Floor plate precursor cells express her9. A: Dorsal view of a 6 hpf (shield stage) whole-mount embryo probed for her9 expression. The animal pole is toward the top. Dorsal cells close to the blastoderm margin (arrows) express her9. B: Sagittal section at the dorsal margin of 6 hpf embryo probed for her9 (blue) and ntl (red). Dorsal is to the right. Some cells are both her9⁺ and ntl⁺ (arrow). C: Sagittal section at the dorsal margin of a 7 hpf embryo. Some her9⁺ cells (arrowhead) are anterior to the ntl expression domain, while others appear to coexpress her9 and ntl (arrow). D: Whole-mount, dorsal view, at 8 hpf (75% epiboly) showing her9⁺ cells at the midline (arrowhead). E: Sagittal section at the dorsal midline, 8 hpf, showing her9 expression in presumptive floor plate cells (arrowheads) overlying ntl⁺ notochord precursors. F: Transverse section across the midline, 8 hpf, approximately at the level of the lower arrowhead in (E). her9⁺ cells are superficial (arrowhead) and lateral to (arrow) ntl⁺ notochord precursors. These are presumptive floor plate and hypochord precursors, respectively. G: Transverse section, 11 hpf (3-somite stage), showing her9 expression in floor plate cells (arrowhead) overlying the notochord (nc) and hypochord cells (arrow) underneath the notochord. Neural plate cells also express her9 at this stage (asterisk). Scale bar = 80 µm in A,D; 40 µm in E; 20 µm in B,C,F,G.

her9 floor plate expression requires Nodal but not Notch signaling. A: Mixed wild-type and Tg[hsp70:XdnSu(H)myc] expressing embryos heat-shocked at shield stage and then probed for her9 expression at late gastrulation. All embryos express her9 apparently normally. B: Whole-mount, dorsal view, of embryo treated with DAPT at shield stage shows her9 expression in the midline (arrowhead) at 80% epiboly (8.5 hpf). C: Whole-mount, dorsal view, of a cyc embryo probed for her9 at 80% epiboly. her9 is expressed in presumptive hypochord precursors (filled arrowhead), but expression in presumptive floor plate precursors is absent (open arrowhead). D: Transverse section at the 1-somite stage of wild-type embryo showing expression in the floor plate (arrowhead), hypochord precursors (arrow) and neural plate cells (asterisk). E: cyc mutant embryos do not express her9 in the floor plate (open arrowhead), but hypochord (arrow) and neural plate (asterisk) expression appears normal. Scale bar = 200 µm in A; 80 µm in B,C; 20 µm in D,E.

her9 overexpression down-regulates ntl and partially rescues the cyc mutant floor plate. A-D: Wild-type embryos injected with hsp70:her9myc and heat shocked at shield stage, fixed at YPC stage (9.5 hpf), and probed for ntl expression (blue) and myc labeling (brown). A: Whole-mount, dorsal view and (B) transverse section at high magnification showing that her9myc expressing cells at the midline do not express ntl (arrowheads). C,D: Control wild-type embryos injected with hsp70:GFP and heat shocked at shield stage, fixed at YPC stage and probed for ntl expression (blue) and GFP labeling (brown). C: Whole-mount, dorsal view and (D) transverse section showing that GFP expressing cells at the midline do express ntl (arrowheads). E-G: Whole-mount, side views of 24 hpf embryos probed for col2a expression. Anterior is to the left. E: Floor plate and hypochord express col2a in wild-type embryos (arrows). F: Floor plate is absent in cyc mutant embryos (asterisk), but hypochord develops normally (arrow). G: cyc mutant embryos injected with hsp70:her9myc and heat shocked at shield stage develop floor plate cells (arrowheads). Scale bar = 40 µm in E-G; 100 µm in A,C; 20 µm in B,D.