Fgf3 and Fgf10a work in concert to promote maturation of the epibranchial placodes in zebrafish

Abstract

Essential cellular components of the paired sensory organs of the vertebrate head are derived from transient thickenings of embryonic ectoderm known as cranial placodes. The epibranchial (EB) placodes give rise to sensory neurons of the EB ganglia that are responsible for relaying visceral sensations form the periphery to the central nervous system. Development of EB placodes and subsequent formation of EB ganglia is a multistep process regulated by various extrinsic factors, including fibroblast growth factors (Fgfs). We discovered that two Fgf ligands, Fgf3 and Fgf10a, cooperate to promote EB placode development. Whereas EB placodes are induced in the absence of Fgf3 and Fgf10a, they fail to express placode specific markers Pax2a and Sox3. Expression analysis and mosaic rescue experiments demonstrate that Fgf3 signal is derived from the endoderm, whereas Fgf10a is emitted from the lateral line system and the otic placode. Further analyses revealed that Fgf3 and Fgf10a activities are not required for cell proliferation or survival, but are required for placodal cells to undergo neurogenesis. Based on these data, we conclude that a combined loss of these Fgf factors results in a failure of the EB placode precursors to initiate a transcriptional program needed for maturation and subsequent neurogenesis. These findings highlight the importance and complexity of reiterated Fgf signaling during cranial placode formation and subsequent sensory organ development.

Figure 1. Fgf3 and Fgf10a are expressed during epibranchial placode formation.

(A-C) In situ hybridization reveals presence of fgf3 transcript in the mesoderm at 14 hpf (A), and then in the endoderm at 18 (B) and 22 hpf (C). (D-F) In situ hybridization reveals presence of fgf10a transcript in the anterior and posterior lateral line (arrows) and the anterior portion of the otic vesicle at 14 (D),18 (E) and 22 hpf (F). Otic vesicle is outlined by a dotted line in (A-F). Abbreviations: ov, otic vesicle; me, mesoderm; en, endoderm; All, anterior lateral line; Pll, posterior lateral line. Scale bar: 50 µm.

Figure 2. Fgf3 and Fgf10a are required for maturation of epibranchial placodes and development of the epibranchial ganglia.

(A-C) Confocal projections showing Pax2a expression in wild-type (A) fgf3+/- (B) and fgf3-/- (C) embryos at 24 hpf. (D-F) Confocal projections showing Pax2a expression in 24-hour old wild-type (D) fgf3+/- (E) and fgf3-/- (F) embryos injected with fgf10a-MO. Note the significant loss of Pax2a expression in the EB placodes (F; arrowheads). (G) Pax2a+ cell number in the facial, glossopharyngeal/vagal, and otic placodes for conditions in (A-F). Note the complete loss of Pax2a+ cells in the facial placode and a 4.5 fold reduction in the glossopharyngeal/vagal placode in fgf3-/-;fgf10a-MO embryos (ANOVA multiple comparison with Bonferroni’s correction; *P<0.05; ***P<<0.001; Error bars: standard error of mean; n=11 embryos per condition). (H-M) Confocal projections of 3 dpf wild-type (H), fgf3+/- (I) and fgf3-/- (J) embryos and wild-type (K), fgf3+/- (L) and fgf3-/- (M) embryos injected with fgf10a-MO. All embryos contain TgBAC(phox2b:EGFP) which marks EB ganglia. Note the loss of EGFP expression in the glossopharyngeal and three small vagal ganglia in fgf3-/- embryos (J) and the complete loss of EGFP expression in all EB ganglia in fgf3-/-;fgf10a-MO with the exception of a few EGFP+ cells in the region of the large vagal ganglion (M). Asterisk marks hindbrain neurons also expressing TgBAC(phox2b:EGFP). Abbreviations: f, facial placode (A) or facial ganglia (H); ov, otic vesicle; g+v, glossopharyngeal/vagal placode; g, glossopharyngeal ganglia; v1-v3, small vagal ganglia 1-3; v4, large vagal ganglion. Scale bars: 50 µm (A); 25 µm (H).

Figure 3. Fgf10a and endodermally derived Fgf3 cooperate during EB placode formation.

(A-C) Confocal projections of Pax2a expression in control (A), casanova-MO injected (B), or casanova/fgf10a-MO coinjected (C) embryos at 24 hpf. (D-F) Confocal projections of 72 hpf TgBAC(phox2b:EGFP) in control (D), casanova-MO (E), or casanova/fgf10-MO (F) embryos. Note a complete loss of Pax2a and EGFP expression in EB placodes and ganglia, respectively, in casanova/fgf10a-MO embryos (C and F). Abbreviations: f, facial placode (A) or facial ganglia (D); ov, otic vesicle; g+v, glossopharyngeal/vagal placode; cas, casanova; g, glossopharyngeal ganglia; v1-v3, small vagal ganglia 1-3; v4, large vagal ganglion. Scale bars: 50 µm (A); 25 µm (D).

Figure 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).

Figure 5. Fgf3 and Fgf10a are required for placode maturation and neurogenesis.

(A-F) Uninjected and fgf10a-MO injected progeny from fgf3+/-;TgBAC(neurog1-DSRed) crosses were collected at 36 hpf and immunolabeled for Pax2a and DSRed to visualize EB placodes and migrating neuroblasts, respectively. Wild-type (A) and fgf3+/- (B) panels shows neurog1:DSRed+ cells undergoing neurogenesis (magenta) at the dorsal aspect of the mature Pax2a+ EB placodes (green; dotted line). fgf3-/- embryos show a loss of properly formed Pax2a+ EB placodes in the region of the prospective glossopharyngeal and three small vagal ganglia and a concurrent loss of neurog1:DSRed+ cells in this region (C; bracket); however, the facial and large vagal placode/ganglia are still present (arrowheads). Analysis of fgf10a-MO injected wild-type (D) and fgf3+/- (E) embryos reveal ectopic neurogenesis as marked by neurog1:DSRed+ cells ventral to the posterior most aspect of the vagal placode (arrow). fgf3-/-;fgf10-MO embryos show a complete loss Pax2a expression in EB placodes and an absence of neurogenesis, except for a few neurog1:DSRed+ cells near the region of the large vagal ganglia (F; asterisk). Abbreviation: ngn:dsred, TgBAC(neurog1-DSRed); f, facial placode; g, glossopharyngeal placode; v, vagal placodes. Scale bar: 50 µm.

Figure 6. A model for EB placode development in zebrafish.

Before 12 hpf, Fgf3 and Fgf8 specify cells of the posterior placodal area that will give rise to the otic and EB placodes. Shortly after, between 14 and 22 hpf, Fgf10a, expressed by the anterior lateral line precursors and the forming otic vesicle, and Fgf3, expressed by the endoderm, are required for EB placode development by promoting the expression of Pax2a and Sox3 in a subset of cells of the Foxi1+ placodal competent ectoderm. By 24 hpf, EB placodes are fully developed. Abbreviations: NT, neural tube; MHB, midbrain hindbrain boundry; f, facial placode; ov, otic vesicle; All, anterior lateral line; g+v, glossopharyngeal/vagal placode.