Fast skeletal muscle-specific expression of a zebrafish myosin light chain2 gene and characterization of its promoter by direct injection into skeletal muscle

A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the z ebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. O ntogenetically, the MLC2f mRNA appears around 16 hours postfertilization (h pf) in the first few well-formed anterior somites. At later stages, the MLC 2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the pro moter of the zebrafish MLC2f gene was isolated and linked to the chloramphe nicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constru cts were analyzed by direct injection into the zebrafish skeletal muscle, a nd significant CAT activity was observed; in contrast, little or no CAT act ivity was generated from a similarly injected prolactin gene promoter/CAT g ene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-b inding sites and E-boxes were identified, suggesting that MLC2f can be regu lated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the tran scription start site, which contains a single MEF2-binding site, is suffici ent to drive a high level of CAT activity in injected muscle. Internal dele tion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044 -bp construct had no effect on induced CAT activity. These observations sug gest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensa ted for by the presence of the upstream MEF2: elements. This study also dem onstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.