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

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. Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom 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. We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts.


Background
Embryonic and postnatal myogenesis and adult muscle regeneration are regulated by a family of basic helixloop-helix myogenic regulatory factors (MRFs) comprising Myf5, Mrf4, Myod and Myogenin (Myog). Following myogenic specification in the embryo, the MRFs are expressed in a sequential manner to ensure commitment, proliferation, differentiation and fusion to give rise to multinucleated skeletal myofibres. Single and combinatorial mouse knockout models of the MRFs have established a genetic hierarchy where Myf5, Mrf4 and Myod control lineage commitment and proliferation of myogenic progenitors, and Myod, Mrf4 and Myog regulate in terminal differentiation [1]. Notably, amongst the single MRF knockout mice, only Myog-null homozygous animals die at birth due to severe skeletal muscle defects [1][2][3]. Thus, unlike the other myogenic bHLH factors, Myog has no redundant or compensatory mechanisms to replace its function during development. Myoblasts lacking this gene accumulate in the muscle-forming areas throughout the body and fail to form normal myofibers in vivo, pointing to its critical role in terminal differentiation of myoblasts [2][3][4]. While Myog-null embryos have some disorganised residual primary fibres, major differences between mutant and wild-type embryos become apparent during the initiation of secondary myofibre formation [2,4]. Unexpectedly, conditional ablation of Myog during the perinatal and postnatal period does not result in noticeable defects in muscle morphology or histology, suggesting that Myog −/− myoblasts can still contribute to muscle growth [5,6]. Additionally, conditional ablation of Myog in a Duchenne muscular dystrophy mouse model (mdx [7]) did not result in an adverse phenotype, confirming that Myog is dispensable for adult muscle regeneration in this disease [8]. Nevertheless, although Myog-null muscle stem cells (MuSCs) proliferate and differentiate in culture as efficiently as wild-type cells, the muscle gene expression program is profoundly altered in the absence of Myog [6].
Adult muscle regeneration depends on MuSCs, characterised by the expression of Pax7 [9][10][11][12][13]. Upon muscle injury, MuSCs activate the expression of Myod, proliferate to generate myoblasts that differentiate and fuse to form myofibres. Different reporter mouse lines have been generated to fluorescently label the Pax7 + muscle progenitor population, either from the endogenous locus [14][15][16] or as transgenes [15,17,18], thereby allowing imaging and isolation of Pax7-expressing cells. Additionally, inducible reporters in which expression of the Cre recombinase under the control of the Pax7 promoter recombines a membrane or cytoplasmic fluorophore [13,19,20] have been used for permanent marking of the myogenic lineage [21][22][23] and for live imaging [24]. Although several reporter mouse lines have been generated to identify differentiating myoblasts based on the expression of Myosin light chain [25], Myog [26][27][28] and Muscle creatine kinase [29], they are based on lacZ (βgalactosidase activity, [30]) or cat (chloramphenicol acetyltransferase, [31]) expression and thus only allow endpoint measurements on fixed samples.
Terminal myoblast differentiation is characterised by the expression of Myog and the cyclin-dependent kinase inhibitor p21 and cell cycle withdrawal [32][33][34]. Experiments using the nucleotide analogue BrdU have shown that MYOG-positive cells can undergo DNA replication [32], but it is still unclear how many divisions they can execute before definitively leaving the cell cycle.
Here, we took advantage of the CRISPR/Cas9 system, which allows precise genome editing [35], to generate a knock-in mouse line expressing a nuclear localised tandem-dimer Tomato (tdTOM) protein under the control of the endogenous Myog promoter, while retaining expression of MYOG protein. We show that heterozygous Myog ntdTom mice exhibit robust reporter gene expression in fixed and live myogenic cells thus allowing in vitro and intravital microscopy studies of the dynamics of muscle differentiation and cell cycle withdrawal.

Mouse maintenance
Animals were handled according to national and European Community guidelines and an ethics committee of the Institut Pasteur (CETEA, Comité d'Ethique en Expérimentation Animale) in France approved protocols (Licence 2015-0008). Except when indicated otherwise, males and females of 2-4 months were used.
Generation of the Myog-ntdTomato construct for CRISPR-Cas9-mediated homologous recombination A fragment of 1000 bp from the last exon of Myog was amplified by PCR from murine gDNA (primers 1 and 2, Supplementary Table 1), introducing SalI and NotI restriction sites. This fragment was subcloned into the donor plasmid encoding for tdTOM (kind gift from Dr. Festuccia, Institut Pasteur). A fragment of 760 bp from the 3′UTR of the Myog gene just after the STOP codon was amplified by PCR from murine gDNA (primers 3 and 4). This amplification also introduced a mutation in the PAM sequence necessary for CRISPR-Cas9 genome editing. Using the PacI and SpeI restriction sites added, the fragment was subcloned into the PacI and XbaI digested tdTOM plasmid. Oligos containing a T2A (primers 5 and 6) [36] peptide and a triple NLS sequence from SV40 large T [37] were annealed and subcloned into a blunt pBluescript SK (+) plasmid. This plasmid was subsequently digested with NotI and KpnI and the T2A-NLS fragment was cloned into the tdTOM plasmid. tdTOM was amplified by PCR from the initial plasmid (primers 7 and 8) adding KpnI and FseI sites and subcloned into the donor vector after the 3xNLS sequence. An FNF cassette containing two FRT sites and the NeoR/KanR gene under the control of the PGK promoter was amplified by PCR (primers 9 and 10) adding FseI and PacI sites. This fragment was subcloned into the donor vector.

Targeting of mouse embryonic stem cells
The Myog-ntdTOM donor construct (linearised by PvuI digestion) and the pU6 vector were electroporated in C57BL/6J mouse embryonic stem cells. Following G418 (300 μg/ml) selection, positive clones were determined by PCR using primers 13 Figure S1), and these primers were subsequently used for genotyping. F2 animals were backcrossed to C57BL/6 animals to eliminate the Tg(ACTFLPe) allele, and Myog ntdTom/+ animals were selected for further characterisation.

Embryo immunofluorescence
For tissue immunofluorescence, embryos were collected in PBS and fixed in 4%PFA 0.1% Triton X-100 in PBS for 2 h at 4°C. After 3 PBS washes, embryos were cryopreserved in 30% sucrose in PBS and embedded in OCT tissue freezing media (Leica, 14020108926) for cryosectioning. Cryosections were allowed to dry for 30 min at room temperature and washed once with PBS. Tissue samples were blocked in 3% BSA, 10% goat serum, 0.5% Triton X-100 for 1 h at room temperature. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. After three washes with PBST (PBS 0.1% Tween20 (Sigma Aldrich, P1379)), secondary antibodies were diluted in blocking solution and incubated for 45 min at room temperature. Finally, samples were incubated with 1 μg/ml Hoechst 33342 for 5 min at room temperature to visualise nuclei, washed three times in PBS and mounted in 70% glycerol in PBS for imaging.
For whole-mount immunofluorescence, embryos were collected in PBS and fixed in 4% PFA 0.1% Triton X-100 for 2 h at 4°C. After two PBS washes, samples were dehydrated in 50% methanol in PBS and kept in 100% methanol at − 20°C until used. Samples were rehydrated in PBS and incubated in blocking buffer (15% goat serum, 1.5% BSA, 0.5% TritonX-100 in 1X PBS) for 1 h at RT in 2-ml Eppendorf tubes. Embryos were then incubated with primary antibodies in the blocking buffer for 5-7 days at 4°C with rocking. Embryos were washed extensively for 2-4 h in PBST and incubated in Fab' secondary antibodies for 2 days at 4°C with rocking. Embryos were washed as above, dehydrated in 50% methanol in PBS, twice in 100% methanol and then cleared with BABB and mounted for imaging [40].
Injured TA muscles were fixed upon harvesting in 4% PFA for 2 h at 4°C, washed with PBS and equilibrated with 30% sucrose in PBS overnight. Samples were mounted in OCT tissue freezing media and cryosectioned between 8 and 12 μm. When endogenous tdTOM was scored, cryosections were rehydrated in PBS and counterstained with Hoechst 33342.
In case of MYOG plus tdTOM detection, tissue sections were processed for histology as described [41]. Briefly, sections were post-fixed in 4% PFA for 10 min at RT and washed with PBS prior to immunostaining. Heat-induced epitope retrieval was performed in a citrate solution pH 6.0 during 6 min in a pressure cooker. Sections were then incubated with 30% H 2 O 2 for 5 min at RT. Samples were then permeabilised with 0.2% Triton-X100, washed in PBS and blocked in blocking buffer (15% goat serum, 1.5% BSA, 0.5% TritonX-100 in 1X PBS). Primary antibodies against MYOG and DsRED (recognising tdTOM) were incubated overnight at 4°C. After washing with PBST, sections were incubated with appropriate secondary antibodies and 1 μg/ml Hoechst 33342 in blocking buffer for 45 min at RT (Table 1).

RNA extraction
RNA from cells isolated by FACS was extracted using a Trizol-based kit (Zymo Research, R2061) and reverse transcribed using SuperScriptIII (Invitrogen, 18080093). RT-qPCR to assess for mRNA relative expression was performed with SYBR green master mix (Roche, 04913914001) in Applied biosciences machine. Data analysis was performed using the 2 −ΔΔCT method [42] and mRNA expression was normalised with Rpl13 (primers 23 and 24, Supplementary Table 1).

In vitro videomicroscopy
MuSCs were plated on a microscopy culture chamber (IBIDI, 80826) and cultured in growth media supplemented as above. The plate was incubated at 37°C, 5% CO 2 and 3% O 2 in a Pecon incubation chamber. A Zeiss Observer.Z1 connected to a Plan-Apochromat 20x/0.8 M27 objective and Hamamatsu Orca Flash 4 camera piloted with Zen software (Carl Zeiss) was used.

Static imaging
The following systems were used for image acquisition: Zeiss SteREO Discovery V20 for macroscopic observations of whole embryos and Zeiss LSM800 or LSM700 laser-scanning confocal microscopes for tissue sections and whole-mount immunostaining of cleared embryos. End point in vitro culture samples were imaged with a Zeiss Observer.Z1.

Intravital microscopy
Intravital imaging of Pax7 CreERT2 ; R26 YFP ; Myog ntdTom mice at different timepoints during regeneration was performed on an upright Nikon NiE A1R MP microscope piloted with NIS software (Nikon). The microscope was equipped with a × 25 NA 1.1 PlanApo LambdaS objective, GaAsP PMT detectors and a Spectra-Physics Insight Deepsee laser. Laser frequency  was tuned to 960 nm to allow simultaneous excitation of YFP and tdTOM fluorophores. For image acquisition, the skin over the upper hindlimb was shaved and incised to expose approximately 1 cm 2 of the muscle and imaged directly. During the imaging period, mice were anaesthetised with 1.5% isofluorane and maintained in an incubation chamber at 37°C.

Image analysis
Cell tracking was performed using the Manual Tracking feature of the TrackMate plug-in [43] in Fiji [44]. ZEN software (Carl Zeiss), Fiji [44] and Imaris (Bitplane) were used for image analysis. Figures were assembled in Adobe Photoshop and Illustrator (Adobe Systems).

Data analysis and statistics
Data analysis and statistics were performed using R [45], and figures were produced using the package ggplot2 [46]. For comparison between two groups, two-tailed paired and unpaired Student's t tests were performed to calculate p values and to determine statistically significant differences (see figure legends).

Generation and characterisation of a Myog ntdTom mouse
Using the CRISPR-Cas9 system, a sgRNA was designed to target the region of the STOP codon of the Myog gene for homologous recombination. The recombination template consisted of two homology arms corresponding to Myog sequences flanking the STOP codon, a tdTom coding sequence and a Neo resistance cassette flanked by frt sites (Fig. 1a). The tdTOM protein was preceded by a T2A peptide sequence [36] to allow cleavage from the MYOG protein following translation, and a triple NLS sequence [37] to ensure nuclear localisation.
First, we evaluated the endogenous tdTOM fluorescence in heterozygous Myog ntdTom/+ embryos between E9.75 and E11.5 at the level of the somites, i.e. transient embryonic structures arising from the segmentation of the paraxial mesoderm. Endogenous tdTOM fluorescence followed a similar pattern to that described for Myog transcripts [47] (Fig. 1b), with expression levels being lower in the caudal (more recently formed) somites.
We then collected tissue samples from E14.5 foetuses and performed RT-qPCR and Western blot analysis to confirm that Myog mRNA and protein levels were similar in wild-type, heterozygous and homozygous animals. Primer pairs were designed to amplify specifically the 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 expressed similar levels of total Myog mRNA, and confirmed that no Myog wild-type transcript could be detected in the homozygous embryos. As expected, the levels of Myog ntdTom mRNA were the highest in homozygous samples, decreased to roughly 50% in the case of the heterozygous and were not detected in wild-type embryos (Fig. 1c). At the protein level, we noted similar expression levels of MYOG in embryos from all three genotypes, whereas the tdTOM protein was absent in wild-type samples (Fig. 1d). Therefore, we conclude that MYOG protein was generated from transcripts that originated from both alleles.
To investigate the expression of the targeted allele with higher resolution, we assessed the temporal expression dynamics of MYOG and tdTOM proteins by whole-mount immunostaining at E10.5 and compared the expression of MYOG and tdTOM in wild-type, heterozygous and homozygous embryos (Fig. 2a, Additional files 1, 2, 3). We confirmed that tdTOM followed the expression pattern of MYOG in the epaxial and hypaxial domains of all somites, indicating that both proteins have similar spatiotemporal expression dynamics. To assess the co-expression of MYOG and tdTOM at the single cell level in heterozygous embryos, cryosections at the level of extraocular, tongue and limb muscles were examined during primary (E12.5) and secondary (E14.5) myogenesis when small oligo-nucleated and larger multi-nucleated myofibres are generated, respectively. Quantification of protein expression in different muscles confirmed an average co-localisation of both proteins in 97% and 95% of the cells at E12.5 (Fig. 2b) and E14.5 ( Figure  S2A), respectively.
(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 CRISPR-Cas9-mediated recombination in mouse embryonic stem cells. First-generation Myog ntdTom-FNF mice were then crossed with a Tg(ACTF LPe) +/+ deleter strain to excise the FNF cassette. b Endogenous fluorescence from Myog ntdTom/+ 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 tdTom allele specifically from E14.5 Myog +/+ , Myog ntdTom/+ and Myog ntdTom/ntdTom 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 of MYOG and tdTOM proteins from E14.5 Myog +/+ , Myog ntdTom/+ and Myog ntdTom/ntdTom 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 To assess the fidelity of the reporter mouse in adult myoblasts, Myog ntdTom animals were crossed with Tg: Pax7-nGFP mice, where GFP marks all MuSCs [15]. MuSCs were isolated by FACS based on GFP expression, then differentiated in vitro for 5 days. In agreement with our results in the embryo, MYOG and tdTOM expression co-localised in about 95% of the cells (Fig. 2c). Additionally, no significant differences were observed in total Myog RNA levels between wild-type, heterozygous and homozygous animals ( Figure S2B).
As indicated above, the KI strategy was designed to be non-disruptive and allow normal MYOG protein expression from the recombined alleles. Given that Myog-null mice are lethal at birth, and our Myog ntdTom/ntdTom knock-in mice are viable, we propose that sufficient levels of MYOG are produced from the targeted allele. Nonetheless, a decrease in MYOG intensity was detected by immunofluorescence in homozygous Myog ntdTom/ntd-Tom embryos and in vitro myoblast cultures from homozygous animals (Fig. 2a, Figure S2C). As this decrease was not observed in heterozygous samples, we decided to use heterozygous animals in our subsequent experiments.
In summary, tdTOM faithfully recapitulates the expression of MYOG protein in embryonic and adult muscle, and its insertion at the Myog locus does not impair significantly the expression of this gene at the mRNA and protein level.

Myog ntdTom mice allow isolation of differentiating myoblasts during development and regeneration
To assess the expression of tdTOM in homeostatic conditions by flow cytometry, we isolated the mononuclear population from limb muscles of Myog ntdTom/+ mice at foetal (embryonic day (E) 18.5), postnatal (postnatal day (p) 21) and adult (10 weeks) stages. tdTOM fluorescent cells were detected at foetal and early postnatal stages where myogenesis was still taking place. In adult muscles in homeostasis, the majority of MuSCs are quiescent and therefore no MYOG + mono-nucleated cells are detectable [48]. As expected, virtually no tdTOM + cells were detected in muscles of adult Myog ntdTom/+ animals (Fig. 3a).
To determine if tdTOM followed the expression dynamics of MYOG during adult muscle regeneration, we performed an injury of the Tibialis anterior (TA) muscle of Tg:Pax7-nGFP; Myog ntdTom/+ mice by intramuscular injection of the snake venom toxin cardiotoxin [49]. We next performed FACS analysis to determine whether the tdTOM + mononucleated fraction could be isolated following tissue injury. As expected, only a few tdTOM + cells were detected at 3 days post-injury, when myogenic cells are known to be maximally proliferating [23,24,50] (Fig. 3b). tdTOM + cells were most abundant at 5 and 10 days post-injury, corresponding to the increased shift towards differentiation of the transiently amplifying myoblast population during this period. As the major features of the regeneration process are completed by 3-4 weeks, the proportion of tdTOM + cells decreased by 21 days post-injury, corresponding to the progressive return to quiescence of the myogenic population (Fig. 3b). Finally, to verify whether the tdTOM + cells isolated by FACS corresponded to myoblasts that expressed MYOG, we isolated the tdTOM + population from regenerating TA muscle at 5 days post-injury. Fixation of cells immediately after sorting and staining for MYOG and tdTOM showed that 95% of isolated cells were positive for MYOG (Fig. 3c), thereby confirming that tdTOM followed the expression dynamics of MYOG also during adult muscle regeneration and that its expression allows the isolation of MYOG + cells by FACS after injury.
Taken together, our results show that the Myog ntdTom KI mouse allows efficient isolation of the MYOG + population at different stages during development as well as from regenerating muscle.

Dynamics of Myog expression during terminal differentiation
To assess if Myog-expressing myoblasts can execute a cell division, we took advantage of the tdTOM reporter to monitor Myog expression by live videomicroscopy of primary myoblasts in vitro. MuSCs from adult Tg:Pax7-nGFP; Myog ntdTom/+ mice were isolated by FACS based on GFP fluorescence and plated for in vitro differentiation. After 3 days of culture, live imaging was initiated and images were acquired every 9 min for 48 h (Fig. 4a, Additional File 4).
By manually tracking individual cells and monitoring their differentiation status based on tdTOM fluorescence, we observed that up to 35% of MYOG + cells  (Fig. 4b, c). Having established that MYOG + cells remain competent for cell division, we sought to quantify the number of divisions that they performed. Amongst all the MYOG + cells tracked, none divided more than once. In contrast, all MYOG − cells divided during the imaging period and performed 1.5 divisions on average during this time (Fig. 4c). Therefore, using the Myog ntdTom reporter mouse, we show that about one third of MYOG + cells can undergo one more cell division when tracking tdTOM expression.
Finally, we assessed the potential of the Myog ntdTom mouse to monitor tissue regeneration by intravital imaging. Pax7 CreERT2/+ ; R26 YFP/+ ; Myog ntdTom/+ mice were  Fig. 4 Myog + cells can undergo cell division. a MuSCs were isolated based on GFP fluorescence from Tg:Pax7-nGFP Tg/+ ; Myog ntdTom/+ 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 Student's t test; **p value = 0.005 to 0.01. d Recombination of Pax7-expressing cells in Pax7 CreERT2 ; R26 YFP ; Myog ntdTom 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 used to permanently label Pax7-expressing cells and their progeny upon tamoxifen administration and simultaneously trace the differentiated fraction by following tdTOM expression. We induced muscle injury by cardiotoxin injection in the upper hindlimb and monitored the regeneration process by live intravital imaging at different time points (Fig. 4d). As expected, few MYOG + cells were detected at 3 days post-injury, when extensive proliferation of YFP + myogenic progenitor cells was taking place (Fig. 4d, left panels). Two days later, the population of tdTOM + myoblasts had significantly expanded and tdTOM + cells could be observed throughout the regenerating area (Fig. 4d, right panels, white arrows), recapitulating the results of our flow cytometry analysis.
Taken together, these experiments demonstrate that tdTOM is a robust reporter that allows monitoring of MYOG + cells by in vitro and intravital imaging.

Discussion
Myog is a critical regulator of myoblast differentiation and fusion, being an essential factor for embryonic muscle development. In the present study, we generated and characterised a novel mouse line to fluorescently label MYOG + cells by expression of the robust nuclear localised tdTOM protein from the endogenous Myog locus.
To characterise the properties of tdTOM + cells, we assessed its co-localisation with MYOG during embryonic and foetal development, in adult primary myogenic cells in vitro and during adult muscle regeneration in vivo. Given that in all conditions virtually all cells were positive for both markers (> 95% cells), we conclude that tdTOM reliably labels MYOG + cells from development to adulthood. Furthermore, total Myog levels from Myog ntdTom/+ animals were comparable to that of the WT, and homozygous Myog ntdTom/ntdTom mice are viable. Moreover, the expression of tdTOM allowed us to isolate the MYOG + population from developing embryos as well as adult regenerating muscle indicating the utility of this reporter mouse in isolating living differentiating myoblasts that were previously inaccessible for direct investigation.
In addition, studies on the cell cycle dynamics of Myog + cells have been hampered by the lack of a fluorescent reporter. Here, by means of live microscopy and single-cell tracking of differentiating primary myoblasts, we demonstrated that about one third of MYOG + cells can divide in vitro and undergo a maximum of one additional cell division during the tracking period. Therefore, the majority of cells that express detectable levels of MYOG exit the cell cycle.
Several studies have performed intravital imaging of muscle tissue [24,[51][52][53]; however, only two of them dynamically monitored the process of muscle regeneration [24,51]. These two studies focused on the progenitor population by labelling PAX7 + cells, but they did not report on the dynamics of differentiation. Here, we carried out proof-of-concept experiments by intravital imaging of adult regenerating muscle and showed that tdTOM fluorescence is sufficient to follow MYOG + cells throughout the regeneration process.

Conclusion
In this study, we describe the creation of a new mouse line where tdTOM is expressed from the endogenous Myog locus. tdTOM faithfully recapitulates MYOG expression during embryonic development and adult muscle regeneration and it can be used to isolate this population by flow cytometry. Additionally, heterozygous tdTOM expression is sufficient for monitoring Myog dynamics by in vivo intravital imaging. Therefore, the Myog ntdTom line can be of great benefit to study the dynamics of lineage progression of muscle progenitors in embryonic and adult stages.