DIFFERENT MRF4 KNOCKOUT ALLELES DIFFERENTIALLY DISRUPT MYF-5 EXPRESSION - CIS-REGULATORY INTERACTIONS AT THE MRF4 MYF-5 LOCUS/

Three different null alleles of the myogenic bHLH gene MRF4/herculin/M yf-6 were created recently. The three alleles were similar in design b ut were surprisingly different in the intensity of their phenotypes, w hich ranged from complete viability of homozygotes to complete lethali ty. One possible explanation for these differences is that each mutati on altered expression from the nearby Myf-5 gene to a different extent . This possibility was first raised by the observation that the most s evere MRF4 knockout allele expresses no Myf-5 RNA and is a development al phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viabilit y correlate with the intensity of rib skeletal defects, and the most e xtreme version of this rib defect is the hallmark phenotype of Myf-5 n ull animals. In the present study we tested this hypothesis for the tw o milder MRF4 alleles. By analyzing compound heterozygous animals carr ying either the intermediate or the weakest MRF4 knockout allele on on e chromosome 10 and a Myf-5 knockout allele on the other chromosome, w e found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed in creased mortality of the normally viable MRF4 allele, together with in tensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quan titative differences in Myf-5 function if Myf-5 products drop below he mizygous levels. The mechanistic basis for cis interactions at the MRF 4/Mfy-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in th e three MRF knockout alleles, with a basal promoter and a lacZ reporte r. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-actin g interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos. (C) 1997 Academic Press.