Fig 2: 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).

Fig 3: Nine to fourteen somite mouse embryos, cultured for 24 h. (A) Control embryo, exposed to the solvent dimethyl sulfoxide (DMSO) only, reveals age-appropriate telencephalic hemispheres (green asterisk) and upper limb buds (green arrowheads); for comparison (green arrow), see corresponding hematoxylin stained histological sections (green colored areas). (B) Treatment with 2.5 μM of the pan-FGFR inhibitor PD173074 considerably impairs the development of telencephalic hemispheres (red asterisk) and upper limb buds (red arrowheads); for comparison (red arrow), see corresponding hematoxylin stained histological sections (red colored areas). (C) Application of 0.5 μM PD173074 did not cause any obvious abnormalities. (D) Embryos incubated with 40 μM of the pan-FGFR inhibitor SU5402 exhibit general growth disturbances (also see Table 1). (E,F) Treatment with 40 μg/ml of anti-FGFR3 neutralizing antibodies or 10 μM of the pan-BMPR inhibitor LDN193189 were well tolerated. Scale bars: 500 μm for stereomicrographs (A–F), 200 μm for light micrographs (A,B).

Fig 4: 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 5: FGFRs were expressed and modulated by FGF23 + Klotho, but not Klotho or FGF23 alone (B-DNA analysis). Cells were cultured in 12-well plates in differentiation medium (alpha-MEM/10% FBS/ascorbic acid [50 μg/mL]/BGP [10 mM]) and supplemented with huFGF23R176Q (F) (1 and 1,000 ng/mL), murine Klotho (KL) (50 ng/mL), their combination, or FGF2 (bFGF) for 14 days. a FGFR “IIIc” forms are preferentially expressed in MC3T3.E1 cells exposed to differentiation medium alone. b FGFR1(IIIc). c FGFR2(IIIc). d FGFR3(IIIc). Gene panel FGFR1(IIIb), FGFR1(IIIc), FGFR2(IIIb), FGFR2(IIIc), FGFR3(IIIc) and FGFR4. Bars represent mean ± SEM. *P < 0.05, **P < 0.01 compared with differentiation medium (Diff. Medium)