(A–F) Dorsal view of 12 ss control (A–C) and meox1 morphant embryos (D–F). Embryos were submitted to double fluorescent in situ hybridization for meox1 (green) and etv2 (red). In control and morphant embryos, meox1; etv2 double-positive cells are detected within the somite compartment (arrowheads). (C,F) Overlay of meox1 (green), etv2 (red), and DAPI (blue). (D–F) Knockdown of meox1 results in ectopic formation of double-positive cells within the somite (arrowheads). We observed 3–4 etv2 positive cells per somite in the meox1 morphants compared to 1–2 etv2-positive cells per somite in the siblings (n=3). (G–H) Time-lapse imaging of a 22 hpf Tg(etv2.1:eGFP)zf372; Tg(phldb1:mCherry) embryo, injected with meox1 morpholino and mOrange2:CAAX mRNA to delineate cell boundaries. Knockdown of meox1 results in an extension of the period that the dermomyotome can generate Etv2:GFP+ cells (arrowheads). (I–K) Cross section of 12 ss Tg(etv2.1:eGFP)zf372 embryo. In absence of meox1 (J), ectopic Etv2:GFP+ cells are visible in epithelialized layer of the somites, compared to controls (I). In embryos coinjected with mib and meox1 morpholinos, the number of Etv2:GFP+ cells within the somite compartment (dotted line) is substantially increased (arrowheads) (K), suggesting that Notch signaling is dispensable for SDEC specification. (L–N) Lateral view of 12 ss embryos analyzed by FISH for meox1 (green), etv2 (red), and DAPI (blue). In notch3+/- heterozygote controls (L) and notch3-/- mutant embryos (M), etv2+ SDECs are detected in the somites. (N) notch3-/- mutant embryos co-injected with meox1 morpholino results in ectopic formation of etv2; meox1 double positive cells (arrowheads). We observed 2–4 etv2-positive cells per somite in the notch3 mutants and >6 etv2-positive cells in the notch3 mutants; Mib morphants (n=3). (O) qRT-PCR in 24 hpf notch3-/- mutant embryos and sibling controls. Genetic ablation of notch3 results in decreased expression of muscle progenitor markers pax3a and pax7b; increased expression of muscle differentiation genes, myod and myog, and endothelial markers, etv2, and fli1. Asterisks denote a statistically significant difference (p<0.05, unpaired, two-tailed Student’s t-test; n=3.) (P,Q) notch3-/- mutant embryos show premature expression of MyoHII in 48 hpf embryos (Q) compared to sibling controls (P). (R) Summary cartoon for the role of Notch signaling in the maintenance of bipotent-muscle progenitors (bipotent muscle progenitors in purple and green; muscle cells in green; SDECs in purple). s, somites; LPM, lateral plate mesoderm; SDECs, somite-derived endothelial cells.