Havis E, Coumailleau P, Bonnet A, Bismuth K, Bonnin MA, Johnson R, Fan CM, Relaix F, Shi DL, Duprez D.

R01AR060042 NIAMS NIH HHS , R01HD35596 NICHD NIH HHS , R01 AR060042 NIAMS NIH HHS , R01 HD035596 NICHD NIH HHS

	Fig. 1. Sim2 is expressed in chick limb muscle cells not expressing MyoD. (A-F) Transverse sections of HH22 chick embryos at forelimb level were co-hybridised with cSim2 (green) and cFgfR4 (red) fluorescent probes (A-C) or with cSim2 (digoxigenin, purple) and cPax3 (fluorescein, orange) probes (D-F). (A-D) Arrowheads indicate ventral muscle masses; arrows show dorsal muscle masses. (F) Arrowheads indicate examples of cSim2+ and cPax3+ cells in ventral muscle masses. (G,H) Limb sections of HH22 chick embryos, incubated with BrdU 1 hour before fixation, were hybridised with cSim2 (E) and cPax3 (F) digoxigenin-labelled probes (blue) and then incubated with the BrdU antibody (brown). Arrowheads indicate the cSim2+ (E) and cPax3+ (F) cells, which are also BrdU positive. (I-N) Transverse sections of HH26 chick forelimbs were labelled by double in situ hybridisation with cMyoD (green) and cSim2 (red) fluorescent probes. (I,K,M) In ventral muscle masses, arrowheads indicate the cSim2 expression domains not overlapping with that of cMyoD, whereas arrows indicate the overlapping cSim2 and cMyoD expression domains. (J,L,N) High magnifications of ventral muscle masses show that in overlapping expression domains, cSim2 mRNA and cMyoD mRNA are not expressed in the same cells.
	Fig. 2. Sim2 expression is not regulated by Notch signalling in chick limbs. (A) Delta1/RCAS-expressing cells were grafted into the presumptive right forelimb buds of HH17 chick embryos. (B-H) Delta1-grafted right (B,D,F,H) and control left (C,E,G) forelimbs from HH26 chick embryos were cut transversely and analysed for cDelta1, cSim2, cFgfr4 and cMyoD expression. Adjacent sections of experimental and control forelimbs were hybridised with cDelta1 (B), cSim2 (C,D), cFgfr4 (E,F) and cMyoD (G,H) digoxigenin-labelled probes. cSim2 expression and that of the progenitor marker cFgfr4 are not affected by Notch signalling misexpression (C-F), whereas cMyoD expression is downregulated (G,H). a, anterior; p, posterior; D, dorsal; V, ventral.
	Fig. 3. Sim2 expression is increased in forelimbs of MyoD–/– mutant mice. (A-D) Forelimb transverse sections of E12 wild-type (A,C) and MyoD–/– (B,D) were hybridised with the mMyog (A,B) and mSim2 (C,D) probes. A,C and B,D are adjacent sections, respectively. (C,D) Arrows indicate the extended mSim2 expression domain in MyoD–/– (D) and the absence of mSim2 expression in control limbs (C). a, anterior; p, posterior; D, dorsal; V, ventral. (E) Relative levels of Pax3, Myf5 Sim2 and Sim2s mRNAs in forelimbs from E11.5 and E12.5 MyoD–/– mutant mice compared with wild-type littermates. The mRNA levels of wild-type and MyoD–/– mice were normalised to that of HPRT in each experiment. The error bars represent s.e.m. **P<0.01; ***P<0.001.
	Fig. 4. Sim2 expression is not regulated by Myf5. (A-C) Adjacent transverse sections from chick embryos electroporated with mMyf5 expression vector in neural tubes were hybridised with probes for mMyf5 (A), cSim2 (B) and cMyoD (C). cSim2 expression is not activated by mMyf5, while cMyoD is. (D-G) Adjacent and transverse sections of hindlimbs from E11.5 wild-type (D,F) and Myf5–/– (E,G) mice were hybridised with the mMyoD (D,E) and mSim2 (F,G) probes. mMyoD expression is reduced in hindlimbs of E11.5 Myf5–/– compared with control mice (D,E), indicating a delay of muscle differentiation in Myf5–/–. On adjacent sections, mSim2 expression is increased in dorsal and ventral muscle masses of Myf5–/– hindlimbs (G, arrows), compared with mSim2 in control hindlimbs (F, arrows). a, anterior; p, posterior; D, dorsal; V, ventral.
	Fig. 5. mMyoD expression is transiently upregulated in limbs of E11.5 Sim2–/– mice. (A-H) Transverse sections of forelimbs (A-F) and hindlimbs (G,H) from E11.5 wild-type (A-C,G) and Sim2–/– (D-F,H) mice were hybridised with the mMyoD probe. mMyoD expression appears increased in limbs of Sim2–/– mutant mice (D-F,H) compared with control limbs (A-C,G). (B,C,E,F) High magnifications of the dorsal and ventral muscle masses of control (A) and Sim2–/– mutant (D) forelimbs. Arrows indicate the increased expression of mMyoD in Sim2–/– forelimbs (E,F) compared with control forelimbs (B,C). (G,H) In hindlimbs, the ventral muscle mass was delineated in the Sim2–/– limbs (H) and copied to control limbs (G); arrows indicate the absence of mMyoD expression in control limbs compared with Sim2–/– limbs. a, anterior; p, posterior; D, dorsal; V, ventral. (I) Relative levels of MyoD mRNAs in forelimbs from E11.5 Sim2–/– compared with wild-type littermates. The mRNA levels were normalised to that of HPRT. Error bars indicate ±s.e.m. **P<0.01.
	Fig. 6. mSim2 overexpression inhibits MyoD expression in chick and Xenopus embryos. (A-J) Chick embryos were electroporated at interlimb regions with mSim2 and GFP expression vectors (A-E) or with GFP expression vector only (F-H). Transverse sections (A-C,F-H) or whole-mount embryos (D,E,I,J) were hybridised with the cMyoD probe and then incubated with GFP antibody to visualise the electroporated regions. (B,C,G,H) High magnifications of the boxed regions in A,F. mSim2 overexpression in somites leads to a loss of cMyoD expression in electroporated regions (A-E) compared with control electroporated with GFP only (F-H), in which electroporated cells express cMyoD. Arrows indicate the loss of cMyoD expression where GFP is expressed in mSim2-electroporated somites (A-E) and cMyoD expression in control electroporated somites (F-J). (K-M) Eight-cell stage Xenopus embryos were injected bilaterally (K,L) or unilaterally (M) with mSim2 and β-gal mRNAs (K,M) or with β-gal mRNAs as control (L) and were processed for whole-mount in situ hybridisation to xMyoD expression. At early gastrula stages, xMyoD expression is inhibited by mSim2 overexpression in β-gal-positive regions (K, arrows), whereas normal xMyoD expression is observed in control embryos injected with β-gal only (L, arrows). At tail bud stages, mSim2 overexpression also leads to a loss of xMyoD expression in the most anterior somites on the injected sides, compared with the uninjected sides (M, arrows and brackets).
	Fig. 7. mSim2 represses the transcriptional activity of MyoD promoter in vitro and is recruited to the core element in vivo in E11.5 mouse limbs. (A-C) Primary myoblasts were co-transfected with GFP-, mMyoD- or mSim2-expression vectors and MyoD reporter constructs, in which the luciferase reporter gene was cloned downstream of two regulatory elements of the mMyoD promoter, the core (A) and the DRR (B) or a minimal promoter (C). mSim2 represses the luciferase activity of the core (A) and the DRR (B), but not that of the TATA promoter (C). Luciferase activity was measured in the same conditions for the experiments in A, B and C. The same arbitrary units (A.U.) therefore apply to all panels. (D) Schematic representation (not in scale) of the regulatory elements of the mouse MyoD promoter and of the position of the three amplified fragments used for ChIP experiments. (E) ChIP assays were performed from limbs of E11.5 mice with antibodies against Sim2 or Ach4, or without any antibody (no Ab) as a negative control. ChIP products were analysed by PCR to study the presence of mSim2 on regulatory regions of mouse MyoD promoter. We could detect the binding of mSim2 to the core element. PCR amplifications were performed on chromatin isolated before immunoprecipitation (Input) as positive control.
	Fig. 8. Differential increase of dorsal versus ventral muscle masses in chick forelimbs. In situ hybridisation to whole-mount (A,B) or to tissue sections (C,D) of forelimbs of HH22 embryos using the cPax3 (A,C) or cSim2 (B,D) probes. (A) Dorsal and ventral views of the same forelimbs hybridised with the cPax3 probe. (B) Ventral view of forelimbs hybridised with the cSim2 probe. (C,D) Transverse and adjacent sections of HH22 forelimbs were hybridised with the cPax3 (C) and cSim2 (D) probes. (E-I) Double immunohistochemistry using the Pax7 and MF20 antibodies on transverse sections of forelimbs at the level of the mid-forearm, at different stages of development: HH22 (E,F), HH23 (G) and HH29 (H,I). D, dorsal; u, ulna; r, radius; V, ventral.
	Fig. 9. mSim2 expression is upregulated in dorsal regions of embryonic limbs deficient for Lmx1b activity. (A-C) In situ hybridisation experiments on transverse HH29 chick limb sections with the cLmx1b (A), cSim2 (B) and cMyoD (C) probes show that cLmx1b expression in dorsal limb regions is excluded from the cMyoD expression domain and from the cSim2 expression domain in ventral limb muscles. (D-I) E10.5 forelimbs (D,E) and E11.5 hindlimbs (F-I) from wild-type (D,F,H) and Lmx1b mutant mice (E,G,I) were hybridised with the mSim2 probe (D-I). mSim2 expression is upregulated in dorsal limb regions of Lmx1b-deficient limbs (E,G,I, arrows), compared with wild-type limbs (D,F,H). In all pictures, the dorsal and ventral limb regions are separated by a line. Arrows indicate the extension of the mSim2 expression domain in dorsal limb regions in Lmx1b mutants. a, anterior; p, posterior; D dorsal; V, ventral.
	Fig. 10. MyoD regulation during limb embryonic myogenesis. (A) Gene network involved in the initiation of MyoD expression in limbs during embryonic myogenesis. Pax3, Myf5 and Pitx2 cell-autonomously activate the expression of Myf5 and MyoD, whereas Sim2 cell-autonomously represses MyoD expression. (B) Sim2 recruitment to the core element of MyoD promoter in E11.5 mouse limbs. (C) Sim2 expression is enhanced in ventral limb muscle progenitors. Lmx1b expressed in dorsal limb regions and excluded from myogenic cells is involved in the non cell-autonomous repression of Sim2 expression in dorsal limb regions.

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