F. Muller et al., ACTIVATOR EFFECT OF COINJECTED ENHANCERS ON THE MUSCLE-SPECIFIC EXPRESSION OF PROMOTERS IN ZEBRAFISH EMBRYOS, Molecular reproduction and development, 47(4), 1997, pp. 404-412
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.