ACTIVATOR EFFECT OF COINJECTED ENHANCERS ON THE MUSCLE-SPECIFIC EXPRESSION OF PROMOTERS IN ZEBRAFISH EMBRYOS

The transient expression of reporter gene constructs in embryos provid es a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have a nalysed the expression pattern of several muscle-specific and ubiquito us 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 s equences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb m yosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to rest rict lacZ expression to the skeletal muscle fibres of prim-6 stage zeb rafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments oi a carp beta-actin regulator y region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expre ssing cells on an expression map. beta-galactosidase activity increase d similarly whether the MyLC enhancer was ligated to the promoter/repo rter construct directly or when coinjected as a separate fragment whil st skeletal muscle specificity was retained. The coinjection of two di fferent forms of the beta-actin regulatory elements also showed a mark ed effect on the MyHC promoter activity. The coinjection of putative e nhancers with minimal promoter constructs and subsequent analysis of t he transient expression pattern in the developing embryos provides a r apid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo. (C) 1997 Wiley-Liss, Inc.