Yoon J K, Olson E N, Arnold H H, Wold B J
Division of Biology, 156-29, California Institute of Technology, Pasadena, California, 91125, USA.
Dev Biol. 1997 Aug 15;188(2):349-62. doi: 10.1006/dbio.1997.8670.
Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were 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 nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null 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 of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified 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 quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting 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.
最近产生了肌源性bHLH基因MRF4/herculin/Myf-6的三种不同无效等位基因。这三种等位基因在设计上相似，但令人惊讶的是它们的表型强度不同，范围从纯合子的完全存活到完全致死。对这些差异的一种可能解释是，每个突变对附近Myf-5基因表达的改变程度不同。这种可能性首先是通过观察到最严重的MRF4敲除等位基因不表达Myf-5 RNA，并且是Myf-5无效突变的发育拟表型而提出的。此外，对两个较弱等位基因的初步研究表明，它们在活力上的差异与肋骨骨骼缺陷的严重程度相关，而这种肋骨缺陷的最极端形式是Myf-5无效动物的标志性表型。在本研究中，我们对两个较温和的MRF4等位基因检验了这一假设。通过分析在一条10号染色体上携带中间或最弱的MRF4敲除等位基因，而在另一条染色体上携带Myf-5敲除等位基因的复合杂合动物，我们发现这两个MRF4等位基因显然都通过顺式作用机制下调Myf-5表达。复合杂合子显示正常存活的MRF4等位基因的死亡率增加，同时两个MRF4等位基因的肋骨缺陷加剧，并且肌节中Myf-5表达的缺陷增加。表型的等位基因特异性梯度也表明，如果Myf-5产物降至半合子水平以下，肋骨形态发生对Myf-5功能的定量差异非常敏感。通过将包含整个MRF4结构基因的DNA片段（包括在三个MRF敲除等位基因中缺失的所有序列）与一个基础启动子和一个lacZ报告基因融合，进一步研究了MRF4/Myf-5位点顺式相互作用的机制基础。转基因胚胎在肌节中显示出特定的LacZ表达模式，该模式与Myf-5 RNA的表达模式非常相似。因此，Myf-5和MRF4之间的顺式作用相互作用可能在调节野生型胚胎早期肌节中这些基因的表达中起重要作用。
