Elevated levels of FGF3 can counteract the effect of elevated RA signalling on sensory development.

<p>Tg(<i>hsp70:fgf3</i>) embryos were treated with DMSO (A,G,M,S) or 20 nM RA from 20S and the first heat shock applied at the same time. Embryos were heat shocked never (A,G,M,S), once (1×HS; C,I,O,U), twice (2×HS; D,J,P,V), three times (3×HS; E,K,Q,W) or four times (4×HS; F,L,R,X) and sorted as non-green non-transgenic siblings (B,H,N,T) or green transgenic embryos (C–X). Non-green non-transgenic siblings were pooled; different HS treatments did not affect the phenotype (B,H,N,T). (A,G,M,S) Tg(<i>hsp70:fgf3</i>) embryos treated with DMSO show normal expression of the sensory marker <i>sox2</i> (A) and the non-neurogenic marker <i>otx1b</i> (G) and the otic markers <i>neurog1</i> (M) and <i>neurod1</i> (S). (B) In non-green sibling embryos treated with 20 nM RA, the expression of <i>sox2</i> extends across the ventral OV floor. (C–F) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and given 1×HS (C) or 2×HS (D), the expression of <i>sox2</i> is present in two extended domains at the anterior and posterior of the OV. (E) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and 3×HS the expression of <i>sox2</i> is normal or slightly reduced. (F) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and 4×HS the expression of <i>sox2</i> is slightly reduced or normal; the posterior domain of expression is shifted laterally. (H) In non-green sibling embryos treated with 20 nM RA, the expression of <i>otx1b</i> is reduced. (I–L) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and given 1×HS (I), 2×HS (J), 3×HS (K) or 4×HS (L), the expression of <i>otx1b</i> is greatly reduced. (N) In non-green sibling embryos treated with 20 nM RA, the expression of <i>neurog1</i> is reduced in the OV. (O,P) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and given 1×HS (O) or 2×HS (P), the expression of <i>neurog1</i> is reduced in the OV. (Q) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and 3×HS the expression of <i>neurog1</i> is normal but slightly shifted towards posterior. (R) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and 4×HS the expression of <i>neurog1</i> is increased but shifted to a more posterior position in the floor of the OV. (S,T) In non-green sibling embryos treated with 20 nM RA, the expression of otic <i>neurod1</i> is slightly increased, while expression is lost in prospective posterior lateral line and vagal ganglion cells posterior to the OV (asterisk). (U–W) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and given 1×HS (U), 2×HS (V) or 3×HS (W) the expression of <i>neurod1</i> is increased in the OV. In embryos heatshocked three times neuroblasts emerge in a more posterior position in the OV floor (W). (X) In Tg(<i>hsp70:fgf3</i>) embryos treated with 20 nM RA and 4×HS the expression of <i>neurod1</i> is increased and otic neuroblasts emerge from a more posterior position in the floor of the OV. In addition, <i>neurod1</i>-positive cell populations re-emerge below the OV. All panels are lateral views with anterior to the left. For numbers see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004858#pgen-1004858-t001" target="_blank">Table 1</a>. Scale bar: 50 µm.