Fig 2: Expressions of thin filament and sarcoplasmic reticulum Ca2+ handling proteins are unaltered in S-D muscles. Representative immunoblots of myosin filament-linked protein titin (A), thin filament proteins actin (C), troponin (Tn) T, TnI (D), sarcoplasmic reticulum Ca2+ handling proteins ryanodine receptor (RyR) 1, DHPR-a2 subunit, and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 (F). The expression levels of titin (B), actin, TnT, TnI (E), RyR1, DHPR, and SERCA1 (G) normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content. Results are expressed as a percentage of CNT value. Bars show the mean and SEM results from 5 to 6 muscles per group. *P < 0.05 vs. CNT.

Fig 3: Prx1-LPM lineage contribution to neck and pectoral girdle.See also Figure 4—figure supplement 1, 2. (A–D) X-gal stainings of Prx1Cre;R26R reporter mice at E9.5 (n = 3) (A) and E18.5 (n = 3) (C–D), and immunostaining for GFP and the Pax7/Myod/My32 myogenic markers in Prx1Cre;R26mTmG E12.5 embryo (n = 2) (B). Note Prx1-derived cells in postcranial LPM (A, black arrowheads) and Prx1-derived cells among, but not in, cucullaris myofibres (B–D). (E–F’’) Immunostaining for ß-gal, Tnnt3 and Tcf4 on coronal cryosections of E18.5 Prx1Cre;R26R mice (n = 2) showed ß-gal+ cells constituting the pectoral girdle (E, level C in Figure 1) and in MCT fibroblasts (F-F’’, white arrowheads), but not in trapezius myofibres. acp, scapular acromion process; atp, acromiotrapezius; ccl, cucullaris anlage; ccp, scapular coracoid process; cl, clavicle; epm, epaxial musculature; hh, humeral head; lb, forelimb bud; lbm, limb muscle anlagen; LPM, lateral plate mesoderm; ltd, latissimus dorsi; PA1-6, pharyngeal arches 1–6; S3, somite 3; scp, scapular muscles; stm, sternocleidomastoid; stp, spinotrapezius. Scale bars: in A for A, B 500 µm; in C for C-D 2000 µm, for E 500 µm; in F’’ for F-F’’ 20 µm.

Fig 4: Cell-autonomous role of Isl1 in esophagus myogenic progenitors.(A) Structures and levels analyzed in the study. (B) Schematic summary of the chimera experiment. (C–E) Immunostainings on transverse cryosections of a E18.5 Isl1Cre;R26mTmG fetus for the GFP reporter, Myod/Myog/Tnnt3 (myogenic markers) and SMA (smooth muscle actin) in the esophagus and tongue. Note that Isl1-derivatives include the esophagus striated muscle but not the esophagus smooth muscle layers and tongue muscle (n = 2). (F–K) Immunostainings on transverse cryosections of E16.5 WT (ES: WT;nlacZ) and Isl1-/- (ES: Isl1-/-;nlacZ) chimeras for the ß-gal reporter, Myod/Myog (myogenic markers) and SMA (smooth muscle actin) in the esophagus and tongue (n = 3 each condition). Insets (bottom, right), higher magnifications. White arrowheads indicate examples of ß-gal colocalization with SMA or Myod/Myog. (L–M) Percentage of chimerism and of ß-gal+ cell contribution to the indicated populations in the esophagus and tongue of WT (ES: WT) and Isl1-/- (ES: Isl1-/-) chimeras (n = 3 each condition, #1–3; three different section levels scored). The total number of cells counted on three different section levels are reported in columns. Note that the Isl1-/- ES-derived cells do not form ESM progenitors but contribute to both esophagus smooth muscle layers and tongue. Scale bars: C, 100 µm; K, 50 µm.

Fig 5: Regulation of esophagus striated muscle patterning involves MET/HGF signaling.(A–C) RT-qPCR analysis for Isl1 and Myf5 at E15.5 (A), E17.5 (B) and 3 weeks postnatal (C) in different esophagus portions and stomach as indicated in the schematic view (top, right). The low level of Isl1 expression in the posterior esophagus at fetal stages might reflect contamination from the stomach at the esophagus interface (n = 3 each condition). (D–E) RT-qPCR analysis for Isl1 and Met at E15.5, E17.5 and 3 weeks postnatal in Tg:Pax7-nGFP+ cells isolated by FACS from the masseter or esophagus. All data points are plotted and presented as the mean ± SEM (error bars) (n = 3 each condition). (F–H) Immunostainings on transverse cryosections of E16.5 control, MetD/D and Hgf-/- fetuses for Myod/Myog (myogenic progenitors) and SMA (smooth muscle actin). E-Cad labels the esophagus lumen epithelium. Higher magnifications are shown in (F’–H’) (n = 3 each condition). (I–K) Immunostainings on transverse cryosections of E16.5 control, MetD/D and Hgf-/- fetuses for Tnnt3 (myofiber marker) and E-Cad. Higher magnifications are shown in (I’–K’). Note the absence of ESM formation in both Met and Hgf mutants (n = 3 each condition). Scale bars: F, 100 µm; F’, 50 µm.