Meadows SM , Warkman AS , Salanga MC , Small EM , Krieg PA .

GM08659 NIGMS NIH HHS , HL63926 NHLBI NIH HHS , HL74184 NHLBI NIH HHS , T32 GM008659 NIGMS NIH HHS , P01 HL063926 NHLBI NIH HHS , R01 HL074184 NHLBI NIH HHS

	Fig. 1. MASTR is a previously uncharacterized member of the myocardin-related family of proteins. (A) Alignment illustrating the domains of the four known Xenopus myocardin family proteins and also mouse MASTR. Percentages indicate identity within each domain relative to myocardin or between Xenopus and mouse MASTR. (B) Co-IP experiments demonstrate that MASTR forms a physical complex with SRF. HA-tagged SRF and radiolabeled in vitro-translated MASTR, δMASTR, and EGFP are shown (Left). Only full-length MASTR protein coprecipitated with SRF (Right). (C–F). In situ hybridization analysis of MASTR expression during Xenopus development. MASTR transcripts are detected specifically in skeletal muscle tissues at all stages. Expression in hypaxial muscle (hy) and jaw muscle (jm) is indicated. (G–J) Transcriptional expression of MyoD. Intensity of MyoD expression differs from MASTR, but the location of transcripts is identical. (K–N) Expression of the striated muscle marker, cardiac α-actin. In addition to marking all tissues expressing MASTR, cardiac α-actin is expressed in the heart (h). (O) RT-PCR analysis showing MASTR expression is first detected in the gastrula (St 10.5) coincident with the appearance of MyoD and Myf5 mRNA. The muscle differentiation marker cardiac α-actin is not detected until St 12.5. Ornithine decarboxylase (ODC) was used as a loading control. (P) RT-PCR analysis of MASTR transcripts in adult frog tissues. MASTR is expressed in representative skeletal muscles (abdominal rectus, quadriceps, and soleus) but not in heart, smooth muscle (intestine), or other tissues.
	Fig. 2. Inhibition of MASTR function reduces expression of skeletal muscle markers. (A–C) Embryos expressing a dominant-negative form of the MASTR protein (DN-MASTR) were assayed at St 22/23 for muscle markers as shown. Injected sides are indicated by arrowheads. (D) Dose-dependent reduction in cardiac α-actin expression after expression of DN-MASTR. (E–G) MO knockdown of MASTR expression results in reduced muscle marker expression as indicated. MO-treated sides are indicated by the arrowheads. (H) Transverse section through the trunk of St 22 embryo treated with MO on the right-hand side and assayed for cardiac α-actin expression. The general structure of the somite is normal, but marker expression is reduced on the treated side. (I) MO-treated embryo shows no reduction in MyoD expression. (J) Two nonoverlapping MO sequences directed against MASTR mRNA (MO1 and MO2) were equally effective in reducing expression of cardiac α-actin. (K) Rescue of the MO-induced phenotype. Embryos were injected with MO2 alone or with MO2 plus MASTR mRNA. Addition of MASTR mRNA achieved partial rescue of MO inhibition in a dose-dependent manner.
	Fig. 3. MASTR induces ectopic expression of muscle markers. (A) Uninjected control embryo at St 22 (lateral view) shows no expression of αMHC marker. (B) Embryo injected with 500 pg of MASTR mRNA shows extensive ectopic expression of αMHC. (C) Control embryo (dorsal view) showing expression of the muscle marker, α-tropomyosin in the somites. (D) Embryo injected with 500 pg of MASTR mRNA shows limited ectopic expression of α-tropomyosin immediately lateral to the somites (arrows). (E and F) Uninjected control embryo (E) and embryo injected with 500 pg of MASTR mRNA (F) assayed for expression of cardiac α-actin. Injected embryo shows extensive ectopic expression of cardiac α-actin. (G) Transverse section through trunk of St 22 embryo injected with MASTR mRNA and assayed for cardiac α-actin transcripts. Arrows indicate that ectopic marker expression is limited to the lateral mesoderm tissue and is absent from epidermis and endodermal tissue. (H) MASTR activates expression of muscle markers in nonmuscle (animal cap) tissue. Embryos were injected with 500 pg of mRNA encoding either myocardin or MASTR, and animal cap explants were isolated and then analyzed by RT-PCR at St 12.5. Myocardin serves as a positive control. Muscle markers are indicated on the left-hand side of the figure. Muscle tissues expressing the various markers are indicated at the right. Lanes labeled St 12.5 WE and St 47 WE are positive control samples from whole embryos at St 12.5 and 47 respectively. ODC transcripts serve as a loading control. (I and J) Mutant MASTR lacking SRF interaction domains (δMASTR) fails to activate muscle marker expression. Experiments were conducted exactly as described for H above but analyzed by quantitative PCR. In the case of both αMHC and α-tropomyosin, a low level of transcript is detected in the untreated animal cap. Results are expressed relative to marker transcripts induced by MASTR.
	coope Fig. 4. MASTR rates with MyoD or Myf5 to activate expression of muscle markers. Embryos were injected with mRNA encoding either myocardin or MASTR alone or together with mRNA encoding MyoD or Myf5. Animal cap explants were harvested at St 12.5 for RT-PCR analysis. Muscle markers are indicated on the left-hand side of the figure. Muscle tissues expressing the various markers are indicated at the right. ODC served as a loading control. (A) Coexpression of MASTR and MyoD results in increased transcription of muscle markers over either factor alone. This is particularly evident for cardiac α-actin, fsTnI, and MLC3f. Coexpression of myocardin with MyoD does not result in an equivalent activation of marker expression. (B) Coexpression of MASTR with Myf5 also activates transcription of muscle markers. Methods are identical to those described in A. No cooperation is observed between MASTR and the MADS factor, MEF2A. (C–G) Embryos were injected with mRNA encoding MASTR or MyoD alone or in combination and then assayed at St 22/23 for expression of muscle markers. Control (C), MASTR mRNA-injected, 100 pg (D), MyoD mRNA-injected, 100 pg (E), and MASTR plus MyoD mRNA-injected (F) embryos (100 pg of each) assayed for cardiac α-actin expression. Minor activation of cardiac α-actin expression is indicated by arrows in D and E. As shown in F, coexpression of MASTR and MyoD resulted in high levels of ectopic expression of cardiac α-actin D. (G) Coexpression of MASTR and MyoD results in high levels of ectopic expression of α-tropomyosin. Quantitation of the results of whole-embryo coexpression studies is presented in SI Table 3.

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