Fig 8. Mesenchymal Fat1 is required for expansion of the myogenic component of Gdnf expression domain but dispensable for Gdnf expression in plexus mesenchyme.

(A) Top: principle of the genetic paradigm used to follow the Prx1-cre lineage, using the R26^{Lox-STOP-Lox-YFP} reporter line combined with Prx1-cre. In tissues in which cre is not expressed, YFP expression is prevented by the STOP cassette. In CRE-expressing mesenchymal cells, STOP cassette excision allows YFP expression. Bottom: scheme of a cross section of a Prx1-cre; R26^YFP/+; Gdnf^LacZ/+ embryo, highlighting in green the cells in which YFP expression is activated, in red, the cells expressing Gdnf^LacZ, and in white or gray, the other non-recombined tissues. (B, C) Cross sections of E12.5 Prx1-cre; Fat1^Flox/+; Gdnf^LacZ/+; R26^YFP/+ (B) and Prx1-cre; Fat1^Flox/Flox; Gdnf^LacZ/+; R26^YFP/+ (C) embryos stained with antibodies against GFP (YFP; to reveal the domain of Prx1-cre activity, in green), with an anti-β-galactosidase antibody (for Gdnf^LacZ, red), visualized at four successive rostro-caudal positions spanning from the brachial plexus to the caudal half of the CM muscle. For each level, the inserts below represent a high-magnification view of the area indicated in the yellow dotted boxes, showing red only, green only, and overlay. (D) Visual summary of the two components of Gdnf^LacZ expression domain, spanning the sections shown in (B) and (C): at the plexus level, Gdnf^LacZ is expressed in YFP⁺ cells derived from Prx1-cre mesenchyme, whereas in the CM and LD muscles (emerging from the plexus and extending dorsally and caudally), Gdnf^LacZ-positive cells do not express YFP, as they are from the myogenic rather than the mesenchymal lineage. At the point where the first myogenic patches emerge from the plexus, such myogenic patches (red only, yellow dotted line in [B], second section) can be surrounded by mesenchymal-Gdnf cells (red + green = yellow, white dotted lines). The overall analysis shows that Prx1-cre-mediated Fat1 ablation does not affect Gdnf expression in the plexus mesenchyme but causes non–cell-autonomous reduction in the myogenic component of Gdnf expression domain through a reduction of the number of Gdnf^LacZ-expressing myogenic progenitors. Scale bars: (B, C) low magnification: 200 μm; inserts: 20 μm. CM, cutaneous maximus; cre, cre recombinase; LD, latissimus dorsi; Lox, recombination sites for the CRE recombinate; Lox-STOP-Lox, cassette in which STOP signal for transcription/translation is flanked by Lox sites; Prx1-cre, transgene driving cre expression in the mesenchyme; R26, Rosa26 locus; YFP, yellow fluorescent protein.