Abstract
Wnt proteins are secreted cytokines and several Wnts are expressed in the developing somites and surrounding tissues. Without proper Wnt stimulation, the organization of the dermomyotome and myotome can become defective. These Wnt signals received by somitic cells can lead to activation of Pax3/Pax7 and myogenic regulatory factors (MRFs), especially Myf5 and MyoD. However, it is currently unknown whether Wnts activate Myf5 and MyoD through direct targeting of their cis-regulatory elements or via indirect pathways. To clarify this issue, in the present study, we tested the regulation of MyoD cis-regulatory elements by Wnt3a secreted from human embryonic kidney (HEK)-293T cells. We found that Wnt3a activated the MyoD proximal 6.0k promoter (P6P) only marginally, but highly enhanced the activity of the composite P6P plus distal enhancer (DE) reporter through canonical and non-canonical pathways. Further screening of the intervening fragments between the DE and the P6P identified a strong Wnt-response element (WRE) in the upstream −8 to −9k region (L fragment) that acted independently of the DE, but was dependent on the P6P. Deletion of a Pax3/Pax7-targeted site in the L fragment significantly reduced its response to Wnt3a, implying that Wnt3a activates the L fragment partially through Pax3/Pax7 action. Binding of β-catenin and Pax7 to their target sites in the DE and the L fragment respectively was also demonstrated by ChIP. These observations demonstrated the first time that Wnt3a can directly activate MyoD expression through targeting cis-elements in the DE and the L fragment.
Highlights
Trunk skeletal muscle (SKM) cells in vertebrates are derived from precursors in the embryonic somites, and a subset of somitic cells become myogenic stem cells after being specified by local signals to express Pax3 and Pax7 [1]
Wnt3a activate MyoD expression in myoblasts and somite explants Wnt3a is post-translationally modified and these modifications are critical to its function; recombinant proteins expressed in bacteria might not be an effective source for treating mammalian cells
To examine the effect of Wnt3a on MyoD expression, C2C12 myoblasts were kept in control (Py) or Wnt3a-containing medium and their MyoD mRNA level was determined by quantitative reverse transcription–PCR (qRT-PCR)
Summary
Trunk skeletal muscle (SKM) cells in vertebrates are derived from precursors in the embryonic somites, and a subset of somitic cells become myogenic stem cells after being specified by local signals to express Pax and Pax7 [1]. They are further confined within the myogenic lineage by the expression of either MyoD or Myf that drives them to become myoblasts. Since mice carrying null mutations in either the Myf or the MyoD gene have apparently
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