Abstract
The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, we show that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity.
Highlights
Basic helix-loop-helix transcription factors are crucial regulators, in some instances referred to as ‘master regulators’, of cell-type identities
A pan-zebrafish-Pbx antibody (Pöpperl et al, 2000) identified ubiquitous nuclear Pbx expression in early paraxial mesoderm, and ubiquitous Pbx expression was maintained during somite development and early muscle differentiation, consistent with prior reports of broad pbx mRNA expression during early zebrafish development (Pöpperl et al, 2000; Vlachakis et al, 2000; Waskiewicz et al, 2002), and confirming that Pbx proteins are present at the right time and place to potentially regulate early muscle development
During somitogenesis stages in control embryos, myod was expressed adjacent to the notochord in adaxial cells as well as in more-lateral cells in the posterior of each somite. myog showed a similar pattern of expression, which was delayed relative to myod (Weinberg et al, 1996; Groves et al, 2005) (Fig. 1A,D,G,J)
Summary
Basic helix-loop-helix (bHLH) transcription factors are crucial regulators, in some instances referred to as ‘master regulators’, of cell-type identities. Homeodomain transcription factors have been termed ‘master regulators’, but typically of organ or positional identities. The Hox proteins control segmental identities, and Pax controls eye development (Pearson et al, 2005; Gehring, 1996). The mechanism is not known, one possibility is that positional-identity factors modulate the activity of cell-typeidentity factors. As suggested by Westerman et al (Westerman et al, 2003), homeodomain proteins might act in their region of expression to directly modulate the activity of bHLH proteins and confer specific characteristics on a cell-type identity. Synergistic interactions of bHLH and homeodomain proteins have been described at some promoters (Westerman et al, 2003), the model in which homeodomain proteins can instruct the bHLH proteins to modulate a cell-type program to generate, for example, a particular type of neuron or muscle cell has not yet been demonstrated
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