Fig. 4

Craniofacial outcomes of zfhx4 F0-generation knock-out (F0-KO) and translation-blocking morpholino knock-down (MO-KD) in zebrafish larvae (zfl).

A Schematic overview of the human primary (green) and secondary palate (light blue) (ventral view). B Schematic overview of the zfl neurocranium with medial (green) and lateral ethmoid (light blue) (ventral view). Both structures are homologous structures to the human palate as indicated by the corresponding colors. C Ventral view of the zfl cartilage structures of the neurocranium (green and light blue) and viscerocranium (dark blue). The eyes are outlined in light gray. D Ventral view of the craniofacial region of whole-mount Alcian blue stained zfl at 4 dpf as represented schematically in C (for a different zfl than that shown in Fig. 3A). Comparison of craniofacial phenotypes between F0-KO zfl against controls (UI_CRISPR and Ctrl-Scr). Scale bar = 200 µm. E Ventral view of the craniofacial region of whole-mount Alcian blue-stained zfl at 4 dpf as represented schematically in C. Comparison of craniofacial phenotypes between MO-KD-injected zfl against controls (UI_MO and Ctrl-MO). Scale bar = 200 µm. F Length of Meckel’s cartilage and ethmoid plate measured as indicated by the pink lines of 4 dpf old, Alcian blue-stained F0-KO- and Ctrl-Scr-injected zfl (mean length Meckel’s: F0-KO: 96.9 μm, Ctrl-Scr: 107.7 μm, difference between means: 10.8 ± 5.4 μm; mean length ethmoid: F0-KO: 103.4 μm, Ctrl-Scr: 108.9 μm, difference between means 5.5 ± 3.1 μm) and MO-KD- and Ctrl-MO-injected zfl (mean length Meckel’s: MO-KD: 79.5 μm, Ctrl-MO: 102.9 μm, difference between means: 23.5 ± 6.3 μm; mean length ethmoid: MO-KD: 91.1 μm, Ctrl-MO: 101.5 μm, difference between means: 10.4 ± 3.2 μm). Comparisons were performed using unpaired, two-tailed Welch’s t tests. G Measurement of the Meckel’s-palatoquadrate (M-PQ) angle as represented by the pink angle, which is a reliable parameter to assess craniofacial outcomes as suggested by Raterman et al. [43]. Comparison of M-PQ angles in 4 dpf old, Alcian blue-stained F0-KO- and Ctrl-Scr-injected zfl (mean M-PQ angle: F0-KO: 45.6°, Ctrl-Scr: 43.1°, difference between means 2.5° ± 1.1°) and MO-KD- and Ctrl-MO-injected zfl (mean M-PQ angle: MO-KD: 58.8°, Ctrl-MO: 46.4°, difference between means 12.5° ± 2.0°). Comparisons were performed using unpaired, two-tailed Student’s t tests. ns, p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. m Meckel’s, pq Palatoquadrate, ch Ceratohyal, hs Hyosympletic, dpf days post-fertilization, UI_CRISPR/MO uninjected wild-type zfl, Ctrl-MO Control MO-injected zfl, Ctrl-Scr Scrambled control-injected zfl.