Fig 2: Impact of FGFR inhibition on the neurogenesis of epibranchial placodes (EP) in 9–14 somite mouse embryos, cultured for 24 h. Box plots indicate the numbers of Neurogenin (Ngn)2+ (A–C) or Ngn1+ (D–F) neuroblasts in EP1 (A,D), EP2 (B,E), or EP3 (C,F) following exposure to either dimethyl sulfoxide (DMSO) only (control; gray), or increasing doses of pan-FGFR inhibitor SU5402 (light blue), or 0.5 µM of pan-FGFR inhibitor PD173074 (dark blue), or increasing concentrations of anti-FGFR3 neutralizing antibodies (yellow). Significant differences between each treatment group and the controls are indicated by gray asterisks (*P < 0.05, **P < 0.001; Mann–Whitney test). Additionally, PD173074-treated embryos were compared to groups exposed to either highest levels of SU5402 or anti-FGFR3 antibodies (significant differences: #P < 0.05, ##P < 0.001; or specific P values, respectively). Whiskers, lower and upper extremes; box limits, 25th and 75th percentiles; red center lines, medians; black dots, data points; open circles, outliers.

Fig 3: Impact of FGFR and BMPR inhibition on epibranchial placode morphogenesis and neurogenesis in 9–14 somite mouse embryos, cultured for 24 h. Anti-Neurogenin-2 (Ngn2) immunohistochemistry (brown precipitate); shown are maximum numbers of Ngn2+ neuroblasts found per section. (A–C) Control placodes reveal high-grade thickened pseudostratified epithelium (2–4 cell nuclei) with 12–16 Ngn2+ neuroblasts. (D–F) Pan-FGFR inhibitor SU5402 (40 µM) causes moderate reductions in placode thickness and statistically significant decreases in neuroblast numbers in the epibranchial placodes 1 and 2, but not 3 (also see Figure 4). (G–I) Pan-FGFR inhibitor PD173074 (0.5 µM) elicits strong decreases in the thickness of the epibranchial placodes 1 and 2 as well as statistically significant decreases in neuroblast numbers in all three epibranchial placodes (also see Figure 4). (J–L) High doses of PD173074 (2.5 µM) result in the complete absence of high-grade thickened epibranchial placodes. Only single Ngn2+ neuroblasts can be detected, if at all (arrowhead in panel L). (M–O) Anti-FGFR3 neutralizing antibodies (40 µg/ml) reduce Ngn2+ neuroblast numbers, but do not have negative effects on placode thickness. (P–R) Following pan-BMPR inhibition (10 µM LDN193189), epibranchial placode 1 reveals reduced numbers of Ngn2+ neuroblasts, but normal morphology (P); epibranchial placode 2 lacks any obvious impairment (Q); epibranchial placode 3 simultaneously presents slight reductions in Ngn2+ neuroblast numbers and placode thickness (R). p1, p2, pharyngeal pouches 1 and 2. Scale bars: 20 µm.

Fig 4: Neurogenesis in the lateral line placodes of Q-VD-OPh-treated mice. (A,C,F,G) Reconstructions of serially sectioned mouse embryos (section interval evaluated=10 µm) show ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), immunopositive cells (brown dots), developing cranial nerves (partly numbered brown contours in G). (B,D,E) Micrographs taken from serially sectioned mouse embryos. (A) Sox2+ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #021). (B) Sox2+ mantle (m) and support (s) cells in a neuromast primordium of a middle lateral line placode. Note the apical cavity (arrowhead). (C) Ngn1+ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #039, mirror-imaged right side). (D) Ngn1+ neuroblasts in an anterodorsal lateral line placode. (E,F) Ngn2+ neuroblasts are present in epibranchial placodes, but absent from lateral line placodes (embryo #001). (G) Tubb3+ neurons constitute the anlagen of the cranial nerves 7 and 8 (VII/VIII), 9 (IX), 10 (X), 11 (XI) and 12 (XII). Tubb3+ candidates for vestigial lateralis ganglia (asterisks) reside in close proximity to the middle lateral line placode (embryo #014). All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 5 µm. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; ot, otic anlage; ov, optic vesicle; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.

Fig 5: Developmental profiles of the epibranchial placodes of C57BL/6N mice (embryonic days (E) 8.5 to E11.5) as reflected by earlier reconstructions of Ngn2+ intraplacodal neuroblasts (Washausen and Knabe, 2013) recounted for present purposes (n = 11 mouse embryos with 22 body sides). (A–C) Using CurveExpert Professional (Hyams Development, Chattanooga, TN, United States), regression curves were generated that represent the developmental profiles of the epibranchial placodes 1, 2, and 3 (EP1, red; EP2, blue; EP3, green). Confidence bands (medium red, blue, or green) most likely contain 95% of all values (black dots). Prediction bands (faint red, blue, or green) indicate the range in which 95% of all future values will fall. Here, embryos possessing 9–14 pairs of somites were included in our whole embryo cultures (WECs). This range is indicated by the left of the two gray columns and is correlated with the embryonic age plotted on the x-axis. The right gray column marks the period within which embryos were removed from the culture after 24 h. Asterisks indicate the respective medians of Ngn2+ neuroblasts for EP1, EP2, and EP3 of DMSO control embryos (Table 2). (D–I) Box plots display the relative changes in the numbers of Ngn2+ (E–G,I) or Ngn1+ (D,H) neuroblasts following exposure to either the pan-FGFR inhibitor PD173074 (D,I), to anti-FGFR3 neutralizing antibodies (F–H), or to the pan-BMPR inhibitor LDN193189 (E), respectively (see Table 2, also for n values). Significant differences between EP1 (red), EP2 (blue), and/or EP3 (green) are indicated by asterisks (*P < 0.05, **P < 0.001; Mann–Whitney test). Whiskers, lower and upper extremes; box limits, 25th and 75th percentiles; red, blue, or green center lines, medians; open circles, outliers. (D,E) Plausibility checks demonstrate that EP2 and EP3, which almost synchronously produce neuroblasts, by no means always react in the same way to certain inhibitors. (D,F–I) Conversely, EP1, which slightly precedes EP2 and EP3 in terms of neuroblast production, does not always deviate in its behavior toward certain inhibitors from EP2 and/or EP3 (for details, see text).