends on MuSCs, char- donor plasmid encoding for tdTOM (kind gift from Dr. acterised by the expression of Pax7 [9–13]. Upon muscle Festuccia, Institut Pasteur). A fragment of 760 bp from injury, MuSCs activate the expression of Myod, prolifer- the 3′UTR of the Myog gene just after the STOP codon ate to generate myoblasts that differentiate and fuse to was amplified by PCR from murine gDNA (primers 3 form myofibres. Different reporter mouse lines have and 4). This amplification also introduced a mutation in been generated to fluorescently label the Pax7 muscle the PAM sequence necessary for CRISPR-Cas9 genome progenitor population, either from the endogenous locus editing. Using the PacI and SpeI restriction sites added, [14–16] or as transgenes [15, 17, 18], thereby allowing the fragment was subcloned into the PacI and XbaI imaging and isolation of Pax7-expressing cells. Addition- digested tdTOM plasmid. Oligos containing a T2A ally, inducible reporters in which expression of the Cre (primers 5 and 6) [36] peptide and a triple NLS sequence recombinase under the control of the Pax7 promoter from SV40 large T [37] were annealed and subcloned recombines a membrane or cytoplasmic fluorophore [13, into a blunt pBluescript SK (+) plasmid. This plasmid 19, 20] have been used for permanent marking of the was subsequently digested with NotI and KpnI and the myogenic lineage [21–23] and for live imaging [24]. Al- T2A-NLS fragment was cloned into the tdTOM plasmid. though several reporter mouse lines have been generated tdTOM was amplified by PCR from the initial plasmid to identify differentiating myoblasts based on the expres- (primers 7 and 8) adding KpnI and FseI sites and sub- sion of Myosin light chain [25], Myog [26–28] and cloned into the donor vector after the 3xNLS sequence. Muscle creatine kinase [29], they are based on lacZ (β- An FNF cassette containing two FRT sites and the galactosidase activity, [30]) or cat (chloramphenicol ace- NeoR/KanR gene under the control of the PGK pro- tyltransferase,[31]) expression and thus only allow end- moter was amplified by PCR (primers 9 and 10) adding point measurements on fixed samples. FseI and PacI sites. This fragment was subcloned into Terminal myoblast differentiation is characterised by the donor vector. the expression of Myog and the cyclin-dependent kinase The highest scoring sgRNA sequence to target the inhibitor p21 and cell cycle withdrawal [32–34]. Experi- Myog STOP codon region was determined using a guide ments using the nucleotide analogue BrdU have shown design tool (crispr.mit.edu, Zhang Lab). Primers contain- that MYOG-positive cells can undergo DNA replication ing this targeting sequence (primers 11 and 12) were [32], but it is still unclear how many divisions they can annealed and subcloned into the pU6-(BbsI) CBh-Cas9- execute before definitively leaving the cell cycle. T2A-mCherry vector (Addgene #64324) digested with Here, we took advantage of the CRISPR/Cas9 sys- BbsI. tem, which allows precise genome editing [35], to generate a knock-in mouse line expressing a nuclear Targeting of mouse embryonic stem cells localised tandem-dimer Tomato (tdTOM) protein The Myog-ntdTOM donor construct (linearised by PvuI under the control of the endogenous Myog promoter, digestion) and the pU6 vector were electroporated in while retaining expression of MYOG protein. We C57BL/6J mouse embryonic stem cells. Following G418 ntdTom show that heterozygous Myog mice exhibit ro- (300 μg/ml) selection, positive clones were determined bust reporter gene expression in fixed and live myo- by PCR using primers 13, 14 and 15 (Supplementary genic cells thus allowing in vitro and intravital Table 1), yielding a 1.7 kb band for the WT and 1.2 kb microscopy studies of the dynamics of muscle differ- for the mutant. Two positive clones were expanded and entiation and cell cycle withdrawal. 8–10 embryonic stem cells were injected into BALB/c blastocysts to generate chimeric mice in the Mouse Gen- Materials and methods etics Engineering Facility at the Institut Pasteur. Germ- Mouse maintenance line transmission was verified by PCR and F1 mice were Animals were handled according to national and Euro- crossed to Tg(ACTFLPe)9205Dym [38] animals to excise pean Community guidelines and an ethics committee of the FNF cassette. Excision of the FNF cassette and pres- ntdTom the Institut Pasteur (CETEA, Comité d’Ethique en Ex- ence of the Myog allele was verified by PCR using périmentation Animale) in France approved protocols primers 16, 17 and 18 (Supplementary Table 1) (Flp- ntdTom (Licence 2015-0008). Except when indicated otherwise, recombined Myog allele, 236 bp and WT allele males and females of 2–4 months were used. 600 bp, Figure S1), and these primers were subsequently Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 3 of 13 used for genotyping. F2 animals were backcrossed to incubated with 1 μg/ml Hoechst 33342 for 5 min at room C57BL/6 animals to eliminate the Tg(ACTFLPe) allele, temperature to visualise nuclei, washed three times in PBS ntdTom/+ and Myog animals were selected for further and mounted in 70% glycerol in PBS for imaging. characterisation. For whole-mount immunofluorescence, embryos were collected in PBS and fixed in 4% PFA 0.1% Triton X-100 Isolation and culture of MuSCs for 2 h at 4 °C. After two PBS washes, samples were Foetal and adult muscles were dissected and minced in dehydrated in 50% methanol in PBS and kept in 100% ice-cold DMEM as described in [39]. Samples were then methanol at − 20 °C until used. Samples were rehydrated incubated in DMEM, 0.08% Collagenase D (Sigma, in PBS and incubated in blocking buffer (15% goat 11088882001), 0.2% Trypsin (ThermoFisher, 15090) and serum, 1.5% BSA, 0.5% TritonX-100 in 1X PBS) for 1 h 10 μg/ml of DNAseI (Sigma, 11284932) for 25 min at at RT in 2-ml Eppendorf tubes. Embryos were then in- 37 °C under gentle agitation for 5 rounds of digestion. cubated with primary antibodies in the blocking buffer After each round, samples were allowed to sediment for for 5–7 days at 4 °C with rocking. Embryos were washed 5 min, the supernatant was collected in 4 ml of foetal bo- extensively for 2–4 h in PBST and incubated in Fab’ sec- vine serum (FBS) on ice and fresh digestion buffer was ondary antibodies for 2 days at 4 °C with rocking. Em- added to the remaining muscle pellet. The collected su- bryos were washed as above, dehydrated in 50% pernatants were centrifuged for 15 min at 550g at 4 °C, methanol in PBS, twice in 100% methanol and then resuspended in DMEM 2% FBS, and filtered through a cleared with BABB and mounted for imaging [40]. 40-μm strainer (Corning, 352235) before cell sorting. Cells were isolated based on size, granulosity and GFP Adult muscle injury, histology and immunofluorescence or tdTOM fluorescence using an Aria III (BD Biosci- Muscle injury was done as described previously [22]. ences) flow cytometer. Cells were collected directly in Mice were anaesthetised with 0.5% Imalgene/2% Rom- MuSC growth media (38.5% DMEM (Fisher Scientific, pun, and the TA muscle was injected with 50 ml of car- 31966047), 38.5% F12 (Fisher Scientific, 31765035), 20% diotoxin (10 mM; Latoxan, L8102) diluted in 0.9% NaCl. FBS (ThermoFisher, 10270), 2% Ultroser (Pall, 15950- Injured TA muscles were fixed upon harvesting in 4% 017), 1% penicillin/streptomycin (GIBCO, 15140-122)). PFA for 2 h at 4 °C, washed with PBS and equilibrated Matrigel® (1 mg/ml, Corning, 354248) coated dishes with 30% sucrose in PBS overnight. Samples were (30 min at 37 °C) were used to culture MuSCs in growth mounted in OCT tissue freezing media and cryosec- media at 3% O ,5%CO , 37 °C for the indicated times. tioned between 8 and 12 μm. When endogenous tdTOM 2 2 For immunostaining, cells were fixed in 4% parafor- was scored, cryosections were rehydrated in PBS and maldehyde (PFA, Electron Microscopy Sciences, 15710) counterstained with Hoechst 33342. in PBS for 15 min at room temperature (RT), permeabi- In case of MYOG plus tdTOM detection, tissue sec- lised in 0.5% Triton X-100 (Merck, T8787) for 5 min at tions were processed for histology as described [41]. RT and blocked with 10% goat serum (GIBCO). Cells Briefly, sections were post-fixed in 4% PFA for 10 min at were incubated with the indicated primary antibodies in RT and washed with PBS prior to immunostaining. PBS 2% goat serum buffer overnight following by 45- Heat-induced epitope retrieval was performed in a cit- min incubation with secondary antibodies and 1 μg/ml rate solution pH 6.0 during 6 min in a pressure cooker. Hoechst (ThermoFisher, H1399). Sections were then incubated with 30% H O for 5 min 2 2 at RT. Samples were then permeabilised with 0.2% Embryo immunofluorescence Triton-X100, washed in PBS and blocked in blocking For tissue immunofluorescence, embryos were collected buffer (15% goat serum, 1.5% BSA, 0.5% TritonX-100 in in PBS and fixed in 4%PFA 0.1% Triton X-100 in PBS for 1X PBS). Primary antibodies against MYOG and DsRED 2 h at 4 °C. After 3 PBS washes, embryos were cryopre- (recognising tdTOM) were incubated overnight at 4 °C. served in 30% sucrose in PBS and embedded in OCT tis- After washing with PBST, sections were incubated with sue freezing media (Leica, 14020108926) for appropriate secondary antibodies and 1 μg/ml Hoechst cryosectioning. Cryosections were allowed to dry for 30 33342 in blocking buffer for 45 min at RT (Table 1). min at room temperature and washed once with PBS. Tis- sue samples were blocked in 3% BSA, 10% goat serum, Western blot 0.5% Triton X-100 for 1 h at room temperature. Primary Embryos were collected in ice-cold PBS and subse- antibodies were diluted in blocking solution and incubated quently snap frozen in dry ice. Embryonic tissues were overnight at 4 °C. After three washes with PBST (PBS ground to a fine powder using a mortar and pestle on 0.1% Tween20 (Sigma Aldrich, P1379)), secondary anti- dry ice and lysed in RIPA buffer (150 M NaCl, 50 mM bodies were diluted in blocking solution and incubated for Tris pH 8, 5 mM EDTA, 1% NP-40 (Sigma, I8896), 0.5% 45 min at room temperature. Finally, samples were sodium deoxycholate, 0.1% SDS supplemented with 1X Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 4 of 13 Table 1 Antibodies used for immunostaining Species Concentration Reference Primary antibodies Myog Mouse monoclonal (IgG1) 1:20 DHSB, clone F5D, supernatant Living colors® Ds-Red Rabbit polyclonal 1:500 Takara Bio, 632496 Myosin heavy chain (MyHC) Mouse monoclonal 1:30 DHSB, MF-20, supernatant (IgG2b) Secondary antibodies Alexa Fluor®488 AffiniPure Goat Anti-Mouse IgG1(γ1) Goat 1:500 Jackson ImmunoResearch Labs, 115-545- Alexa Fluor® 555 F(ab')2 Fragment of Goat Anti-Rabbit Goat 1:500 Thermo Fisher Scientific, A-21430 IgG Alexa Fluor® 633 Goat Anti-Mouse IgG2b (γ2b) Goat 1:500 Thermo Fisher Scientific, A-21146 protease (Sigma, S8820) and phosphatase inhibi- In vitro videomicroscopy tors (Roche, 4906845001)). Fifteen microgrammes of MuSCs were plated on a microscopy culture chamber protein extracts were run on a 4–12% Bis-Tris Gel (IBIDI, 80826) and cultured in growth media supple- NuPAGE (Invitrogen, NP0322) and transferred on a mented as above. The plate was incubated at 37 °C, 5% PVDF Amersham Hybond-P transfer membrane (GE CO and 3% O in a Pecon incubation chamber. A Zeiss 2 2 Healthcare, RPN303F). The membrane was blocked with Observer.Z1 connected to a Plan-Apochromat 20x/0.8 5% milk (Dominique Dutscher, 711160) in Tris-Buffer M27 objective and Hamamatsu Orca Flash 4 camera Saline 0.2% Tween (Sigma, P9416) (TBS-T) for 1 h at piloted with Zen software (Carl Zeiss) was used. room temperature and probed with specific primary antibodies overnight at 4 °C. After three washes in TBS- Static imaging T, the membrane was incubated with HRP or The following systems were used for image acquisition: fluorophore-conjugated secondary antibodies and re- Zeiss SteREO Discovery V20 for macroscopic observa- vealed by chemiluminescence (Pierce ECL2 western tions of whole embryos and Zeiss LSM800 or LSM700 blotting substrate, Thermo Scientific, 80196) or fluores- laser-scanning confocal microscopes for tissue sections cence (Bio-Rad, Chemidoc MP) (Table 2). and whole-mount immunostaining of cleared embryos. End point in vitro culture samples were imaged with a Zeiss Observer.Z1. RNA extraction RNA from cells isolated by FACS was extracted using a Trizol-based kit (Zymo Research, R2061) and reverse Intravital microscopy CreERT2 YFP ntdTom transcribed using SuperScriptIII (Invitrogen, 18080093). Intravital imaging of Pax7 ; R26 ; Myog RT-qPCR to assess for mRNA relative expression was mice at different timepoints during regeneration was performed with SYBR green master mix (Roche, performed on an upright Nikon NiE A1R MP micro- 04913914001) in Applied biosciences machine. Data scope piloted with NIS software (Nikon). The micro- −ΔΔCT analysis was performed using the 2 method [42] scope was equipped with a × 25 NA 1.1 PlanApo and mRNA expression was normalised with LambdaS objective, GaAsP PMT detectors and a Rpl13 (primers 23 and 24, Supplementary Table 1). Spectra-Physics Insight Deepsee laser. Laser frequency Table 2 Antibodies used for Western blot Species Concentration Reference Primary antibodies Myog Mouse monoclonal (IgG1) 1:500 DHSB, clone F5D, supernatant Living colors® Ds-Red Rabbit polyclonal 1:1000 Takara Bio, 632496 Secondary antibodies Mouse-HRP Goat polyclonal 1:5000 Pierce, 31430 Rabbit-HRP Goat polyclonal 1:5000 Pierce, 31460 GAPDH-Rhodamine Human IgG Fab fragment 1:5000 Bio-Rad, 12004168 Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 5 of 13 Myog E3 tdTOM Neo Myog 3’ UTR Donor construct 5’UTR E1 E2 E3 3’UTR Myog locus CRISPR-Cas9 mediated recombination ntdTom-FNF 5’UTR E1 E2 E3 tdTOM Neo 3’UTR Myog locus Flippase mediated excision ntdTom Myog locus 5’UTR E1 E2 E3 tdTOM 3’UTR E9.5 E10.5 E11.5 C D ntdTom/+ ntdTom/ntdTom WT Myog Myog 5’UTR E1 E2 E3 tdTOM 3’UTR tdTOM MYOG GAPDH Total Myog Myog WT Myog tdTOM *** ** 2.5 Quantification ** * ** tdTOM MYOG 2.0 Genotype 5 1.5 *** WT Genotype Het WT Homo 1.0 3 Het 2 Homo 0.5 0.0 Fig. 1 (See legend on next page.) T2A 3xNLS FRT FRT Stop T2A 3xNLS T2A FRT 3xNLS FRT FRT T2A 3xNLS FRT WT Het Homo WT Het Homo WT Het Homo WT Het Homo WT Het Homo 2^−ddCT Expression normalized to GAPDH Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 6 of 13 (See figure on previous page.) Fig. 1 Generation of a Myog knock-in mouse line. a Scheme depicting the endogenous Myog locus, the donor construct and the result of the ntdTom-FNF CRISPR-Cas9-mediated recombination in mouse embryonic stem cells. First-generation Myog mice were then crossed with a Tg(ACTF +/+ ntdTom/+ LPe) deleter strain to excise the FNF cassette. b Endogenous fluorescence from Myog embryos at different stages. An overlay between the brightfield and fluorescent images is shown. Scale bar, 1000 μm. c Scheme showing the primer pairs amplifying the wild-type allele (2), the ntdTom allele (3) and both alleles (1) in the targeted Myog locus. RT-qPCR analysis of the levels of total Myog mRNA, the wild-type allele and the +/+ ntdTom/+ ntdTom/ntdTom tdTom allele specifically from E14.5 Myog , Myog and Myog embryos. n = 4 embryos per genotype. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; ***p value < 0.005, **p value = 0.0005 to 0.01, *p value = 0.01 to 0.05. d Western blot assessing the levels +/+ ntdTom/+ ntdTom/ntdTom of MYOG and tdTOM proteins from E14.5 Myog , Myog and Myog embryos (n = 4 embryos per genotype). Bar graph shows the quantification of protein expression levels normalised to GAPDH. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; ***p value < 0.005, *p value = 0.01 to 0.05 was tuned to 960 nm to allow simultaneous excitation of Myog transcripts [47] (Fig. 1b), with expression levels be- YFP and tdTOM fluorophores. ing lower in the caudal (more recently formed) somites. For image acquisition, the skin over the upper hind- We then collected tissue samples from E14.5 foetuses limb was shaved and incised to expose approximately 1 and performed RT-qPCR and Western blot analysis to cm of the muscle and imaged directly. During the im- confirm that Myog mRNA and protein levels were simi- aging period, mice were anaesthetised with 1.5% iso- lar in wild-type, heterozygous and homozygous animals. fluorane and maintained in an incubation chamber at Primer pairs were designed to amplify specifically the 37 °C. wild-type allele or the tdTom allele, and one primer set amplified both (Fig. 1c). This analysis showed that Myog heterozygous and homozygous knock-in (KI) embryos Image analysis expressed similar levels of total Myog mRNA, and con- Cell tracking was performed using the Manual Tracking firmed that no Myog wild-type transcript could be de- feature of the TrackMate plug-in [43] in Fiji [44]. ZEN tected in the homozygous embryos. As expected, the software (Carl Zeiss), Fiji [44] and Imaris (Bitplane) were ntdTom levels of Myog mRNA were the highest in homozy- used for image analysis. Figures were assembled in gous samples, decreased to roughly 50% in the case of Adobe Photoshop and Illustrator (Adobe Systems). the heterozygous and were not detected in wild-type em- bryos (Fig. 1c). At the protein level, we noted similar ex- Data analysis and statistics pression levels of MYOG in embryos from all three Data analysis and statistics were performed using R [45], genotypes, whereas the tdTOM protein was absent in and figures were produced using the package ggplot2 wild-type samples (Fig. 1d). Therefore, we conclude that [46]. For comparison between two groups, two-tailed MYOG protein was generated from transcripts that orig- paired and unpaired Student’s t tests were performed to inated from both alleles. calculate p values and to determine statistically signifi- To investigate the expression of the targeted allele cant differences (see figure legends). with higher resolution, we assessed the temporal ex- pression dynamics of MYOG and tdTOM proteins by Results whole-mount immunostaining at E10.5 and compared ntdTom Generation and characterisation of a Myog mouse the expression of MYOG and tdTOM in wild-type, Using the CRISPR-Cas9 system, a sgRNA was designed heterozygous and homozygous embryos (Fig. 2a, Add- to target the region of the STOP codon of the Myog itional files 1, 2, 3). We confirmed that tdTOM gene for homologous recombination. The recombination followed the expression pattern of MYOG in the ep- template consisted of two homology arms corresponding axial and hypaxial domains of all somites, indicating to Myog sequences flanking the STOP codon, a tdTom that both proteins have similar spatiotemporal expres- coding sequence and a Neo resistance cassette flanked sion dynamics. To assess the co-expression of MYOG by frt sites (Fig. 1a). The tdTOM protein was preceded and tdTOM at the single cell level in heterozygous by a T2A peptide sequence [36] to allow cleavage from embryos, cryosections at the level of extraocular, the MYOG protein following translation, and a triple tongue and limb muscles were examined during pri- NLS sequence [37] to ensure nuclear localisation. mary (E12.5) and secondary (E14.5) myogenesis when First, we evaluated the endogenous tdTOM fluores- small oligo-nucleated and larger multi-nucleated myo- ntdTom/+ cence in heterozygous Myog embryos between fibres are generated, respectively. Quantification of E9.75 and E11.5 at the level of the somites, i.e. transient protein expression in different muscles confirmed an embryonic structures arising from the segmentation of average co-localisation of both proteins in 97% and the paraxial mesoderm. Endogenous tdTOM fluores- 95% of the cells at E12.5 (Fig. 2b) and E14.5 (Figure cence followed a similar pattern to that described for S2A), respectively. Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 7 of 13 tdTom/tdTom tdTom/+ Myog Myog WT E10.5 st st st interlimb 1 somites interlimb 1 somites interlimb 1 somites Quantification E12.5 - + MYOG tdTOM + - MYOG tdTOM + + MYOG tdTOM HOECHST MYOG tdTOM MyHC Tg:Pax7-nGFP; Quantification ntdTom/+ D0 D5 Myog MuSC isolation Fix + Stain + Plating - + MYOG tdTOM + - MYOG tdTOM + + MYOG tdTOM HOECHST MYOG tdTOM Limb Fig. 2 (See legend on next page.) EOM MAS Tongue Back Limb Limb Tongue EOM MYOG tdTOM MyHC MYOG tdTOM % cells % cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 8 of 13 (See figure on previous page.) +/+ ntdTom/+ Fig. 2 tdTOM expression recapitulates endogenous Myog expression. a Whole-mount immunofluorescence from Myog , Myog and ntdTom/ntdTom Myog embryos at E10.5 stained for tdTOM, MYOG and MyHC proteins. Scale bar, 300 μm on upper panels and 100 μm on lower ntdTom/+ insets. b Immunofluorescence of extraocular (EOM), tongue and limb muscle cryosections of Myog embryos at E12.5. The bar graph shows the quantification of tdTOM- and MYOG-positive cells. n = 3 embryos, > 100 cells per muscle and per embryo were counted. Scale bar, 40 μm. c ntdTom MuSCs were isolated from limb muscles of Tg:Pax7-nGFP; Myog adult animals and plated for in vitro differentiation for 5 days. Bar graph shows the quantification of tdTOM- and MYOG-positive cells (n = 3, > 200 cells per animal counted). Scale bar, 100 μm on the left image, 50 μm on the right inset To assess the fidelity of the reporter mouse in adult performed an injury of the Tibialis anterior (TA) muscle ntdTom ntdTom/+ myoblasts, Myog animals were crossed with Tg: of Tg:Pax7-nGFP; Myog mice by intramuscular Pax7-nGFP mice, where GFP marks all MuSCs [15]. injection of the snake venom toxin cardiotoxin [49]. We MuSCs were isolated by FACS based on GFP expression, next performed FACS analysis to determine whether the then differentiated in vitro for 5 days. In agreement with tdTOM mononucleated fraction could be isolated fol- our results in the embryo, MYOG and tdTOM expres- lowing tissue injury. As expected, only a few tdTOM sion co-localised in about 95% of the cells (Fig. 2c). Add- cells were detected at 3 days post-injury, when myogenic itionally, no significant differences were observed in total cells are known to be maximally proliferating [23, 24, Myog RNA levels between wild-type, heterozygous and 50] (Fig. 3b). tdTOM cells were most abundant at 5 homozygous animals (Figure S2B). and 10 days post-injury, corresponding to the increased As indicated above, the KI strategy was designed to be shift towards differentiation of the transiently amplifying non-disruptive and allow normal MYOG protein expres- myoblast population during this period. As the major sion from the recombined alleles. Given that Myog-null features of the regeneration process are completed by 3– ntdTom/ntdTom + mice are lethal at birth, and our Myog 4 weeks, the proportion of tdTOM cells decreased by knock-in mice are viable, we propose that sufficient 21 days post-injury, corresponding to the progressive re- levels of MYOG are produced from the targeted allele. turn to quiescence of the myogenic population (Fig. 3b). Nonetheless, a decrease in MYOG intensity was detected Finally, to verify whether the tdTOM cells isolated by ntdTom/ntd- by immunofluorescence in homozygous Myog FACS corresponded to myoblasts that expressed MYOG, Tom + embryos and in vitro myoblast cultures from homo- we isolated the tdTOM population from regenerating zygous animals (Fig. 2a, Figure S2C). As this decrease TA muscle at 5 days post-injury. Fixation of cells imme- was not observed in heterozygous samples, we decided diately after sorting and staining for MYOG and tdTOM to use heterozygous animals in our subsequent showed that 95% of isolated cells were positive for experiments. MYOG (Fig. 3c), thereby confirming that tdTOM In summary, tdTOM faithfully recapitulates the ex- followed the expression dynamics of MYOG also during pression of MYOG protein in embryonic and adult adult muscle regeneration and that its expression allows muscle, and its insertion at the Myog locus does not im- the isolation of MYOG cells by FACS after injury. ntdTom pair significantly the expression of this gene at the Taken together, our results show that the Myog mRNA and protein level. KI mouse allows efficient isolation of the MYOG popu- lation at different stages during development as well as ntdTom Myog mice allow isolation of differentiating from regenerating muscle. myoblasts during development and regeneration To assess the expression of tdTOM in homeostatic con- Dynamics of Myog expression during terminal ditions by flow cytometry, we isolated the mononuclear differentiation ntdTom/+ population from limb muscles of Myog mice at To assess if Myog-expressing myoblasts can execute foetal (embryonic day (E) 18.5), postnatal (postnatal day a cell division, we took advantage of the tdTOM re- (p) 21) and adult (10 weeks) stages. tdTOM fluorescent porter to monitor Myog expression by live videomi- cells were detected at foetal and early postnatal stages croscopy of primary myoblasts in vitro. MuSCs from ntdTom/+ where myogenesis was still taking place. In adult muscles adult Tg:Pax7-nGFP; Myog mice were isolated in homeostasis, the majority of MuSCs are quiescent by FACS based on GFP fluorescence and plated for and therefore no MYOG mono-nucleated cells are de- in vitro differentiation. After 3 days of culture, live tectable [48]. As expected, virtually no tdTOM cells imaging was initiated and images were acquired ntdTom/+ were detected in muscles of adult Myog animals every 9 min for 48 h (Fig. 4a, Additional File 4). (Fig. 3a). By manually tracking individual cells and monitoring To determine if tdTOM followed the expression dy- their differentiation status based on tdTOM fluores- namics of MYOG during adult muscle regeneration, we cence, we observed that up to 35% of MYOG cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 9 of 13 Fetal Postnatal Adult Quantification tdTOM cells ** ** tdTOM+ tdTOM+ tdTOM+ 3.1 2.5 0.19 PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed Fetal Post Adult natal tdTom/+ Myog D0 D3 D5 D10 D21 Muscle injury FACS D3 D5 D10 D21 + + + + + + + + tdT tdTOM OM tdT tdTO OM M tdT tdTO OM M tdT tdTO OM M 0.47 0.47 3.19 3.19 2.25 2.25 0.66 0.66 PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed Quantification sorted cells Sorted tdTOM cells (D5 post-injury) + + HOECHST MYOG tdTOM MYOG tdTOM Fig. 3 Myog cells can be isolated based on tdTOM fluorescence. a FACS profiles of the mononuclear cell fraction isolated from limb muscles of ntdTom/+ + foetal (E18.5), postnatal (p21) and adult Myog animals. Bar graph shows the percentage of tdTOM cells at the different stages. n >4 animals per condition. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; **p value = 0.0005 to 0.01. b FACS profiles of the ntdTom/+ mononuclear cell fraction isolated from cardiotoxin injured TA muscles from Tg:Pax7-nGFP; Myog mice at different timepoints (D3, D5, D10, D21 post-injury). c Immunostaining of the tdTOM cell fraction at 5 DPI isolated as in b for MYOG (3 h post-plating). Bar graph shows the quantification of tdTOM- and MYOG-positive cells (n = 3, > 200 cells per animal counted). Data represent mean ± s.d. Scale bar, 50 μm ntdTom underwent cell division during the imaging period this time (Fig. 4c). Therefore, using the Myog re- + + (Fig. 4b, c). Having established that MYOG cells remain porter mouse, we show that about one third of MYOG competent for cell division, we sought to quantify the cells can undergo one more cell division when tracking number of divisions that they performed. Amongst all tdTOM expression. + ntdTom the MYOG cells tracked, none divided more than once. Finally, we assessed the potential of the Myog In contrast, all MYOG cells divided during the imaging mouse to monitor tissue regeneration by intravital im- CreERT2/+ YFP/+ ntdTom/+ period and performed 1.5 divisions on average during aging. Pax7 ; R26 ; Myog mice were DAPI DAPI DAPI DAPI % tdTOM cells over total cell population % cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 10 of 13 Tg:Pax-nGFP; D0 D3 D5 ntdTom/+ Myog 0h 48h MuSC isolation Live imaging 21:07 21:16 21:40 21:58 22:07 B C Cell tracking Quantification Cell Status % of cells that have divided Number of divisions ** tdTOM division 100 tdTOM tdTOM division - + - + tdTOM tdTOM tdTOM tdTOM CreERT2/+ YFP/+ Pax7 ; R26 ; D3 D5 ntdTom/+ Myog D-1 Tamoxifen (IP) D0 Muscle injury D3 Intravital imaging D5 YFP tdTOM YFP tdTOM + Tg/+ ntdTom/+ Fig. 4 Myog cells can undergo cell division. a MuSCs were isolated based on GFP fluorescence from Tg:Pax7-nGFP ; Myog mice. Cells were plated for 3 days before initiating live imaging. Black arrowheads point to a tdTOM cell dividing and its daughter cells. Images were acquired every 9 min. Scale bar, 25 μm. b Representative tracking output from experiment in a. c The left bar graph shows the percentage of − + tdTOM and tdTOM cells that have undergone at least one cell division. The right bar graph indicates the average number of divisions that − + tdTOM and tdTOM underwent during the tracking period (n = 4, 100 cells tracked in total). Data represent mean ± s.d. Two-tailed unpaired CreERT2 YFP ntdTom Student’s t test; **p value = 0.005 to 0.01. d Recombination of Pax7-expressing cells in Pax7 ; R26 ; Myog reporter mice was induced 1 day before injury of the upper hindlimb muscle. Images were acquired by intravital imaging at 2 timepoints during muscle regeneration (1 mouse/timepoint). White arrows indicate double-positive YFP and tdTOM cells. Scale bar, 100 μm tdTOM Time % of cells Average number of divisions Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 11 of 13 used to permanently label Pax7-expressing cells and progenitor population by labelling PAX7 cells, but they their progeny upon tamoxifen administration and simul- did not report on the dynamics of differentiation. Here, taneously trace the differentiated fraction by following we carried out proof-of-concept experiments by intravi- tdTOM expression. We induced muscle injury by cardi- tal imaging of adult regenerating muscle and showed otoxin injection in the upper hindlimb and monitored that tdTOM fluorescence is sufficient to follow MYOG the regeneration process by live intravital imaging at dif- cells throughout the regeneration process. ferent time points (Fig. 4d). As expected, few MYOG cells were detected at 3 days post-injury, when extensive Conclusion proliferation of YFP myogenic progenitor cells was tak- In this study, we describe the creation of a new mouse line ing place (Fig. 4d, left panels). Two days later, the popu- where tdTOM is expressed from the endogenous Myog lation of tdTOM myoblasts had significantly expanded locus. tdTOM faithfully recapitulates MYOG expression and tdTOM cells could be observed throughout the re- during embryonic development and adult muscle regener- generating area (Fig. 4d, right panels, white arrows), re- ation and it can be used to isolate this population by flow capitulating the results of our flow cytometry analysis. cytometry. Additionally, heterozygous tdTOM expression Taken together, these experiments demonstrate that is sufficient for monitoring Myog dynamics by in vivo in- ntdTom tdTOM is a robust reporter that allows monitoring of travital imaging. Therefore, the Myog line can be of MYOG cells by in vitro and intravital imaging. great benefit to study the dynamics of lineage progression of muscle progenitors in embryonic and adult stages. Discussion Myog is a critical regulator of myoblast differentiation Supplementary Information and fusion, being an essential factor for embryonic The online version contains supplementary material available at https://doi. muscle development. In the present study, we generated org/10.1186/s13395-021-00260-x. and characterised a novel mouse line to fluorescently label MYOG cells by expression of the robust nuclear Additional file 1 Related to Fig. 2a. Whole mount immunofluorescence +/+ from a Myog embryo at E10.5 stained for tdTOM, MYOG and MyHC localised tdTOM protein from the endogenous Myog proteins. locus. Additional file 2 Related to Fig. 2a. Whole mount immunofluorescence To characterise the properties of tdTOM cells, we ntdTom/+ from a Myog and embryo at E10.5 stained for tdTOM, MYOG and assessed its co-localisation with MYOG during embryonic MyHC proteins. and foetal development, in adult primary myogenic cells Additional file 3 Related to Fig. 2a. Whole mount immunofluorescence ntdTom/ntdTom from a Myog embryo at E10.5 stained for tdTOM, MYOG and in vitro and during adult muscle regeneration in vivo. MyHC proteins. Given that in all conditions virtually all cells were positive Additional file 4 Related to Fig. 4a. In vitro division of a tdTOM cell. for both markers (> 95% cells), we conclude that tdTOM Additional file 5: Figure S1. Genotyping of the ntdTom allele. A. reliably labels MYOG cells from development to adult- ntdTom/ntdTom ntdTom/+ Genotyping of ear-clip samples from Myog , Myog and ntdTom/+ +/+ hood. Furthermore, total Myog levels from Myog Myog animals. Myog-ntdTom allele was verified by PCR using primers 16, 17 and 18 (Flp-recombined Myog-ntdTom allele, 236 bp and WT allele animals were comparable to that of the WT, and homozy- 600 bp). ntdTom/ntdTom gous Myog mice are viable. Moreover, the ex- Additional file 6: Figure S2. tdTOM expression recapitulates pression of tdTOM allowed us to isolate the MYOG endogenous Myog expression. A. Immunofluorescence of ntdTom/+ population from developing embryos as well as adult re- extraocular (EOM), tongue and limb muscles of Myog embryo at E14.5. Bar graph shows the quantification of tdTOM and MYOG positive generating muscle indicating the utility of this reporter cells. n = 3 embryos, > 100 cells per muscle and per embryo were mouse in isolating living differentiating myoblasts that counted. Scale bar, 40 μm. B. RT-qPCR assessing the levels of total Myog +/+ were previously inaccessible for direct investigation. mRNA, the wild-type allele and the tdTom allele specifically from Myog , ntdTom/+ ntdTom/ntdTom Myog and Myog adult myoblasts using the primer set In addition, studies on the cell cycle dynamics of described in Fig. 1c. n = 3 animals per genotype. Data represents mean ± Myog cells have been hampered by the lack of a fluores- s.d. Two-tailed unpaired Student’s t-test; * p-value = 0.01 to 0.05. C. +/+ cent reporter. Here, by means of live microscopy and MuSCs from limb muscles were isolated from Tg:Pax7-nGFP; Myog , ntdTom/+ ntdTom/ntdTom Tg:Pax7-nGFP; Myog and Tg:Pax7-nGFP; Myog animals single-cell tracking of differentiating primary myoblasts, and plated for in vitro differentiation for 5 days. Cells were stained for we demonstrated that about one third of MYOG cells MYOG and tdTOM proteins. Scale bar, 100 μm. can divide in vitro and undergo a maximum of one add- Additional file 7: Supplementary Table 1. Primer sequences itional cell division during the tracking period. There- fore, the majority of cells that express detectable levels Abbreviations of MYOG exit the cell cycle. Myog: Myogenin; MRF: Myogenic regulatory factor; MuSC: Muscle stem cell; Several studies have performed intravital imaging of BrdU: 5-Bromo-2′-deoxyuridine; sgRNA: Single guide RNA; CRISPR: Clustered regularly interspaced short palindromic repeats; NLS: Nuclear localisation muscle tissue [24, 51–53]; however, only two of them sequence; GFP: Green fluorescent protein; RT-qPCR: Real-time quantitative dynamically monitored the process of muscle regener- polymerase chain reaction; KI: Knock-in; FACS: Fluorescence-assisted cell ation [24, 51]. These two studies focused on the sorting; TA: Tibialis anterior; YFP: Yellow fluorescent protein Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 12 of 13 Acknowledgements 7. Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked muscular We gratefully acknowledge S. Paisant for help with the maintenance of dystrophy (mdx) in the mouse. Proc National Acad Sci. 1984;81:1189–92 mouse embryonic stem cell lines, the Institut Pasteur Mouse Genetics https://doi.org/10.1073/pnas.81.4.1189. Engineering Platform, the UtechS Photonic BioImaging (Imagopole) at 8. Meadows E, Flynn JM, Klein WH. Myogenin regulates exercise capacity but Institut Pasteur supported by the French National Research Agency (France is dispensable for skeletal muscle regeneration in adult mdx mice. Plos One. BioImaging; ANR-10–INSB–04; Investments for the Future), the Center for 2011;6:e16184 https://doi.org/10.1371/journal.pone.0016184. Translational Science (CRT)-Cytometry and Biomarkers Unit of Technology 9. 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Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse

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Abstract

Background: Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. Results: Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the ntdTom endogenous Myogenin locus. Expression of tdTOMATO in Myog mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN myoblasts. Conclusions: We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts. Keywords: Myogenin, Knock-in mouse, tdTOMATO, Intravital imaging, Skeletal muscle Background single MRF knockout mice, only Myog-null homozygous Embryonic and postnatal myogenesis and adult muscle animals die at birth due to severe skeletal muscle defects regeneration are regulated by a family of basic helix- [1–3]. Thus, unlike the other myogenic bHLH factors, loop-helix myogenic regulatory factors (MRFs) compris- Myog has no redundant or compensatory mechanisms to ing Myf5, Mrf4, Myod and Myogenin (Myog). Following replace its function during development. Myoblasts lack- myogenic specification in the embryo, the MRFs are ing this gene accumulate in the muscle-forming areas expressed in a sequential manner to ensure commit- throughout the body and fail to form normal myofibers ment, proliferation, differentiation and fusion to give rise in vivo, pointing to its critical role in terminal differenti- to multinucleated skeletal myofibres. Single and com- ation of myoblasts [2–4]. While Myog-null embryos have binatorial mouse knockout models of the MRFs have some disorganised residual primary fibres, major differ- established a genetic hierarchy where Myf5, Mrf4 and ences between mutant and wild-type embryos become Myod control lineage commitment and proliferation of apparent during the initiation of secondary myofibre for- myogenic progenitors, and Myod, Mrf4 and Myog regu- mation [2, 4]. Unexpectedly, conditional ablation of late in terminal differentiation [1]. Notably, amongst the Myog during the perinatal and postnatal period does not result in noticeable defects in muscle morphology or −/− histology, suggesting that Myog myoblasts can still * Correspondence: shahragim.tajbakhsh@pasteur.fr Stem Cells & Development Unit, Institut Pasteur, 25 rue du Dr. Roux, 75015 contribute to muscle growth [5, 6]. Additionally, condi- Paris, France tional ablation of Myog in a Duchenne muscular dys- UMR CNRS 3738, Institut Pasteur, Paris, France trophy mouse model (mdx [7]) did not result in an Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 2 of 13 adverse phenotype, confirming that Myog is dispensable Generation of the Myog-ntdTomato construct for CRISPR- for adult muscle regeneration in this disease [8]. Never- Cas9-mediated homologous recombination theless, although Myog-null muscle stem cells (MuSCs) A fragment of 1000 bp from the last exon of Myog was proliferate and differentiate in culture as efficiently as amplified by PCR from murine gDNA (primers 1 and 2, wild-type cells, the muscle gene expression program is Supplementary Table 1), introducing SalI and NotI re- profoundly altered in the absence of Myog [6]. striction sites. This fragment was subcloned into the Adult muscle regeneration depends on MuSCs, char- donor plasmid encoding for tdTOM (kind gift from Dr. acterised by the expression of Pax7 [9–13]. Upon muscle Festuccia, Institut Pasteur). A fragment of 760 bp from injury, MuSCs activate the expression of Myod, prolifer- the 3′UTR of the Myog gene just after the STOP codon ate to generate myoblasts that differentiate and fuse to was amplified by PCR from murine gDNA (primers 3 form myofibres. Different reporter mouse lines have and 4). This amplification also introduced a mutation in been generated to fluorescently label the Pax7 muscle the PAM sequence necessary for CRISPR-Cas9 genome progenitor population, either from the endogenous locus editing. Using the PacI and SpeI restriction sites added, [14–16] or as transgenes [15, 17, 18], thereby allowing the fragment was subcloned into the PacI and XbaI imaging and isolation of Pax7-expressing cells. Addition- digested tdTOM plasmid. Oligos containing a T2A ally, inducible reporters in which expression of the Cre (primers 5 and 6) [36] peptide and a triple NLS sequence recombinase under the control of the Pax7 promoter from SV40 large T [37] were annealed and subcloned recombines a membrane or cytoplasmic fluorophore [13, into a blunt pBluescript SK (+) plasmid. This plasmid 19, 20] have been used for permanent marking of the was subsequently digested with NotI and KpnI and the myogenic lineage [21–23] and for live imaging [24]. Al- T2A-NLS fragment was cloned into the tdTOM plasmid. though several reporter mouse lines have been generated tdTOM was amplified by PCR from the initial plasmid to identify differentiating myoblasts based on the expres- (primers 7 and 8) adding KpnI and FseI sites and sub- sion of Myosin light chain [25], Myog [26–28] and cloned into the donor vector after the 3xNLS sequence. Muscle creatine kinase [29], they are based on lacZ (β- An FNF cassette containing two FRT sites and the galactosidase activity, [30]) or cat (chloramphenicol ace- NeoR/KanR gene under the control of the PGK pro- tyltransferase,[31]) expression and thus only allow end- moter was amplified by PCR (primers 9 and 10) adding point measurements on fixed samples. FseI and PacI sites. This fragment was subcloned into Terminal myoblast differentiation is characterised by the donor vector. the expression of Myog and the cyclin-dependent kinase The highest scoring sgRNA sequence to target the inhibitor p21 and cell cycle withdrawal [32–34]. Experi- Myog STOP codon region was determined using a guide ments using the nucleotide analogue BrdU have shown design tool (crispr.mit.edu, Zhang Lab). Primers contain- that MYOG-positive cells can undergo DNA replication ing this targeting sequence (primers 11 and 12) were [32], but it is still unclear how many divisions they can annealed and subcloned into the pU6-(BbsI) CBh-Cas9- execute before definitively leaving the cell cycle. T2A-mCherry vector (Addgene #64324) digested with Here, we took advantage of the CRISPR/Cas9 sys- BbsI. tem, which allows precise genome editing [35], to generate a knock-in mouse line expressing a nuclear Targeting of mouse embryonic stem cells localised tandem-dimer Tomato (tdTOM) protein The Myog-ntdTOM donor construct (linearised by PvuI under the control of the endogenous Myog promoter, digestion) and the pU6 vector were electroporated in while retaining expression of MYOG protein. We C57BL/6J mouse embryonic stem cells. Following G418 ntdTom show that heterozygous Myog mice exhibit ro- (300 μg/ml) selection, positive clones were determined bust reporter gene expression in fixed and live myo- by PCR using primers 13, 14 and 15 (Supplementary genic cells thus allowing in vitro and intravital Table 1), yielding a 1.7 kb band for the WT and 1.2 kb microscopy studies of the dynamics of muscle differ- for the mutant. Two positive clones were expanded and entiation and cell cycle withdrawal. 8–10 embryonic stem cells were injected into BALB/c blastocysts to generate chimeric mice in the Mouse Gen- Materials and methods etics Engineering Facility at the Institut Pasteur. Germ- Mouse maintenance line transmission was verified by PCR and F1 mice were Animals were handled according to national and Euro- crossed to Tg(ACTFLPe)9205Dym [38] animals to excise pean Community guidelines and an ethics committee of the FNF cassette. Excision of the FNF cassette and pres- ntdTom the Institut Pasteur (CETEA, Comité d’Ethique en Ex- ence of the Myog allele was verified by PCR using périmentation Animale) in France approved protocols primers 16, 17 and 18 (Supplementary Table 1) (Flp- ntdTom (Licence 2015-0008). Except when indicated otherwise, recombined Myog allele, 236 bp and WT allele males and females of 2–4 months were used. 600 bp, Figure S1), and these primers were subsequently Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 3 of 13 used for genotyping. F2 animals were backcrossed to incubated with 1 μg/ml Hoechst 33342 for 5 min at room C57BL/6 animals to eliminate the Tg(ACTFLPe) allele, temperature to visualise nuclei, washed three times in PBS ntdTom/+ and Myog animals were selected for further and mounted in 70% glycerol in PBS for imaging. characterisation. For whole-mount immunofluorescence, embryos were collected in PBS and fixed in 4% PFA 0.1% Triton X-100 Isolation and culture of MuSCs for 2 h at 4 °C. After two PBS washes, samples were Foetal and adult muscles were dissected and minced in dehydrated in 50% methanol in PBS and kept in 100% ice-cold DMEM as described in [39]. Samples were then methanol at − 20 °C until used. Samples were rehydrated incubated in DMEM, 0.08% Collagenase D (Sigma, in PBS and incubated in blocking buffer (15% goat 11088882001), 0.2% Trypsin (ThermoFisher, 15090) and serum, 1.5% BSA, 0.5% TritonX-100 in 1X PBS) for 1 h 10 μg/ml of DNAseI (Sigma, 11284932) for 25 min at at RT in 2-ml Eppendorf tubes. Embryos were then in- 37 °C under gentle agitation for 5 rounds of digestion. cubated with primary antibodies in the blocking buffer After each round, samples were allowed to sediment for for 5–7 days at 4 °C with rocking. Embryos were washed 5 min, the supernatant was collected in 4 ml of foetal bo- extensively for 2–4 h in PBST and incubated in Fab’ sec- vine serum (FBS) on ice and fresh digestion buffer was ondary antibodies for 2 days at 4 °C with rocking. Em- added to the remaining muscle pellet. The collected su- bryos were washed as above, dehydrated in 50% pernatants were centrifuged for 15 min at 550g at 4 °C, methanol in PBS, twice in 100% methanol and then resuspended in DMEM 2% FBS, and filtered through a cleared with BABB and mounted for imaging [40]. 40-μm strainer (Corning, 352235) before cell sorting. Cells were isolated based on size, granulosity and GFP Adult muscle injury, histology and immunofluorescence or tdTOM fluorescence using an Aria III (BD Biosci- Muscle injury was done as described previously [22]. ences) flow cytometer. Cells were collected directly in Mice were anaesthetised with 0.5% Imalgene/2% Rom- MuSC growth media (38.5% DMEM (Fisher Scientific, pun, and the TA muscle was injected with 50 ml of car- 31966047), 38.5% F12 (Fisher Scientific, 31765035), 20% diotoxin (10 mM; Latoxan, L8102) diluted in 0.9% NaCl. FBS (ThermoFisher, 10270), 2% Ultroser (Pall, 15950- Injured TA muscles were fixed upon harvesting in 4% 017), 1% penicillin/streptomycin (GIBCO, 15140-122)). PFA for 2 h at 4 °C, washed with PBS and equilibrated Matrigel® (1 mg/ml, Corning, 354248) coated dishes with 30% sucrose in PBS overnight. Samples were (30 min at 37 °C) were used to culture MuSCs in growth mounted in OCT tissue freezing media and cryosec- media at 3% O ,5%CO , 37 °C for the indicated times. tioned between 8 and 12 μm. When endogenous tdTOM 2 2 For immunostaining, cells were fixed in 4% parafor- was scored, cryosections were rehydrated in PBS and maldehyde (PFA, Electron Microscopy Sciences, 15710) counterstained with Hoechst 33342. in PBS for 15 min at room temperature (RT), permeabi- In case of MYOG plus tdTOM detection, tissue sec- lised in 0.5% Triton X-100 (Merck, T8787) for 5 min at tions were processed for histology as described [41]. RT and blocked with 10% goat serum (GIBCO). Cells Briefly, sections were post-fixed in 4% PFA for 10 min at were incubated with the indicated primary antibodies in RT and washed with PBS prior to immunostaining. PBS 2% goat serum buffer overnight following by 45- Heat-induced epitope retrieval was performed in a cit- min incubation with secondary antibodies and 1 μg/ml rate solution pH 6.0 during 6 min in a pressure cooker. Hoechst (ThermoFisher, H1399). Sections were then incubated with 30% H O for 5 min 2 2 at RT. Samples were then permeabilised with 0.2% Embryo immunofluorescence Triton-X100, washed in PBS and blocked in blocking For tissue immunofluorescence, embryos were collected buffer (15% goat serum, 1.5% BSA, 0.5% TritonX-100 in in PBS and fixed in 4%PFA 0.1% Triton X-100 in PBS for 1X PBS). Primary antibodies against MYOG and DsRED 2 h at 4 °C. After 3 PBS washes, embryos were cryopre- (recognising tdTOM) were incubated overnight at 4 °C. served in 30% sucrose in PBS and embedded in OCT tis- After washing with PBST, sections were incubated with sue freezing media (Leica, 14020108926) for appropriate secondary antibodies and 1 μg/ml Hoechst cryosectioning. Cryosections were allowed to dry for 30 33342 in blocking buffer for 45 min at RT (Table 1). min at room temperature and washed once with PBS. Tis- sue samples were blocked in 3% BSA, 10% goat serum, Western blot 0.5% Triton X-100 for 1 h at room temperature. Primary Embryos were collected in ice-cold PBS and subse- antibodies were diluted in blocking solution and incubated quently snap frozen in dry ice. Embryonic tissues were overnight at 4 °C. After three washes with PBST (PBS ground to a fine powder using a mortar and pestle on 0.1% Tween20 (Sigma Aldrich, P1379)), secondary anti- dry ice and lysed in RIPA buffer (150 M NaCl, 50 mM bodies were diluted in blocking solution and incubated for Tris pH 8, 5 mM EDTA, 1% NP-40 (Sigma, I8896), 0.5% 45 min at room temperature. Finally, samples were sodium deoxycholate, 0.1% SDS supplemented with 1X Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 4 of 13 Table 1 Antibodies used for immunostaining Species Concentration Reference Primary antibodies Myog Mouse monoclonal (IgG1) 1:20 DHSB, clone F5D, supernatant Living colors® Ds-Red Rabbit polyclonal 1:500 Takara Bio, 632496 Myosin heavy chain (MyHC) Mouse monoclonal 1:30 DHSB, MF-20, supernatant (IgG2b) Secondary antibodies Alexa Fluor®488 AffiniPure Goat Anti-Mouse IgG1(γ1) Goat 1:500 Jackson ImmunoResearch Labs, 115-545- Alexa Fluor® 555 F(ab')2 Fragment of Goat Anti-Rabbit Goat 1:500 Thermo Fisher Scientific, A-21430 IgG Alexa Fluor® 633 Goat Anti-Mouse IgG2b (γ2b) Goat 1:500 Thermo Fisher Scientific, A-21146 protease (Sigma, S8820) and phosphatase inhibi- In vitro videomicroscopy tors (Roche, 4906845001)). Fifteen microgrammes of MuSCs were plated on a microscopy culture chamber protein extracts were run on a 4–12% Bis-Tris Gel (IBIDI, 80826) and cultured in growth media supple- NuPAGE (Invitrogen, NP0322) and transferred on a mented as above. The plate was incubated at 37 °C, 5% PVDF Amersham Hybond-P transfer membrane (GE CO and 3% O in a Pecon incubation chamber. A Zeiss 2 2 Healthcare, RPN303F). The membrane was blocked with Observer.Z1 connected to a Plan-Apochromat 20x/0.8 5% milk (Dominique Dutscher, 711160) in Tris-Buffer M27 objective and Hamamatsu Orca Flash 4 camera Saline 0.2% Tween (Sigma, P9416) (TBS-T) for 1 h at piloted with Zen software (Carl Zeiss) was used. room temperature and probed with specific primary antibodies overnight at 4 °C. After three washes in TBS- Static imaging T, the membrane was incubated with HRP or The following systems were used for image acquisition: fluorophore-conjugated secondary antibodies and re- Zeiss SteREO Discovery V20 for macroscopic observa- vealed by chemiluminescence (Pierce ECL2 western tions of whole embryos and Zeiss LSM800 or LSM700 blotting substrate, Thermo Scientific, 80196) or fluores- laser-scanning confocal microscopes for tissue sections cence (Bio-Rad, Chemidoc MP) (Table 2). and whole-mount immunostaining of cleared embryos. End point in vitro culture samples were imaged with a Zeiss Observer.Z1. RNA extraction RNA from cells isolated by FACS was extracted using a Trizol-based kit (Zymo Research, R2061) and reverse Intravital microscopy CreERT2 YFP ntdTom transcribed using SuperScriptIII (Invitrogen, 18080093). Intravital imaging of Pax7 ; R26 ; Myog RT-qPCR to assess for mRNA relative expression was mice at different timepoints during regeneration was performed with SYBR green master mix (Roche, performed on an upright Nikon NiE A1R MP micro- 04913914001) in Applied biosciences machine. Data scope piloted with NIS software (Nikon). The micro- −ΔΔCT analysis was performed using the 2 method [42] scope was equipped with a × 25 NA 1.1 PlanApo and mRNA expression was normalised with LambdaS objective, GaAsP PMT detectors and a Rpl13 (primers 23 and 24, Supplementary Table 1). Spectra-Physics Insight Deepsee laser. Laser frequency Table 2 Antibodies used for Western blot Species Concentration Reference Primary antibodies Myog Mouse monoclonal (IgG1) 1:500 DHSB, clone F5D, supernatant Living colors® Ds-Red Rabbit polyclonal 1:1000 Takara Bio, 632496 Secondary antibodies Mouse-HRP Goat polyclonal 1:5000 Pierce, 31430 Rabbit-HRP Goat polyclonal 1:5000 Pierce, 31460 GAPDH-Rhodamine Human IgG Fab fragment 1:5000 Bio-Rad, 12004168 Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 5 of 13 Myog E3 tdTOM Neo Myog 3’ UTR Donor construct 5’UTR E1 E2 E3 3’UTR Myog locus CRISPR-Cas9 mediated recombination ntdTom-FNF 5’UTR E1 E2 E3 tdTOM Neo 3’UTR Myog locus Flippase mediated excision ntdTom Myog locus 5’UTR E1 E2 E3 tdTOM 3’UTR E9.5 E10.5 E11.5 C D ntdTom/+ ntdTom/ntdTom WT Myog Myog 5’UTR E1 E2 E3 tdTOM 3’UTR tdTOM MYOG GAPDH Total Myog Myog WT Myog tdTOM *** ** 2.5 Quantification ** * ** tdTOM MYOG 2.0 Genotype 5 1.5 *** WT Genotype Het WT Homo 1.0 3 Het 2 Homo 0.5 0.0 Fig. 1 (See legend on next page.) T2A 3xNLS FRT FRT Stop T2A 3xNLS T2A FRT 3xNLS FRT FRT T2A 3xNLS FRT WT Het Homo WT Het Homo WT Het Homo WT Het Homo WT Het Homo 2^−ddCT Expression normalized to GAPDH Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 6 of 13 (See figure on previous page.) Fig. 1 Generation of a Myog knock-in mouse line. a Scheme depicting the endogenous Myog locus, the donor construct and the result of the ntdTom-FNF CRISPR-Cas9-mediated recombination in mouse embryonic stem cells. First-generation Myog mice were then crossed with a Tg(ACTF +/+ ntdTom/+ LPe) deleter strain to excise the FNF cassette. b Endogenous fluorescence from Myog embryos at different stages. An overlay between the brightfield and fluorescent images is shown. Scale bar, 1000 μm. c Scheme showing the primer pairs amplifying the wild-type allele (2), the ntdTom allele (3) and both alleles (1) in the targeted Myog locus. RT-qPCR analysis of the levels of total Myog mRNA, the wild-type allele and the +/+ ntdTom/+ ntdTom/ntdTom tdTom allele specifically from E14.5 Myog , Myog and Myog embryos. n = 4 embryos per genotype. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; ***p value < 0.005, **p value = 0.0005 to 0.01, *p value = 0.01 to 0.05. d Western blot assessing the levels +/+ ntdTom/+ ntdTom/ntdTom of MYOG and tdTOM proteins from E14.5 Myog , Myog and Myog embryos (n = 4 embryos per genotype). Bar graph shows the quantification of protein expression levels normalised to GAPDH. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; ***p value < 0.005, *p value = 0.01 to 0.05 was tuned to 960 nm to allow simultaneous excitation of Myog transcripts [47] (Fig. 1b), with expression levels be- YFP and tdTOM fluorophores. ing lower in the caudal (more recently formed) somites. For image acquisition, the skin over the upper hind- We then collected tissue samples from E14.5 foetuses limb was shaved and incised to expose approximately 1 and performed RT-qPCR and Western blot analysis to cm of the muscle and imaged directly. During the im- confirm that Myog mRNA and protein levels were simi- aging period, mice were anaesthetised with 1.5% iso- lar in wild-type, heterozygous and homozygous animals. fluorane and maintained in an incubation chamber at Primer pairs were designed to amplify specifically the 37 °C. wild-type allele or the tdTom allele, and one primer set amplified both (Fig. 1c). This analysis showed that Myog heterozygous and homozygous knock-in (KI) embryos Image analysis expressed similar levels of total Myog mRNA, and con- Cell tracking was performed using the Manual Tracking firmed that no Myog wild-type transcript could be de- feature of the TrackMate plug-in [43] in Fiji [44]. ZEN tected in the homozygous embryos. As expected, the software (Carl Zeiss), Fiji [44] and Imaris (Bitplane) were ntdTom levels of Myog mRNA were the highest in homozy- used for image analysis. Figures were assembled in gous samples, decreased to roughly 50% in the case of Adobe Photoshop and Illustrator (Adobe Systems). the heterozygous and were not detected in wild-type em- bryos (Fig. 1c). At the protein level, we noted similar ex- Data analysis and statistics pression levels of MYOG in embryos from all three Data analysis and statistics were performed using R [45], genotypes, whereas the tdTOM protein was absent in and figures were produced using the package ggplot2 wild-type samples (Fig. 1d). Therefore, we conclude that [46]. For comparison between two groups, two-tailed MYOG protein was generated from transcripts that orig- paired and unpaired Student’s t tests were performed to inated from both alleles. calculate p values and to determine statistically signifi- To investigate the expression of the targeted allele cant differences (see figure legends). with higher resolution, we assessed the temporal ex- pression dynamics of MYOG and tdTOM proteins by Results whole-mount immunostaining at E10.5 and compared ntdTom Generation and characterisation of a Myog mouse the expression of MYOG and tdTOM in wild-type, Using the CRISPR-Cas9 system, a sgRNA was designed heterozygous and homozygous embryos (Fig. 2a, Add- to target the region of the STOP codon of the Myog itional files 1, 2, 3). We confirmed that tdTOM gene for homologous recombination. The recombination followed the expression pattern of MYOG in the ep- template consisted of two homology arms corresponding axial and hypaxial domains of all somites, indicating to Myog sequences flanking the STOP codon, a tdTom that both proteins have similar spatiotemporal expres- coding sequence and a Neo resistance cassette flanked sion dynamics. To assess the co-expression of MYOG by frt sites (Fig. 1a). The tdTOM protein was preceded and tdTOM at the single cell level in heterozygous by a T2A peptide sequence [36] to allow cleavage from embryos, cryosections at the level of extraocular, the MYOG protein following translation, and a triple tongue and limb muscles were examined during pri- NLS sequence [37] to ensure nuclear localisation. mary (E12.5) and secondary (E14.5) myogenesis when First, we evaluated the endogenous tdTOM fluores- small oligo-nucleated and larger multi-nucleated myo- ntdTom/+ cence in heterozygous Myog embryos between fibres are generated, respectively. Quantification of E9.75 and E11.5 at the level of the somites, i.e. transient protein expression in different muscles confirmed an embryonic structures arising from the segmentation of average co-localisation of both proteins in 97% and the paraxial mesoderm. Endogenous tdTOM fluores- 95% of the cells at E12.5 (Fig. 2b) and E14.5 (Figure cence followed a similar pattern to that described for S2A), respectively. Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 7 of 13 tdTom/tdTom tdTom/+ Myog Myog WT E10.5 st st st interlimb 1 somites interlimb 1 somites interlimb 1 somites Quantification E12.5 - + MYOG tdTOM + - MYOG tdTOM + + MYOG tdTOM HOECHST MYOG tdTOM MyHC Tg:Pax7-nGFP; Quantification ntdTom/+ D0 D5 Myog MuSC isolation Fix + Stain + Plating - + MYOG tdTOM + - MYOG tdTOM + + MYOG tdTOM HOECHST MYOG tdTOM Limb Fig. 2 (See legend on next page.) EOM MAS Tongue Back Limb Limb Tongue EOM MYOG tdTOM MyHC MYOG tdTOM % cells % cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 8 of 13 (See figure on previous page.) +/+ ntdTom/+ Fig. 2 tdTOM expression recapitulates endogenous Myog expression. a Whole-mount immunofluorescence from Myog , Myog and ntdTom/ntdTom Myog embryos at E10.5 stained for tdTOM, MYOG and MyHC proteins. Scale bar, 300 μm on upper panels and 100 μm on lower ntdTom/+ insets. b Immunofluorescence of extraocular (EOM), tongue and limb muscle cryosections of Myog embryos at E12.5. The bar graph shows the quantification of tdTOM- and MYOG-positive cells. n = 3 embryos, > 100 cells per muscle and per embryo were counted. Scale bar, 40 μm. c ntdTom MuSCs were isolated from limb muscles of Tg:Pax7-nGFP; Myog adult animals and plated for in vitro differentiation for 5 days. Bar graph shows the quantification of tdTOM- and MYOG-positive cells (n = 3, > 200 cells per animal counted). Scale bar, 100 μm on the left image, 50 μm on the right inset To assess the fidelity of the reporter mouse in adult performed an injury of the Tibialis anterior (TA) muscle ntdTom ntdTom/+ myoblasts, Myog animals were crossed with Tg: of Tg:Pax7-nGFP; Myog mice by intramuscular Pax7-nGFP mice, where GFP marks all MuSCs [15]. injection of the snake venom toxin cardiotoxin [49]. We MuSCs were isolated by FACS based on GFP expression, next performed FACS analysis to determine whether the then differentiated in vitro for 5 days. In agreement with tdTOM mononucleated fraction could be isolated fol- our results in the embryo, MYOG and tdTOM expres- lowing tissue injury. As expected, only a few tdTOM sion co-localised in about 95% of the cells (Fig. 2c). Add- cells were detected at 3 days post-injury, when myogenic itionally, no significant differences were observed in total cells are known to be maximally proliferating [23, 24, Myog RNA levels between wild-type, heterozygous and 50] (Fig. 3b). tdTOM cells were most abundant at 5 homozygous animals (Figure S2B). and 10 days post-injury, corresponding to the increased As indicated above, the KI strategy was designed to be shift towards differentiation of the transiently amplifying non-disruptive and allow normal MYOG protein expres- myoblast population during this period. As the major sion from the recombined alleles. Given that Myog-null features of the regeneration process are completed by 3– ntdTom/ntdTom + mice are lethal at birth, and our Myog 4 weeks, the proportion of tdTOM cells decreased by knock-in mice are viable, we propose that sufficient 21 days post-injury, corresponding to the progressive re- levels of MYOG are produced from the targeted allele. turn to quiescence of the myogenic population (Fig. 3b). Nonetheless, a decrease in MYOG intensity was detected Finally, to verify whether the tdTOM cells isolated by ntdTom/ntd- by immunofluorescence in homozygous Myog FACS corresponded to myoblasts that expressed MYOG, Tom + embryos and in vitro myoblast cultures from homo- we isolated the tdTOM population from regenerating zygous animals (Fig. 2a, Figure S2C). As this decrease TA muscle at 5 days post-injury. Fixation of cells imme- was not observed in heterozygous samples, we decided diately after sorting and staining for MYOG and tdTOM to use heterozygous animals in our subsequent showed that 95% of isolated cells were positive for experiments. MYOG (Fig. 3c), thereby confirming that tdTOM In summary, tdTOM faithfully recapitulates the ex- followed the expression dynamics of MYOG also during pression of MYOG protein in embryonic and adult adult muscle regeneration and that its expression allows muscle, and its insertion at the Myog locus does not im- the isolation of MYOG cells by FACS after injury. ntdTom pair significantly the expression of this gene at the Taken together, our results show that the Myog mRNA and protein level. KI mouse allows efficient isolation of the MYOG popu- lation at different stages during development as well as ntdTom Myog mice allow isolation of differentiating from regenerating muscle. myoblasts during development and regeneration To assess the expression of tdTOM in homeostatic con- Dynamics of Myog expression during terminal ditions by flow cytometry, we isolated the mononuclear differentiation ntdTom/+ population from limb muscles of Myog mice at To assess if Myog-expressing myoblasts can execute foetal (embryonic day (E) 18.5), postnatal (postnatal day a cell division, we took advantage of the tdTOM re- (p) 21) and adult (10 weeks) stages. tdTOM fluorescent porter to monitor Myog expression by live videomi- cells were detected at foetal and early postnatal stages croscopy of primary myoblasts in vitro. MuSCs from ntdTom/+ where myogenesis was still taking place. In adult muscles adult Tg:Pax7-nGFP; Myog mice were isolated in homeostasis, the majority of MuSCs are quiescent by FACS based on GFP fluorescence and plated for and therefore no MYOG mono-nucleated cells are de- in vitro differentiation. After 3 days of culture, live tectable [48]. As expected, virtually no tdTOM cells imaging was initiated and images were acquired ntdTom/+ were detected in muscles of adult Myog animals every 9 min for 48 h (Fig. 4a, Additional File 4). (Fig. 3a). By manually tracking individual cells and monitoring To determine if tdTOM followed the expression dy- their differentiation status based on tdTOM fluores- namics of MYOG during adult muscle regeneration, we cence, we observed that up to 35% of MYOG cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 9 of 13 Fetal Postnatal Adult Quantification tdTOM cells ** ** tdTOM+ tdTOM+ tdTOM+ 3.1 2.5 0.19 PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed Fetal Post Adult natal tdTom/+ Myog D0 D3 D5 D10 D21 Muscle injury FACS D3 D5 D10 D21 + + + + + + + + tdT tdTOM OM tdT tdTO OM M tdT tdTO OM M tdT tdTO OM M 0.47 0.47 3.19 3.19 2.25 2.25 0.66 0.66 PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed PE-T PE-TexasRed exasRed Quantification sorted cells Sorted tdTOM cells (D5 post-injury) + + HOECHST MYOG tdTOM MYOG tdTOM Fig. 3 Myog cells can be isolated based on tdTOM fluorescence. a FACS profiles of the mononuclear cell fraction isolated from limb muscles of ntdTom/+ + foetal (E18.5), postnatal (p21) and adult Myog animals. Bar graph shows the percentage of tdTOM cells at the different stages. n >4 animals per condition. Data represents mean ± s.d. Two-tailed unpaired Student’s t test; **p value = 0.0005 to 0.01. b FACS profiles of the ntdTom/+ mononuclear cell fraction isolated from cardiotoxin injured TA muscles from Tg:Pax7-nGFP; Myog mice at different timepoints (D3, D5, D10, D21 post-injury). c Immunostaining of the tdTOM cell fraction at 5 DPI isolated as in b for MYOG (3 h post-plating). Bar graph shows the quantification of tdTOM- and MYOG-positive cells (n = 3, > 200 cells per animal counted). Data represent mean ± s.d. Scale bar, 50 μm ntdTom underwent cell division during the imaging period this time (Fig. 4c). Therefore, using the Myog re- + + (Fig. 4b, c). Having established that MYOG cells remain porter mouse, we show that about one third of MYOG competent for cell division, we sought to quantify the cells can undergo one more cell division when tracking number of divisions that they performed. Amongst all tdTOM expression. + ntdTom the MYOG cells tracked, none divided more than once. Finally, we assessed the potential of the Myog In contrast, all MYOG cells divided during the imaging mouse to monitor tissue regeneration by intravital im- CreERT2/+ YFP/+ ntdTom/+ period and performed 1.5 divisions on average during aging. Pax7 ; R26 ; Myog mice were DAPI DAPI DAPI DAPI % tdTOM cells over total cell population % cells Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 10 of 13 Tg:Pax-nGFP; D0 D3 D5 ntdTom/+ Myog 0h 48h MuSC isolation Live imaging 21:07 21:16 21:40 21:58 22:07 B C Cell tracking Quantification Cell Status % of cells that have divided Number of divisions ** tdTOM division 100 tdTOM tdTOM division - + - + tdTOM tdTOM tdTOM tdTOM CreERT2/+ YFP/+ Pax7 ; R26 ; D3 D5 ntdTom/+ Myog D-1 Tamoxifen (IP) D0 Muscle injury D3 Intravital imaging D5 YFP tdTOM YFP tdTOM + Tg/+ ntdTom/+ Fig. 4 Myog cells can undergo cell division. a MuSCs were isolated based on GFP fluorescence from Tg:Pax7-nGFP ; Myog mice. Cells were plated for 3 days before initiating live imaging. Black arrowheads point to a tdTOM cell dividing and its daughter cells. Images were acquired every 9 min. Scale bar, 25 μm. b Representative tracking output from experiment in a. c The left bar graph shows the percentage of − + tdTOM and tdTOM cells that have undergone at least one cell division. The right bar graph indicates the average number of divisions that − + tdTOM and tdTOM underwent during the tracking period (n = 4, 100 cells tracked in total). Data represent mean ± s.d. Two-tailed unpaired CreERT2 YFP ntdTom Student’s t test; **p value = 0.005 to 0.01. d Recombination of Pax7-expressing cells in Pax7 ; R26 ; Myog reporter mice was induced 1 day before injury of the upper hindlimb muscle. Images were acquired by intravital imaging at 2 timepoints during muscle regeneration (1 mouse/timepoint). White arrows indicate double-positive YFP and tdTOM cells. Scale bar, 100 μm tdTOM Time % of cells Average number of divisions Benavente-Diaz et al. Skeletal Muscle (2021) 11:5 Page 11 of 13 used to permanently label Pax7-expressing cells and progenitor population by labelling PAX7 cells, but they their progeny upon tamoxifen administration and simul- did not report on the dynamics of differentiation. Here, taneously trace the differentiated fraction by following we carried out proof-of-concept experiments by intravi- tdTOM expression. We induced muscle injury by cardi- tal imaging of adult regenerating muscle and showed otoxin injection in the upper hindlimb and monitored that tdTOM fluorescence is sufficient to follow MYOG the regeneration process by live intravital imaging at dif- cells throughout the regeneration process. ferent time points (Fig. 4d). As expected, few MYOG cells were detected at 3 days post-injury, when extensive Conclusion proliferation of YFP myogenic progenitor cells was tak- In this study, we describe the creation of a new mouse line ing place (Fig. 4d, left panels). Two days later, the popu- where tdTOM is expressed from the endogenous Myog lation of tdTOM myoblasts had significantly expanded locus. tdTOM faithfully recapitulates MYOG expression and tdTOM cells could be observed throughout the re- during embryonic development and adult muscle regener- generating area (Fig. 4d, right panels, white arrows), re- ation and it can be used to isolate this population by flow capitulating the results of our flow cytometry analysis. cytometry. Additionally, heterozygous tdTOM expression Taken together, these experiments demonstrate that is sufficient for monitoring Myog dynamics by in vivo in- ntdTom tdTOM is a robust reporter that allows monitoring of travital imaging. Therefore, the Myog line can be of MYOG cells by in vitro and intravital imaging. great benefit to study the dynamics of lineage progression of muscle progenitors in embryonic and adult stages. Discussion Myog is a critical regulator of myoblast differentiation Supplementary Information and fusion, being an essential factor for embryonic The online version contains supplementary material available at https://doi. muscle development. In the present study, we generated org/10.1186/s13395-021-00260-x. and characterised a novel mouse line to fluorescently label MYOG cells by expression of the robust nuclear Additional file 1 Related to Fig. 2a. Whole mount immunofluorescence +/+ from a Myog embryo at E10.5 stained for tdTOM, MYOG and MyHC localised tdTOM protein from the endogenous Myog proteins. locus. Additional file 2 Related to Fig. 2a. Whole mount immunofluorescence To characterise the properties of tdTOM cells, we ntdTom/+ from a Myog and embryo at E10.5 stained for tdTOM, MYOG and assessed its co-localisation with MYOG during embryonic MyHC proteins. and foetal development, in adult primary myogenic cells Additional file 3 Related to Fig. 2a. Whole mount immunofluorescence ntdTom/ntdTom from a Myog embryo at E10.5 stained for tdTOM, MYOG and in vitro and during adult muscle regeneration in vivo. MyHC proteins. Given that in all conditions virtually all cells were positive Additional file 4 Related to Fig. 4a. In vitro division of a tdTOM cell. for both markers (> 95% cells), we conclude that tdTOM Additional file 5: Figure S1. Genotyping of the ntdTom allele. A. reliably labels MYOG cells from development to adult- ntdTom/ntdTom ntdTom/+ Genotyping of ear-clip samples from Myog , Myog and ntdTom/+ +/+ hood. Furthermore, total Myog levels from Myog Myog animals. Myog-ntdTom allele was verified by PCR using primers 16, 17 and 18 (Flp-recombined Myog-ntdTom allele, 236 bp and WT allele animals were comparable to that of the WT, and homozy- 600 bp). ntdTom/ntdTom gous Myog mice are viable. Moreover, the ex- Additional file 6: Figure S2. tdTOM expression recapitulates pression of tdTOM allowed us to isolate the MYOG endogenous Myog expression. A. Immunofluorescence of ntdTom/+ population from developing embryos as well as adult re- extraocular (EOM), tongue and limb muscles of Myog embryo at E14.5. Bar graph shows the quantification of tdTOM and MYOG positive generating muscle indicating the utility of this reporter cells. n = 3 embryos, > 100 cells per muscle and per embryo were mouse in isolating living differentiating myoblasts that counted. Scale bar, 40 μm. B. RT-qPCR assessing the levels of total Myog +/+ were previously inaccessible for direct investigation. mRNA, the wild-type allele and the tdTom allele specifically from Myog , ntdTom/+ ntdTom/ntdTom Myog and Myog adult myoblasts using the primer set In addition, studies on the cell cycle dynamics of described in Fig. 1c. n = 3 animals per genotype. Data represents mean ± Myog cells have been hampered by the lack of a fluores- s.d. Two-tailed unpaired Student’s t-test; * p-value = 0.01 to 0.05. C. +/+ cent reporter. Here, by means of live microscopy and MuSCs from limb muscles were isolated from Tg:Pax7-nGFP; Myog , ntdTom/+ ntdTom/ntdTom Tg:Pax7-nGFP; Myog and Tg:Pax7-nGFP; Myog animals single-cell tracking of differentiating primary myoblasts, and plated for in vitro differentiation for 5 days. Cells were stained for we demonstrated that about one third of MYOG cells MYOG and tdTOM proteins. Scale bar, 100 μm. can divide in vitro and undergo a maximum of one add- Additional file 7: Supplementary Table 1. Primer sequences itional cell division during the tracking period. There- fore, the majority of cells that express detectable levels Abbreviations of MYOG exit the cell cycle. Myog: Myogenin; MRF: Myogenic regulatory factor; MuSC: Muscle stem cell; Several studies have performed intravital imaging of BrdU: 5-Bromo-2′-deoxyuridine; sgRNA: Single guide RNA; CRISPR: Clustered regularly interspaced short palindromic repeats; NLS: Nuclear localisation muscle tissue [24, 51–53]; however, only two of them sequence; GFP: Green fluorescent protein; RT-qPCR: Real-time quantitative dynamically monitored the process of muscle regener- polymerase chain reaction; KI: Knock-in; FACS: Fluorescence-assisted cell ation [24, 51]. These two studies focused on the sorting; TA: Tibialis anterior; YFP: Yellow fluorescent protein Benavente-Diaz et al. 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