Figures and data in Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis

Figures
Tables
Additional files

4 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement

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Myogenin is required for normal myofibre size and nuclear accretion.

(A) Schematic of trunk muscle processing for analysis, pink represents fish fillet. Colours identify sib (*myog^kg125/+,* blue) or *myog*^-/- (*myog^kg125,* red) samples throughout the figure. (B) qPCR analysis shows downregulation of *myog*, *mymk*, *mymx* and *mrf4* mRNAs in adult *myog*^-/-. Symbol shapes denote paired sib and *myog*^-/- samples, n = 4–6 fish/genotype, paired t-test. (C) Schematic of myofibre isolation for morphometric analysis (top) and representative images (bottom) showing smaller *myog*^-/- myofibre (red brackets) compared to age-matched sib. Scale bar = 100 μm. (D) Measure of absolute myofibre length, n = 3 fish/genotype, n = 110–120 myofibres/fish, unpaired t-test. (E) Representative images and measure of unaltered sarcomere length on freshly isolated myofibres, n = 3 fish/genotype, n = 10 myofibres/fish, unpaired t-test. Scale bar = 10 µm. (F) Representative images of isolated fixed adult myofibres show size reduction in *myog^-/-* (red brackets). Scale bar = 100 µm. (**G–J**) Quantification of number of nuclei/100 µm (G), absolute number of nuclei per myofibre (H), myofibre diameter (I), and nuclear domain size (myofibre volume per nucleus) (J) showing significant changes in myofibres from juvenile (Juv, 1 month-old) and adult (Ad, 8 months-old) stages within (coloured p) or among (black p) genotypes. n = 3 fish/genotype, n = 30–50 adult myofibres/fish, n = 15–20 juvenile myofibres/fish, one-way ANOVA. (K) Relationship of number of nuclei and log_e(Surface Area) indicates different growth mode between sib and *myog*^-/- (i.e. significant slope difference, black p), despite significant correlation of log_e(SA) with nuclear number within genotype (coloured p) (see Materials and methods). (L) Increase in Surface Area (SA) from juvenile to adult stage (=Ad_SA – Juv_SA) indicates reduced growth rate in *myog*^-/-. Data from Figure 1—figure supplement 1E, unpaired t-test. All graphs report mean ± SEM. Statistical significance within (coloured p) or between (black p) genotypes is indicated.

Figure 1—figure supplement 1

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*Myog^kg125* mutant adult and juvenile myofibres are smaller.

(A) Fraction of adult myofibres (%) with indicated length in sib (blue, *myog^kg125/+*) and *myog*^-/- (red, *myog^kg125*). n = 3 fish/genotype, n = 110–120 myofibres/fish, p-value indicates probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² test. (B) Measurement of 1-month-old juvenile myofibre length, n = 3 fish/genotype, unpaired t-test (left) and fraction of juvenile myofibres (%) with indicated length in sib (blue) and *myog*^-/- (red), n = 3 fish/genotype, n = 15–20 juvenile myofibres/fish, p-value indicates probability of rejecting null hypothesis of no difference between *myog^-/-* and sib in χ² test. (right). (C) Measure of absolute adult myofibre length, n = 3 fish/genotype, n = 100 myofibres/fish, unpaired t-test; sib (blue, *myog^fh265/+*), hypomorphic mutant (cyan, *myog^fh265*). (D) Representative images showing sib and *myog*^-/- juvenile myofibres (left), scale bar = 100 µm. Quantification of absolute number of nuclei per juvenile myofibre. n = 3 fish/genotype, n = 15–20 myofibres/fish, unpaired t-test. (**E–F**) Quantification of average surface area/100 µm myofibre length (E) and SADS (Surface Area Domain Size) (F) showing significant changes at juvenile (Juv) and adult (Ad) stages within (coloured p) or among (black p) genotypes. n = 3 fish/genotype, n = 30–50 adult myofibre/fish, n = 15–20 juvenile myofibre/fish, one-way ANOVA.

Figure 2 with 1 supplement

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Lack of Myogenin alters MuSC number and localisation.

Colours identify sib (blue) or *myog^-/-* (red) samples throughout the figure. (A) qPCR analysis showing upregulation of *pax7a*, *pax7b*, *myf5* and unaltered *myod* in adult *myog*^-/- mutant muscle. Symbol shapes denote paired sib and *myog*^-/- samples, n = 6 fish/genotype, paired t-tests. (B) Schematic of myofibre isolation for GFP^+ve cells analysis from *pax7a:GFP;myog*^-/- and sib muscles. (C) Representative images showing GFP^+ve cells on sib (blue arrowhead) or *myog*^-/- (red arrowheads) on isolated myofibres. Scale bar = 100 µm. (D) Representative immunodetection of GFP (green), DP312 (Pax3/Pax7; magenta) and nuclei (white) on GFP^+ve (positive, green panel) cells or GFP^-ve (negative, grey panel) nuclei on freshly isolated adult sib or *myog*^-/- myofibres. Scale bar = 5 µm. Quantification of DP312 immunostaining on *pax7a:GFP* MuSCs confirms the stem cell identity of both sib (n = 36/genotype) and *myog^-/-* (n = 67/genotype) GFP^+ve cells, n = 3 fish/genotype unpaired t-test. (**E,F**) Quantification of absolute number of GFP^+ve cells (*bona fide* MuSC) per myofibre and myonuclear domain size. n = 3 fish/genotype, n = 20–30 myofibres/fish, unpaired t-test. (G) Pie charts showing the fraction of nuclei (left) and fraction of myofibres (right) with the indicated number of nuclei in GFP^+ve cells (left) or cells/myofibre (right) in sib (blue circles) and *myog*^-/- (red circles). Data from Figure 2—figure supplement 1G. p-values indicate probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² tests. (H) Diagram of measurement of GFP^+ve cell distance from nearest myofibre-end (top). Each myofibre was segmented into tenths, where 1/10 and 5/10 corresponded to the segments nearest to and furthest from the myofibre-end, respectively. Quantification of fraction of MuSC located within each myofibre segment (bottom, see Materials and methods and Figure 2—figure supplement 1H). n = 3 fish/genotype, n = 30–50 MuSCs, p-value indicates probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² test. (I) Representative images showing localisation of the GFP^+ve cell on sib (blue rectangle) or *myog*^-/- (red rectangle) mono-MuSC myofibres (with only one GFP^+ve cell). Scale bar = 100 μm (=20 µm in 2x-zoom). (J) Quantification of fraction of MuSC located within each 10 µm segment from mono-MuSC myofibre-end. n = 3 fish/genotype, n = 15–25 MuSCs, black p-value indicates probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² test. Coloured p-values indicate one-way ANOVA analysis of non-random MuSC distribution in 10 µm segments within each genotype. Magenta inset reports fraction of sib and *myog^-/-* MuSC within the 0–9 and 10–19 µm segments, unpaired t-test. Symbols show results for individual fish. All graphs report mean ± SEM.

Figure 2—figure supplement 1

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*pax7a:GFP;myog^kg125* muscle is smaller and displays altered MuSC cellularity and unchanged *Pax3* genes expression.

(A) Schematic of trunk muscle processing for qPCR analysis (left), sib (blue), *myog*^-/- (red). Expression of *pax3a* and *pax3b* mRNAs is unchanged in *myog*^-/- muscle (right). Symbol shapes denote paired sib and *myog*^-/- samples, n = 6 fish/genotype, paired t-test. (B) Representative images of adult *pax7a:GFP;myog^+/-* (sib) and *pax7a:GFP;myog*^-/- (left). Scale bar = 1 cm. Quantification showing reduced length, weight, body mass index and standard weight in *myog*^-/- compared to co-reared sibs (right), n = 7, unpaired t-test. (C) Schematic of *pax7a:GFP* adult trunk muscle processing for MuSC analysis (left). Fraction of GFP^+ve cells (*bona fide* MuSCs) per myofibre-associated nucleus is significantly increased in *myog*^-/- muscle (right). n = 3 fish/genotype, unpaired t-test. (D) Quantification of absolute number of (non-MuSC, GFP^-ve) myonuclei per adult myofibre. n = 3 fish/genotype, n = 20–30 myofibres/fish, unpaired t-test. (**E,F**) Quantification of absolute myofibre volume and myonuclear SADS (Surface Area Domain Size). n = 3 fish/genotype, n = 20–30 myofibres/fish, unpaired t-test. (G) Fraction of myofibres (%) with indicated number of GFP^+ve cells (MuSCs), n = 3 fish/genotype, n = 20–30 myofibres/fish,p-value indicates probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² test. (H) Schematic of GFP^+ve cell distance to nearest myofibre-end measurement (left). Note that while average half-length (in magenta) of sib myofibre (blue diagram) is 500 µm, *myog*^-/- myofibre average half-length is 250 µm (red diagram). To compare absolute distance to myofibre-end, each myofibre was segmented into 50 µm segments and the fraction of GFP^+ve cells (%) within each distance segment from nearest myofibre-end plotted, n = 3 fish/genotype, n = 20–30 MuSCs/fish, p-value indicates probability of rejecting null hypothesis of no difference between *myog*^-/- and sib in χ² test. Note that in sibs almost no GFP^+ve MuSCs were located in the central 50% of their myofibre. All graphs report mean ± SEM.

Figure 3 with 1 supplement

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*Myog^-/-* mutant muscle and MuSCs display enhanced mTORC1 signalling.

(A) Schematic of adult trunk muscle processing for analysis. Colours identify sib (blue) or *myog*^-/- (red) samples throughout the figure. (B) qPCR analysis shows upregulation of *igf1* and downregulation of *mstnb* in *myog*^-/- muscle. Symbol shapes denote paired sib and *myog*^-/- samples, n = 4 fish/genotype, paired t-test. (C) Summary of mTORC1 pathway with analysed components, positive (arrows) or negative (bars) effects are indicated. Dashed lines indicate other molecules involved; red dots represent phosphorylation events. (D) Western blot (left) and quantification (right) of phosphorylated/pan AKT (pAKT, Ser473) ratio and phosphorylated/pan RPS6 (pRPS6, Ser240/244). pRPS6 is increased in *myog*^-/- muscle, n = 3 fish/genotype, unpaired t-test. Actin immunoreactivity was used to normalise pan and phospho signals to protein loading prior to calculation of sample-specific phospho/pan ratio. (E) Representative images of myofibre immunostained for GFP (green), pRPS6 (red) and nuclei (white) fixed freshy immediately after isolation. Arrowheads indicate MuSCs on sib (blue) or *myog*^-/- (red) myofibres. Dashed magenta squares highlight 4x-magnified cells. Scale bar = 100 µm (=5 µm in 4x-zoom). (F) Quantification of pRPS6 intensity shows increase in *myog*^-/- MuSCs. Symbol shapes denote paired sib and *myog*^-/- samples, n = 4 fish/genotype, n = 20–30 MuSCs/fish, paired t-test. (G) qPCR analysis showing downregulation of *tsc1a* and *tsc1b* mRNAs but unaltered *tsc2* mRNA in *myog*^-/- muscle. Symbol shapes denote paired sib and *myog*^-/- samples, n = 4–6 fish/genotype, paired t-test. All graphs report mean ± SEM.

Figure 3—figure supplement 1

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*Myog^-/-* mutant muscle displays unaltered *igfr1a* and *igfr1b* mRNA expression.

Schematic of adult trunk muscle processing for qPCR analysis (left). Summary of mTORC1 pathway with analysed members, positive (arrows) or negative (bars) effects are indicated (centre). Dashed lines indicate other molecules involved, plain arrow indicate direct effect, red dots represent phosphorylation. *Igfr1a* and *igfr1b* are unchanged in *myog*^-/- muscle compared to sib (right). Symbol shapes denote paired sib and *myog*^-/- samples, n = 5 fish/genotype, paired t-test.

Figure 4 with 1 supplement

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*Myog^-/-* mutant MPCs exhibit faster entrance into proliferation phase.

(A) Schematic of adult single myofibre culture and representative immunodetection of GFP (green) and nuclei (white) on cultured MPCs. Colours identify sib (blue) or *myog*^-/- (red) samples throughout the figure. (B) Quantification of GFP^+ve cells obtained by single myofibre culture revealed increased MPCs yield from *myog*^-/- after 4 days in growth medium, n = 3 fish/genotype, n = 8 myofibres/fish. Unpaired t-test on average MPCs/fish/genotype. (C) Fraction of GFP^+ve cells out of total number of cells obtained in B. n = 3 fish/genotype, unpaired t-test. (D) Relative proliferation rate of sib or *myog*^-/- MPCs calculated as the ratio of the average number of GFP^+ve MPCs/plated-myofibre obtained on day 4 in culture divided by the average number of GFP^+ve MuSCs/myofibre, unpaired t-test. (E) Diagram of myofibres culture and EdU pulse regime (top). Quantification of fraction of EdU^+ve MPCs at indicated time points (bottom left), symbol shapes denote MPCs obtained from paired sib and *myog*^-/- samples, n = 3. Measurement of Area Under Curve (AUC, coloured area underneath dashed blue or red lines) to compare overall proliferation dynamics indicates reduction in *myog*^-/- MPCs (bottom right), unpaired t-test. (F) Representative images of sib and *myog*^-/- MPC numbers and EdU incorporation at indicated times in culture, showing detection of EdU (red) and nuclei (blue). (G) qPCR analysis showing expression dynamics of *pax7a*, *pax7b*, *myod* and *myf5* mRNAs in sib and *myog*^-/- MPCs cultured for 2 (n = 4) or 3 days (n = 3), unpaired t-test. (H) qPCR analysis indicating upregulation of *mef2d, mylpfa*, *flnca* and *mybpc1* mRNAs in *myog*^-/- MPCs cultured in growth medium for 2 (n = 4) or 3 days (n = 3), unpaired t-test. (I) Representative immunodetection of MyHC (red) and nuclei (white) on sib or *myog*^-/- MPCs at 3 days in growth medium for 3 days (left). Extent of differentiation (Index (%), see Materials and methods) is higher in *myog*^-/- than in sib MPCs (right). All graphs report mean ± SEM. Statistical significance within (coloured p) or between (black p) genotypes is indicated. Scale bars = 100 µm.

Figure 4—figure supplement 1

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Characteristics of cultured MPCs and mRNA analysis.

(A) Schematic of single myofibre culture and analysis of derived MPCs after 4 days from plating in growth medium (left). Total number of cells (= GFP^+ve + GFP^-ve) is increased in *myog*^-/- (centre). n = 3 fish/genotype, n = 8 myofibre/fish. Unpaired t-test on average number of cells/fish/genotype. Representative immunostaining of Desmin (red) and nuclei (white) confirming myogenic identity of both sib and *myog^-/-* cells from single-plated myofibres at 4 days (right). (B) Schematic of 90–100 myofibres culture in growth medium (GM) and analysis of derived MPCs (left). Representative immunostaining of Desmin (red), GFP (green) and nuclei (white) confirming myogenic identity of both sib and *myog*^-/- cells after 2 days of culture (centre). *Myog*^-/- myofibres yielded higher number of Desmin⁺ MPCs per field of view (FOV). Symbol shapes denote paired sib and *myog*^-/- samples, n = 3 fish/genotype, paired t-test (right). (C) 90–100 myofibres cultured from each genotype yielded over 90% of Desmin⁺ cells, confirming high purity of the MPC population, n = 3 fish/genotype, unpaired t-test. (D) qPCR analysis shows expression dynamics of *cdkn1a* (p21) and *cdkn1ca* (p57) mRNAs in sib and *myog*^-/- MPCs cultured for 2 (n = 4) or 3 days (n = 3), unpaired t-test. Statistical significance within (coloured p) or between (black p) genotypes is indicated. (E) Schematic of 90–100 myofibres culture and analysis of derived MPCs cultured for 5 days in differentiation medium (DM), following expansion for 4 days in growth medium (GM) (left). Representative immunostaining of MyHC (red), nuclei (white) confirming that fusion into multinucleated myotubes occurred only in sib MCPs, whereas differentiated *myog^-/-* cells remained mostly mononucleated, as reported previously (Ganassi et al., 2018) (right). Dashed cyan squares highlight 3x-magnified cells, coloured arrowheads indicate nuclei of each separate differentiated cell. All graphs report mean ± SEM. Scale bars = 100 µm.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. rerio)	myog^fh265, (AB)	Hughes/Moens Labs, Hinits et al., 2011	myog^fh265; RRID:ZFIN_ZDB-GENO-200128-7
Genetic reagent (D. rerio)	myog^kg125, (TL)	Hughes Lab, Ganassi et al., 2018	myog^kg125/+ (sib) myog^-/- (mut); RRID:ZFIN_ZDB-GENO-200128-8
Genetic reagent (D. rerio)	TgBAC(pax7a:GFP)^t32239Tg, (AB)	Nüsslein-Volhard Lab, Mahalwar et al., 2014	pax7a:GFP; RRID:ZFIN_ZDB-GENO-170316-2
Genetic reagent (D. rerio)	myog^kg125;TgBAC (pax7a:GFP)^t32239Tg, (AB)	This paper	pax7a:GFP;myog^kg125	Generated from outcrossing myog^kg125 onto pax7a:GFP
Antibody	Anti-phospho-S6 ribosomal protein (Ser 240/244) (Rabbit monoclonal)	Cell Signalling	#5364; D68F8; RRID:AB_10694233	(1:1000) IF (1:2000) WB
Antibody	Anti-S6 ribosomal protein (pan) (Mouse monoclonal)	Cell Signalling	#2317; 54D2; RRID:AB_2238583	(1:1000)
Antibody	Anti-phospho-Akt (Ser473) (Rabbit monoclonal)	Cell Signalling	#4060; D9E; RRID:AB_2315049	(1:1000)
Antibody	Anti-Akt (pan) (Mouse monoclonal)	Cell Signalling	#2920; 40D4; RRID:AB_1147620	(1:1000)
Antibody	Anti-beta-Actin (Mouse monoclonal)	Sigma Aldrich	#A5316; AC-74; RRID:AB_476743	(1:500)
Antibody	HRP goat-anti mouse IgG(H+L) (Goat polyclonal)	Sigma Aldrich	#AP308P; RRID:AB_92635	(1:5000)
Antibody	HRP goat anti -abbit IgG (H+L) (Goat polyclonal)	Sigma Aldrich	#AP307P; RRID:AB_11212848	(1:5000)
Antibody	Anti-Pax3/Pax7 DP312 (Mouse monoclonal)	Nipam Patel, UC Berkeley, USA	DP312	(1:50)
Antibody	A4.1025 anti-Myosin heavy chain (Mouse monoclonal)	Hughes Lab, Blagden et al., 1997 Developmental Studies Hybridoma Bank (DSHB) Merck	DSHB #A4.1025; Merck #05–716; RRID:AB_309930	(1:5)
Antibody	MF20 anti-Myosin heavy chain (Mouse monoclonal)	Developmental Studies Hybridoma Bank (DSHB)	#MF20; RRID:AB_2147781	(1:300)
Antibody	Anti-Desmin (Mouse monoclonal)	Sigma Aldrich	#D8281; RRID:AB_476910	(1:100)
Antibody	Anti-GFP (Chicken polyclonal)	Abcam	#13970; RRID:AB_300798	(1:400)
Antibody	Alexa Fluor 488 goat anti-chicken IgY (H+L) (Goat polyclonal)	Invitrogen	#A11039; RRID:AB_2534096	(1:1000)
Antibody	Alexa Fluor 555 goat anti-rabbit IgG (H+L) (Rabbit polyclonal)	Invitrogen	#A27039; RRID:AB_2536100	(1:1000)
Other	Hoechst 33342 nuclear stain	Thermo Fisher	#H3570	(10 µg/ml)
Commercial assay or kit	Click-iT Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor 594 dye	Invitrogen	#C10639	Used according to supplier instruction
Software, algorithm	Generalised linear models (GLS)	Adapted from: Pinheiro et al., 2020; Zeileis, 2002; Ho et al., 2011 ; Sekhon, 2011	Growth mode analysis