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Analysis of mef2 function during Drosophila muscle development has shown that a major aspect of its role is in the differentiation pathway downstream of the genes that specify muscle [5–7]. However, Mef2 protein expression precedes muscle differentiation [1]. It is first expressed in the mesoderm at gastrulation, approximately 3 hr after egg laying (AEL) [6]. This is approximately 7 hr before the activation at stage 13 (10 hr AEL) of the expression of many known Mef2 target genes, e.g., Mhc, Mlc1, and wupA ([4, 8]; data not shown). This delay implies that the activity of Mef2 is restrained and that other regulatory proteins operate in the control of muscle differentiation during this period. However, little is known about these other proteins nor about how the gene expression at stage 13 is coordinated. Here, we address these unanswered questions through an analysis of the Him gene in muscle differentiation. Him was described in a computational screen [9], and we isolated it separately in an expression screen [10], but its function has not previously been analyzed. We demonstrate that it is an inhibitor of Mef2 activity and muscle differentiation, and on the basis of this phenotype, we call it Holes in muscle (Him).
Him has a striking, transient pattern of expression during Drosophila embryogenesis. It is first expressed during stage 9 broadly in the mesoderm (Figure 1A). This expression then refines, and at stage 12 it is specifically expressed in the precursors of the somatic musculature and of the heart (Figure 1C). Him expression then rapidly declines in the somatic mesoderm, such that in 90 min it has disappeared from the differentiating somatic muscle (stage 13, Figure 1D). However, it persists in the adult muscle precursors (AMPs), which are set aside in the somatic mesoderm and which remain undifferentiated at this stage, and also in the developing heart. Him protein expression closely resembles that of Him RNA (Figures 1E and 1F). The disappearance of Him coincides with the expression of Myosin, a classic marker of muscle differentiation. Double labeling with a Him-GFP fusion gene (see Experimental Procedures in the Supplemental Data available online) demonstrates that Myosin is expressed only after Him disappears from the developing muscle (Figures 1G and 1H). The expression of Him in the progenitors of the somatic muscle and its disappearance from differentiating muscle are consistent with a role for Him as an inhibitor of muscle differentiation.
We then investigated the significance of the Him/Gro interaction in vivo during embryonic muscle development by overexpressing Him in a gro mutant background. Strikingly, the loss of gro function suppresses the inhibitory effect of Him (Figures 3H–3J), showing that Him requires gro to inhibit muscle differentiation. This result, together with our finding that mef2 can suppress the inhibitory effect of Him (Figure 2), indicates that Drosophila muscle differentiation in vivo is controlled by a balance between the activities of Him and Gro on the one hand and Mef2 on the other (Figures 3K–3M). The effect of overexpression of Him can be balanced by a reduction in Gro or by an increase in Mef2 (Figures 2H–2J).
Taken together, our combination of in vitro and in vivo assays (Figures 3 and 4) reveals key features of Him's mechanism of action. They demonstrate that Him is found in the nucleus and requires its Gro-binding WRPW motif and gro function to inhibit both Mef2 activity and muscle differentiation during development. The previously characterized Drosophila proteins that have a C-terminal Gro-interacting WRPW motif are the Hairy group of HLH domain DNA-binding transcriptional repressors [14]. However, Him is novel and does not have an HLH domain (see Experimental Procedures), suggesting that it does not bind DNA directly. Its mechanism of action may have parallels with Ripply1, which functions in vertebrate somitogenesis [16]. Ripply1 also appears not to be an HLH protein and yet contains a functional Gro-interacting WRPW motif, although in this case near the N-terminus of the protein. Like Ripply1, Him may be part of a transcriptional-repressor protein complex. The precise mechanism by which Him targets Mef2 awaits analysis of this putative complex and the protein partners within it.
Our results also indicate that the inhibition of Mef2 activity by endogenous levels of Him is incomplete prior to stage 13. Thus, in normal muscle development, the Mef2 target gene β3-tubulin is expressed at stage 12, even though we find that overexpression of Him can downregulate its expression then. This implies that in the wild-type embryo, there is some Mef2 activity at stage 12, and such activity is sufficient for β3-tubulin expression. This is consistent with other work that indicates that Mef2 regulates some gene expression at this stage and earlier [3, 4, 10] and suggests that Him can provide one level of control of Mef2 activity during the muscle-differentiation program. Taken together, our results move the molecular analysis of muscle differentiation on from a simple model in which the key events are expression of pivotal positive regulators, for example Mef2. Rather they indicate that muscle differentiation in vivo is controlled by a balance of positive and negative regulators, including Him, Gro, and Mef2, that governs whether muscle precursors differentiate. In this model, one can think of Him and Gro as part of a mechanism holding the cells in a committed, but undifferentiated, state in which a cohort of muscle-differentiation genes is poised to be expressed. This might be a widespread strategy for coordinated gene expression in cell-differentiation programs. For example, it can be compared with melanocyte stem cell differentiation, where cells are primed to rapidly express terminal differentiation markers once Pax3/Groucho-mediated repression is relieved [20].
Experimental Procedures and two figures are available at http://www.current-biology.com/cgi/content/full/17/16/1409/DC1/.
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We thank Barbara Jennings and Mark Wainwright for sharing their groucho expertise, Huw Williams for assistance early in the work, and all those (indicated in Experimental Procedures) who very kindly sent us reagents for this study. This research was supported by the John Ryder Memorial Trust, the Association Francaise contre les Myopathies, the European Union FP6 Network of Excellence MYORES, The Wellcome Trust, the BBSRC, The Royal Society, and the American Heart Association (Z.H.).