Different MRF4 Knockout Alleles Differentially Disrupt Myf-5 Expression: cis-Regulatory Interactions at the MRF4/Myf-5 Locus

Abstract

Three different null alleles of the myogenic bHLH geneMRF4/herculin/Myf-6were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearbyMyf-5gene to a different extent. This possibility was first raised by the observation that the most severeMRF4knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of theMyf-5null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype ofMyf-5null animals. In the present study we tested this hypothesis for the two milderMRF4alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakestMRF4knockout allele on one chromosome 10 and aMyf-5knockout allele on the other chromosome, we found that both of theseMRF4alleles apparently downregulate Myf-5 expression by acis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viableMRF4allele, together with intensified rib defects for bothMRF4alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis forcisinteractions at theMRF4/Myf-5locus was further examined by fusing a DNA segment containing the entireMRF4structural gene, including all sequences deleted in the threeMRFknockout alleles, with a basal promoter and alacZreporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA.cis-acting interactions betweenMyf-5andMRF4may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

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