Fig. 1

Hindbrain boundary cells display Yap/Taz activity. (A-C) Whole-mount double in situhybridization in embryos at the indicated developmental stages with probes targeting: egr2a(A,C), which labels rhombomeres (r) 3 and 5; hoxb1a (B,C), which labels r4; and rfng (A,B), which labels hindbrain boundaries. The expression of rfng is restricted at the interface between adjacent rhombomeres. (D) BrdU staining of a Mu4127 embryo expressing mCherry (magenta) in r3 and r5 at 18 hpf. Cells at the boundaries (at the border of mCherry expression) display green BrdU staining (white arrowheads indicate examples). (E) Immunostaining of a Tg[elA:GFP] embryo at 24 hpf with anti-pH3 (magenta). Nuclei were stained using Draq5 (gray). Cells at the boundaries (at the border of GFP expression) display pH3 staining (see white arrowheads). (F) A still image from a time-lapse analysis of a double transgenic Tg[CAAX:GFP]Mu4127 embryo displaying GFP in the plasma membrane (shown in white) and mCherry in r5 (shown in magenta). T₁-T₃ are enlargements of the region framed in F, corresponding to different times. Upon division, the cell challenges the boundary when it undergoes mitosis. (G) Tg[4xGTIIC:GFP] embryo injected with H2B-mCherry to visualize cell nuclei, which display Yap/Taz-TEAD activity (green) in discrete progenitor domains of the hindbrain at 36 hpf. Cells of the rhombic lip (rl) are devoid of Yap/Taz activity. (H) Tg[4xGTIIC:GFP]Mu4127 embryos showing that TEAD activity is restricted to the boundary cells. Images on the right are enlargements of the r3/r4 (white arrow indicates a Yap/Taz-active r3 cell) and r5/r6 (white arrowhead indicates an r6 cell with Yap/Taz activity) boundaries framed in H. (I,I′) Tg[4xGTIIC:GFP] embryos immunostained using anti-Sox2 antibodies (magenta), showing that green Yap/Taz-TEAD active cells are located in the ventricular zone and express this progenitor cell marker (I′). (J-K″) Whole-mount anti-Yap (J-J″) and anti-Taz (K-K″) immunostaining of Tg[4xGTIIC:GFP] embryos at 25 hpf showing overlapping expression of Yap and Taz (magenta in J,J″,K,K″) with TEAD activity (green cells in J′,J″,K′,K″) in boundary cells. All images are dorsal views with anterior to the left, except for the transverse views in I′,I″. r, rhombomere; rl, rhombic lip; ov, otic vesicle. Scale bars: 50 µm.