mef2 activity levels differentially affect gene expression during Drosophila muscle development

Abstract

Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. In this study we used a strategy combining genomics and developmental genetics to address this issue in vivo during Drosophila muscle development. We found that groups of mef2-regulated genes respond differently to changes in mef2 activity levels: some require higher levels for their expression than others. Furthermore, this differential requirement correlates with when the gene is first expressed during the muscle differentiation program. Genes that require higher levels are activated later. These results implicate mef2 in the temporal regulation of muscle gene expression, and, consistent with this, we show that changes in mef2 activity levels can alter the start of gene expression in a predictable manner. Together these results indicate that Mef2 is not an all-or-none regulator; rather, its action is more subtle, and levels of its activity are important in the differential expression of muscle genes. This suggests a route by which mef2 can orchestrate the muscle differentiation program and contribute to the stringent regulation of gene expression during myogenesis.

Fig. 1.

Different genes respond differently to changes in mef2 activity levels. (A) Hierarchical clustering of expression data for the 97 mef2-regulated genes selected as described. Columns show expression for each gene in wild-type, mef2⁶⁵, mef2⁴²⁴, mef2¹¹³, and mef2^22.21 embryos as indicated. Green represents genes with no change in expression from wild type, red represents genes with no detectable expression, and intermediate shades represent intermediate values. Labeled bars indicate gene cohorts with high, intermediate, and low requirements for mef2. (B) Histograms showing the expression array data for seven selected mef2-regulated genes (Act57B, CG17492, CG5080, Mlc2, Mlc1, CG6972, and Mhc) at stage 13 across the mef2 allelic series. (C) In situ hybridizations for the seven genes across the allelic series at stage 13 (anterior is to left and dorsal is to top in these and all other figures).

Fig. 2.

The mef2 requirement of target genes correlates with the start of their expression. (A) In situ hybridizations of wild-type embryos for Act57B, CG17492, CG5080, Mlc2, Mlc1, CG6972, and Mhc at different stages of development as indicated. Genes that only need low levels of mef2 activity (Act57B and CG17492) are at the top, and those that require the highest levels (CG6972 and Mhc) are at the bottom. (B) Graph, with fitted line from linear regression, showing the correlation between the requirement for mef2 and when the gene is first activated for 16 genes known to be expressed specifically in muscle at stage 13. The requirement for mef2 is taken as the expression of each gene at stage 13 in mef2⁴²⁴ embryos relative to wild type. The start of expression was compiled from in situ hybridizations (A) (refs. and and data not shown).

Fig. 3.

Increased or decreased levels of mef2 activity lead to premature or delayed expression of target genes, respectively. (A) In situ hybridization images of wild-type, twistGal4;twistGal4 > UAS-mef2, and mef2¹¹³ embryos for CG6972, CG5080, and Act57B at different stages of development as indicated. CG6972 and CG5080 expression is premature when mef2 is overexpressed. CG6972, CG5080, and Act57B expression is delayed when mef2 activity is reduced. (B) Quantitative RT-PCR analysis of CG6972, CG5080, and Act57B transcript levels at the indicated stages. Expression relative to stage 13 levels in the wild type is shown as means from triplicate experiments. Red stars denote values that differ by >1 SD in UAS-mef2 embryos relative to wild type, and blue stars denote values that differ by >1 SD in mef2¹¹³ embryos relative to wild type.