Fig. S1

Distinct features of boundary cells.

A-D’’) Double in situ hybridization for detection of the expression of the rfng boundary marker, and either the hoxb1a r4 marker (B) or the egr2 r3 and r5 marker (C-D’’). Note that rfng is not yet expressed at 15hpf, and that, once it is expressed, rfng is stained in two cell rows: one cell row that expresses hoxb1a/egr2a together with rfng, and the other that expresses only rfng (see cells with white arrows in B-C, and D-D’’). E) Tg[CAAX:GFP]Mu4127 and F) Tg[CAAX:GFP] embryos showing that cells at the boundaries display a singular triangular shape (see white arrows). G) Tg[CAAX:GFP]Mu4127 embryos were used for single cell segmentation. Inserts are images of single segmented cells from the framed regions (green and blue) indicated in the embryo. They display dorsal (top) and lateral (bottom) views of manually segmented single cells from the r3/r4 boundary (green cells) and from r4 (blue cells), with the apical side at the left of the image. Note that the green r3/r4 cell has a triangular shape with a large apical side, whereas the blue r4 cell is spindle-shaped. H-H’’) Tg[elA:GFP] embryos expressing GFP in r3 and r5 were assayed using rfng fluorescence in situ hybridization and immunostaining for GFP. Note that rfng is expressed at the border of GFP expression, i.e. in the boundary cells. I-I’’) Tg[elA:GFP] embryos expressing GFP in r3 and r5 were assayed using ephA4 fluorescence in situ hybridization and immunostaining for GFP. Note the complete overlap of ephA4 and GFP expression, as previously described, which reflects the fact that ephA4 is a direct transcriptional target of egr2. Dorsal views with anterior to the top. r, rhombomere.