Abstract
Pax3 is a paired box transcription factor expressed during somitogenesis that has been implicated in initiating the expression of the myogenic regulatory factors during myogenesis. We find that Pax3 is necessary and sufficient to induce myogenesis in pluripotent stem cells. Pax3 induced the expression of the transcription factor Six1, its cofactor Eya2, and the transcription factor Mox1 prior to inducing the expression of MyoD and myogenin. Overexpression of a dominant negative Pax3, engineered by fusing the active transcriptional repression domain of mouse EN-2 in place of the Pax3 transcriptional activation domain, completely abolished skeletal myogenesis without inhibiting cardiogenesis. Expression of the dominant negative Pax3 resulted in a loss of expression of Six1, Eya2, and endogenous Pax3 as well as a down-regulation in the expression of Mox1. No effect was found on the expression of Gli2. These results indicate that Pax3 activity is essential for skeletal muscle development, the expression of Six1 and Eya2, and is involved in regulating its own expression. In summary, the combined approach of expressing both a wild type and dominant negative transcription factor in stem cells has identified a cascade of transcriptional events controlled by Pax3 that are necessary and sufficient for skeletal myogenesis.
Highlights
The final stages of skeletal myogenesis are controlled by a family of basic helix-loop-helix transcription factors, the myogenic regulatory factors (MRFs)1 [1]
Pax3 is a paired box transcription factor expressed during somitogenesis that has been implicated in initiating the expression of the myogenic regulatory factors during myogenesis
The ordered pattern of expression of these transcription factors during P19 cell myogenesis is similar to the order they would be expressed in a mesoderm cell as it matures from paraxial mesoderm to an early somite and into the dermomyotome and into the myotome
Summary
The final stages of skeletal myogenesis are controlled by a family of basic helix-loop-helix transcription factors, the myogenic regulatory factors (MRFs)1 [1]. Several transcription factors are candidate factors for mediating the response to signals from surrounding tissues and inducing MRF expression These include Pax3 [17], Six1 [18], Eya2 [19], Mox1/2 [20], and Gli2/3 [21]. Eya and Dach family members synergize with each other and interact with Six to regulate transcription [44, 45] These molecules were shown to regulate each other’s expression and to induce muscle-specific gene expression in mesoderm explants [32]. The role of Gli molecules in Shh signaling and the expression profiles of Gli and Gli in the developing somite suggest that they are candidates for regulating MRF expression and commitment to the myogenic lineage [51, 52]
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