Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.