Fig. 4

The anterior lateral line is the tissue source of Fgf10a responsible for facial placode development.

(A-D) confocal projections of Tg(pax2a:EGFP) zebrafish embryos (green) analyzed for Pax2a expression (magenta) at 14 (A), 18 (B), 21 (C), and 24 hpf (D). The presumptive facial placode is outlined in yellow as it condenses between 18 and 24 hpf. Insets show co-expression of Pax2a and Tg(pax2a:EGFP) at 14 hpf (A); by 18 hpf, however, Pax2a expression is absent in anterior lateral line precursors, while Tg(pax2a:EGFP) maintains expression in these cells (B). (E-E′′) Live confocal projection of a 12 hpf Tg(pax2a:Kaede) zebrafish embryo (E) with the anterior portion of the pax2a:Kaede+ domain photoconverted from green to red emission (E′) overlay (E′′). (F-F′′) Composite image of the same photoconverted embryo from (E) at 24 hpf analyzed for Pax2a expression (F) and cyan in (F′′) and photoconverted Kaede (F′) and red in (F′′). Note absence of Kaede positive cells in the facial placode. (G, H) In situ hybridization of eya1 in 52 hpf zebrafish embryos reveals proper neuromast deposition in control (G) and a failure of deposition and elongation of the anterior lateral line in fgf3-/-;fgf10-MO embryo (H; arrowheads). (I, J) Lateral views of the 24 hpf fgf3+10 morphant embryo showing the side that received wild-type donor cells (green) as well as the contralateral control side that did not receive donor cells. Pax2a expression is visualized by immunolabeling (magenta). Note partial rescue of the facial placode when wild-type donor cells were present in the presumptive anterior lateral line (J; arrowhead). (K, L) Quantification of Pax2a+ cells reveals a significant increase in the number of Pax2a+ cells in the facial (K) and glossopharyngeal and vagal placodes (L) of the transplanted sides versus contralateral sides (Wilcoxon matched-pairs signed rank test: **P<0.01; error bars: standard error of mean; n=8 embryos). Abbreviations: f, facial placode; g+v glossopharyngeal/vagal placode; ov, otic vesicle. Scale bars: 50 μm (A, I); 25 μm (G).