Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis
(A, C) Whole-mount β-galactosidase staining was performed using X-gal as a substrate on embryos carrying the MLC3F-2E transgene (A), or using Salmon-Gal as substrate on embryos carrying the GdnfLacZ/+ allele (C), in the context of mesenchyme-specific deletion of Fat1, driven by Prx1-cre (S1 Table) at E12.5. Top images show a side view of the whole flank of an embryo. Yellow dotted lines highlight the area occupied by body wall muscles. Lower images are higher magnification of the area in which the CM spreads. Red and white dotted lines correspond (as in Fig 1) to the areas covered by MLC3F-2E+ CM fibers (red, A) and to the area covered by GdnfLacZ+ progenitors (white, C), respectively. (B) Quantification of the expansion rate of differentiated CM fibers. Left graph: For each embryo side, the area covered by differentiated CM fibers was plotted relative to the area occupied by body wall muscles. Right plot: for each embryo, the CM area/body wall area was normalized to the median ratio of control embryos. Blue dots: Fat1Flox/Flox; MLC3F-2E (n = 57, includes the same set of controls as S8C Fig); red dots: Prx1-cre; Fat1Flox/Flox-; MLC3F-2E (n = 23). Underlying data are provided in S1 Data. (D) Quantification of the expansion rate of the area occupied by GdnfLacZ+ progenitors. Left plot: for each embryo side, the area covered by GdnfLacZ+ progenitors was plotted relative to the trunk length. Right plot: for each embryo, the GdnfLacZ+ CM area/trunk length was normalized to the median ratio of control embryos. Blue dots: Fat1Flox/Flox; GdnfLacZ/+ (n = 36, pooling respective littermates); red dots: Prx1-cre; Fat1Flox/Flox-; GdnfLacZ/+ (n = 12). Underlying data are provided in S1 Data. (E) Cross sections of E12.5 Prx1-cre; Fat1Flox/+; GdnfLacZ/+; R26YFP/+ and Prx1-cre; Fat1Flox/Flox; GdnfLacZ/+; R26YFP/+ embryos at equivalent rostro-caudal positions (caudal CM level) were immunostained with antibodies against GFP/YFP (green) to reveal the domain of Prx1-cre activity (green) against β-galactosidase (red), against Myh1 (white on top panels and inserts, green on middle panels), against Pax7 (bottom panels), and with DAPI (blue). The yellow dotted boxes indicate the areas magnified in inserts and in the bottom panels, in equivalent positions of the CM. Images show that lowered Gdnf levels represent a non–cell-autonomous consequence of lack of Fat1 signaling in the mesenchyme of Prx1-cre; Fat1Flox/Flox embryos and result from a reduced number of Pax7-GDNF-expressing progenitors cells rather than from a lower level of GdnfLacZ expression per cell. Scale bars: (A, C) 500 μm; (E) low magnification: 200 μm; inserts: 20 μm; lower panels: 40 μm. CM, cutaneous maximus; cre, cre recombinase; dCM, dorsal cutaneous maximus; GFP, green fluorescent protein; Prx1-cre, mesenchymal cre expression driven by a regulatory enhancer of the paired related homeobox 1 gene; R26, Rosa26 locus; vCM, ventral cutaneous maximus; X-gal, substrate for β-galactosidase; YFP, yellow fluorescent protein.