Functional Dissection of Pax3 in Paraxial Mesoderm Development and Myogenesis

Abstract

The paired box transcription factor Pax3 is well-known as a major regulator of embryonic myogenesis. Before Pax3 expression becomes restricted to the dermomyotome, this transcription factor is also expressed in the developing somites. The role of Pax3 at this early stage is unclear, in particular because of the scarce frequency of Pax3-positive cells in the early mouse embryo. Inducible gene expression in embryonic stem (ES) cells represents an excellent tool to overcome this limitation, since it can provide large quantities of otherwise rare embryonic populations expressing a factor of interest. Here we used engineered mouse ES cells to perform a functional analysis of Pax3 with the aim to identify the molecular determinants involved in the early functions of this transcription factor. We find that Pax3 induction during embryoid body (EB) differentiation results in the up-regulation of genes expressed in the presomitic and somitic mesoderm. Moreover, we show that paraxial mesoderm induced by transient expression of Pax3 is not irreversibly committed to myogenesis, rather requires sustained Pax3 expression. Using a series of deletion mutants of Pax3, which differentially affect its transcriptional activity, we map protein domains necessary for induction of paraxial mesoderm and induction of the myogenic program. The paired, homeo- and transcriptional activation domains were each required for both processes, however the paired-c-terminal RED domain showed a paraxial mesoderm-specific activity that was dispensable for myogenesis. These findings demonstrate and provide mechanistic insight into an early role for Pax3 in the generation of paraxial mesoderm.

PAX7 Targets, CD54, Integrin α9β1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors

“…S1B). In agreement with our previous studies using mouse ES cells (Magli et al, 2012; Magli et al, 2014), these results show that genes associated with paraxial mesoderm development, such as DLL1, MEOX1 and LFNG, are also up-regulated by PAX7 expression, indicating the ability of this transcription factor to induce a population of paraxial mesodermal progenitors (Fig. 1D).…”

Making muscle: skeletal myogenesisin vivoandin vitro

“…As Pax3/7 are also expressed in neural derivatives in vivo (Fougerousse et al, 2002;Gerard et al, 1995;Goulding et al, 1991;Jostes et al, 1990), a purification step using surface markers such as PDGFRα (CD140a) or CD56 was needed to enrich in myogenic cells (Darabi et al, 2008(Darabi et al, , 2011Quattrocelli et al, 2011) (Box 1). Pax3 overexpression acts on the early PDGFRα + population, upregulating transcription factors Tcf15 and Meox1 and preventing the cells from undergoing cardiomyogenesis (Magli et al, 2013(Magli et al, , 2014. Both Pax3-and Pax7-reprogrammed mouse progenitors were shown to engraft when transplanted in a mouse model.…”

The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain

“…Mouse Tbx1 was amplified from cDNA reverse-transcribed from RNA isolated from the pharyngeal arch of mouse embryos (E9.5); all other genes were amplified from plasmid templates encoding their respective cDNAs. Mouse Pax3 deletions were constructed as described (Magli et al, 2013). The human MYOD expression construct was as previously described (Bosnakovski et al, 2008a).…”