At embryonic day 9 (E9), before neural crest-derived neurons have differentiated, neuronal beta-III tubulin expression (detected by the TuJ1 antibody) shows neurons in the ophthalmic lobe of the trigeminal ganglion (arrowheads) projecting towards the eye in a 17-somite wildtype embryo (green channel only in A; green channel superimposed on brightfield in B; higher-power images of the same embryo in A1,B1), but absent in a 17-somite homozygous Splotch^2H (Sp^2H) embryo (green channel only in C; green channel superimposed on brightfield in D; higher-power images of the same embryo in C1,D1). The same difference in phenotype was also seen at the 18-somite-stage (not shown). (E,F) At E9, Ngn1 is expressed in the ophthalmic lobe of the trigeminal ganglion (arrowheads) in both wildtype (E) and homozygous Sp^2H embryos (F). (G,H) At late E9.5, expression of the neuronal marker SCG10 shows neurons in the ophthalmic lobe of the trigeminal ganglion (white arrowheads) and along the ophthalmic nerve projecting towards the eye (black arrowheads), both in wildtype embryos (G), and also, though in reduced numbers, in homozygous Sp^2H embryos (H). The lefthand trigeminal ganglion can be seen, out of focus, in both embryos.
E, embryonic day; ma, mandibular arch; ss, somite-stage.

Panels A-E show transverse sections through the opV placode region of chick embryos a day after electroporating either mPax3En-nGFP or nGFP at the 4–7 somite-stage. The opV placode region can be identified by the expression in untargeted cells of Pax3 and the opV placode-specific markers FGFR4 (A) or Ngn2 (B,C). (A,B) Most mPax3En-nGFP-targeted cells (nuclear GFP, green) in the opV placode region do not express Pax3 itself (red nuclei) or the neuronal differentiation marker Islet1 (white nuclei in A3,B3 for easier visualisation; blue nuclei in merged images in A4,A5 and B4,B5), and do not delaminate. (C) Control nGFP-targeted cells (nuclear GFP, green) in the opV placode region (identified by expression of Ngn2) express Pax3 (red nuclei), differentiate as neurons (white nuclei in C3 for easier visualisation; blue nuclei in merged images in C4,C5), and delaminate into the mesenchyme (arrows indicate examples of delaminated nGFP-targeted Pax3+Islet1+ cells). (D,E) Most mPax3En-nGFP-targeted cells (nuclear GFP, green) in the opV placode region do not express the preplacodal marker gene Eya2. Arrows indicate untargeted Eya2+ cells near Eya2-negative mPax3En-nGFP-targeted ectoderm. (F,G) mPax3En-nGFP (nuclear GFP, green) has no obvious effect on Pax2 expression in the otic vesicle and epibranchial placodes (arrowheads indicate examples of Pax2+ mPax3En-nGFP-targeted cells). (H) Summary graph showing the mean and s.e.m. of the percentage per opV placode of mPax3En-nGFP-targeted versus control nGFP-targeted cells expressing FGFR4, Ngn2, Islet1, and Pax3 itself, and delaminating (see Table 1 for data). The number above each column indicates the number of opV placodes counted for that case.
hb, hindbrain; opV, ophthalmic trigeminal; ov, otic vesicle.

All panels show transverse sections through the epibranchial placode and/or otic vesicle region of chick embryos either 1 day (A-F) or 2 days (G-J) after electroporating cPax3/GFP or GFP at the 6–8 somite-stage. (A,B) Most cPax3/GFP-targeted cells (Pax3, red nuclei) in the epibranchial placode region do not co-express Pax2 (green nuclei). Compare with unelectroporated side in A. White arrowheads in B show Pax3+Pax2-negative cells; yellow arrowheads show Pax3+Pax2+ cells. (C,D) Most cPax3/GFP-targeted cells (Pax3, red nuclei) in the otic vesicle, as well as expressing Ngn2, fail to co-express Pax2 (green nuclei), and the otic vesicle fails to close properly (compare with Fig. 4G,H). White arrowheads in D show Pax3+Pax2-negative cells; yellow arrowheads show Pax3+Pax2+ cells. (E,F) Electroporating GFP (cytoplasmic GFP, green) has no effect on Pax2 expression in the epibranchial placodes: white arrowheads show examples of Pax2+GFP+ cells. (NB All these embryos were fixed for in situ hybridisation on wax sections. Since only the rabbit anti-GFP antibody works well in our hands after this procedure, and the anti-Pax2 antibody was also raised in rabbit, for the GFP controls we performed in situ hybridisaton for Pax2, followed by immunostaining for GFP.)
hb, hindbrain; ov, otic vesicle; ph, pharynx.

All panels show transverse sections through the opV placode region of chick embryos a day after electroporating either mPax3En-nGFP or nGFP at the 4–7 somite-stage. (A,B) Most mPax3En-nGFP-targeted cells (nuclear GFP, green) do not express FGFR4 (arrowheads show examples). (C,D) Control nGFP-targeted cells (nuclear GFP, green), including cells that have delaminated from the ectoderm, express FGFR4 (arrowheads show examples). (E,F) Most mPax3En-nGFP-targeted cells (nuclear GFP, green) do not express Ngn2 (arrowheads show examples). (G,H) Control nGFP-targeted cells (nuclear GFP, green) express Ngn2. Much more extensive Ngn2 expression is seen in controls (compare panels E and G).
hb, hindbrain.

(A-C) Ectopic FGFR4 expression is seen in head ectoderm a day after electroporating cPax3/GFP on the righthand side (black arrowheads), including in the otic vesicle (ov) and nearby pharyngeal ectoderm, where the epibranchial placodes form. Normal FGFR4 expression is seen in the opV placode on the unelectroporated side (white arrowhead) and in the lens (Marcelle et al., 1994). Panels A and B show two different embryos; panel C shows a transverse section through another embryo after wholemount in situ hybridisation for FGFR4. (D) No ectopic FGFR4 expression is seen after electroporating GFP; normal FGFR4 expression is seen in the lens and opV placode (white arrowhead). (E,F) In situ hybridisation directly on sections shows strong FGFR4 expression in cPax3/GFP-targeted cells (Pax3, red nuclei) in the epibranchial placode region, a day after electroporation at the 6–7 somite-stage. (This is a different embryo from those shown in A-C.) (G) FGFR4 expression is not seen in GFP-targeted cells (cytoplasmic GFP, green) in the otic and epibranchial placode region, a day after electroporation at the 7–8 somite-stage. (H) Normal Ngn2 expression is seen in GFP-targeted cells (cytoplasmic GFP, green) in the otic and epibranchial placode region, a day after electroporation at the 7–8 somite-stage. (I,J) Ngn2 expression is seen in cPax3/GFP-targeted cells (Pax3, red nuclei) in surface ectoderm in the vicinity of the otic vesicle, a day after electroporation at the 6–7 somite-stage. (K,L) The neuronal nuclear protein NeuN (red nuclei) is expressed in the vestibuloacoustic and geniculate ganglia, but not in a cluster of cPax3/GFP-targeted cells (Pax3, blue nuclei; GFP, green cytoplasm) at the edge of the geniculate ganglion, 36 hours after electroporation at the 16ss.
gen, geniculate ganglion; h, heart; hb, hindbrain; ov, otic vesicle; va, vestibuloacoustic ganglion.

Model for opV placode development in the chick embryo, based on work presented here and in Lassiter et al. (2007), as well as McCabe and Bronner-Fraser (2008). See text for details.
fb, forebrain; ov, otic vesicle.