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Fig. 4. Blocking Notch signaling produces complementary changes in the expression of markers of the three germ layers at gastrula. Embryos were injected in one cell at the 4-cell stage with 1 ng of deltaSTU mRNA (A–B′,G–G′,L), 20 ng of Control morpholino (Control Mo; C,H–H′,M), 20 ng of Notch Mo (D–E,I–J′,N), or with 20 ng of Notch Mo + 1 ng of notchICD mRNA (F,K,O). They were fixed when sibling control embryos reached stage 11 and were revealed by ISH with the following probes: bra(purple staining, A–F), sox2 (purple staining, G–K), sox17α (purple staining, L–O). When possible, embryos hybridized with bra were also revealed with a chd probe to readily identify the dorsal side (turquoise staining in A′–B′). All photographs of whole embryos were oriented with the injected side towards the right. is: injected side; nis: non-injected side. Dotted red lines delineate the blastopore. Embryos are shown with the corresponding fluorescence image at the left (A,D for A′,D′, respectively) or in the insets, to identify the injected side (green fluorescence), which was revealed by immunofluorescence of GFP (A,G,L), DOG (C,D,E,H,I,M,N) or the Myc-tag epitope fused to NotchICD (F,K,O). (B,C–D′,L–O) Vegetal views, with the dorsal side up. (A,A′,E,G,H,I,J,K) Dorsal views. (B′,B′) Lateral views of the non-injected- and the injected side, respectively, of the embryo shown in (B). In this case, photographs were inverted to facilitate comparisons between both sides. This embryo was co-injected with 1 ng of GFP mRNA as tracer, and the injected side was determined by the fluorescence of GFP before the ISH procedure. (D,D′ and E) Two different embryos injected with Notch Mo. (G′,G′) Contralateral parasagittal sections of an embryo injected with delta-1STU mRNA + BDA as tracer, which was revealed with BCIP (turquoise staining). (H′,H′) Contralateral parasagittal sections of the embryo injected with Control Mo shown in (H). (J′,J′) Contralateral parasagittal sections of the embryo shown in (J), which was injected with Notch Mo and revealed by immunostaining of the fluorescein epitope with BCIP (turquoise staining). Arrows in (A′,B′,E) point to the animal expansion of the bra domain. The asterisk in (B) indicates an expansion of the gap of unstained cells between the bra+ ring and the blastopore, which correlates with an expansion of the suprablastoporal endoderm (see L for comparison). Arrows in (D′) and the green arrowhead in (E) point to the vegetal expansion of the bra domain. Light blue bars in (D′,E,F) indicate the difference in the width of the bra domain between the injected- and the non-injected side. Notice the stretch of thinner and lower bra staining between the red arrowheads in (F), coinciding with the green fluorescence on the bra domain on the inset, indicating that notchICD reversed the effect of Notch Mo. The black arrowheads in (G′,G′,H′,H′,J′,J′) indicate the distance between the blastopore and the vegetal border of the sox2 domain. The black arrowhead in (K) points to supernumerary sox2+ cells in the marginal zone on the injected side that are not present on the non-injected side (white arrowhead), indicating that notchICD mRNA reversed the effect of Notch Mo on the location of the vegetal boundary of sox2. |
