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.
Müller
F
F
Institute for Molecular Genetics, Agricultural Biotechnology Center, Gödöllö, Hungary.
Williams
D W
DW
Kobolák
J
J
Gauvry
L
L
Goldspink
G
G
Orbán
L
L
Maclean
N
N
D000199:Actins / Q000235:genetics; D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D006651:Histocytochemistry; D007763:Lac Operon; D008845:Microinjections; D018482:Muscle, Skeletal / Q000196:embryology / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics; D018994:Myosin Light Chains / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000199:Actins; D018994:Myosin Light Chains; D001616:beta-Galactosidase; D018995:Myosin Heavy Chains