Kirsten R. Dalrymple

Expression of myogenic regulatory factors during the development of mouse tongue striated muscle

While the role of myogenic regulatory factors (MRFs) in skeletal myogenesis has been well evaluated in limb and trunk muscles, very little is known about their role in tongue myogenesis. Here the expression of MRF mRNA in mouse tongue muscle was examined during development from embryonic day (E)11 to birth and compared them with that in hind-limb muscle. Desmin, muscle creatine kinase and troponin C mRNAs were used as markers for myoblast determination, myotubule formation and myofibre maturation, respectively. The mRNA quantities were determined by competitive reverse transcriptase-polymerase chain reaction. The expression profile of desmin mRNA indicated that myoblast determination occurred before E11 in both the tongue and hind-limb muscles; the profile of muscle creatine kinase and troponin C mRNAs indicated that myotubule formation and myofibre maturation began between E11 and 13 in both tongue and hind-limb muscles, but ended 2 days earlier in the tongue than in the hind limb. Expression of myoD and myogenin mRNAs began at E11, increased, and showed peak values earlier in the tongue muscle (E13) than in the hind-limb muscle (E15). Expression of MRF4 mRNA appeared earlier in the tongue (E13) than in the hind-limb muscle (E15) and increased in both muscles after that. These results suggest that myotubule formation and myofibre maturation in the tongue muscle progress faster than in the hind-limb muscle, a result of earlier expression of myoD, myogenin, and MRF4 in response to earlier functional demands such as suckling immediately after birth.

Direct DNA injection into mouse tongue muscle for analysis of promoter function in vivo

The striated muscle of the tongue provides a readily accessible site for the introduction of DNA expression vectors. Parameters were established to use the striated muscle of the tongue as a model system for the examination of gene expression following the direct injection of DNA constructs bearing gene promoter sequences controlling the expression of reporter genes. Plasmid expression vectors were used that contained either constitutive or muscle-specific promoters directing the transcription of reporter genes. Chloramphenicol acetyltransferase (CAT), luciferase, and β-galactosidase (lacZ) were used as the reporter genes to detect the promoter-specific expression of the injected DNA. The expression of the injected plasmids was directly correlated with the mass of injected DNA and the time of incubation following the injection. Maximal levels of reporter gene expression were observed seven days after the injection, and the expression was maintained for more than two months following injection. Simultaneous injection of two individual expression vectors bearing either CAT or luciferase reporter genes resulted in a dose-dependent level of expression for each of the plasmids. The linearity of the coexpression provided a means to normalize DNA uptake and analyze promoter efficiency. The troponin C-fast enhancer linked to its own promoter directed significantly more CAT expression than an enhancerless SV40 promoter-CAT plasmid, demonstrating that different promoter strengths could be determined in the mouse tongue muscle in vivo. This model system represents a convenient means to approach the functional analysis of muscle gene promoters in vivo.

Differential expression of troponin C genes during tongue myogenesis

Determination of muscle fiber type is related to the developmental stage of the tissue. Ordinarily the final distribution of fast and slow fibers in a muscle is determined postnatally. Tongue muscle, however, is composed solely of fast‐twitch fibers that express only troponin C fast mRNA and fast (type II) myosin heavy chain (MHC) proteins in both the adult and the one‐day‐old mouse. The fiber‐type determination of this muscle was examined during fetal development. Both troponin C fast and slow mRNAs were expressed at initial stages of tongue development at embryonic day 13. However, by embryonic day 16 the troponin C fast transcripts predominated. At 17 days of embryonic development, TnC fast mRNA was 10 times more abundant than TnC slow, and at 18 days of development the TnC slow mRNA was barely detectable. The tongue muscle myotubes expressed fast, slow, and embryonic MHC isoforms during early embryonic development. At 18 days of gestation, the MHC isoform expressed by the majority of the myotubes was the fast isoform, whereas the slow isoform was present in very few fibers. RT‐PCR analysis of the MHC transcripts present throughout tongue development demonstrated expression of the mdms or type IIx MHC in both late fetal and postnatal stages of development. In contrast, the type I/β slow MHC mRNA was undetectable in the postnatal and adult tongue. The absence of TnC and MHC slow‐isoform mRNAs in the newborn mouse tongue suggests that slow isoform genes become dominantly repressed with the TnC‐F and MHC type IIx genes remaining transcriptionally active, giving rise to an unusually homogeneous fast‐twitch phenotype. The tongue muscle fibers acquire their specific adult‐type fiber characteristics during fetal development rather than postnatally. Dev. Dyn. 209:36–44, 1997.