Fig. 3: TM9SF3 is essential for the early development of zebrafish.
From: Cells adapt to extracellular acidic pH through TM9SF3-mediated PI(4,5)P2 flop
a Schematic illustration of zebrafish embryogenesis from 6 to 14 hpf. b Ratiometric images of SEpHluorin/mCherry fluorescence intensity in a Tg(EF:Lyn-mCherry-SEpHluorin) embryo from 6 to 14 hpf. The color scale represents relative pH, with blue to purple indicating acidic conditions and yellow to red indicating alkaline. This color scheme is used consistently for all pH analyses in the study. Lateral view; anterior is up. c Ratiometric images of SEpHluorin/mCherry fluorescence intensity in an embryo injected with mCherry-SEpHluorin-GPI encoding mRNA from 6 to 14 hpf. Lateral view; anterior is up. d Whole-mount in situ hybridization showing tm9sf3 mRNA expression in zebrafish embryos at 4 hpf. e Western blot analysis comparing protein expression in WT and tm9sf3−/− zebrafish embryos at 4 hpf. f Immunofluorescence images of Tm9sf3 in WT and tm9sf3−/− zebrafish embryos at 4 hpf. Insets from the upper panel (yellow outline) are enlarged below. Animal pole view. g Confocal single-scanned cell images of isolated blastoderm cells from WT, tm9sf3−/−, and tm9sf3 mRNA-injected tm9sf3−/− zebrafish embryos at 4 hpf incubated with PIP2 probe in Danieau’s buffer adjusted to pH 7.2. h Quantitative analysis of (f). The mean fluorescence intensity of TagRFP on the PM was normalized to the average value in WT cells (set as 1) (WT, n = 181; tm9sf3−/−, n = 153; tm9sf3−/− + Tm9sf3, n = 137). P-values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. **** indicates P < 0.0001. The P value for the comparison between WT and tm9sf3−/− + Tm9sf3 mRNA is 0.1522. i Confocal scanned z-stack images of WT and tm9sf3−/− embryos at 7 hpf, injected with mRNA encoding membrane-anchored EGFP and a secretable PIP2 probe (see Methods) at 7 hpf. Enlarged views of EVL and blastoderm cells are shown. Animal pole view. j Images depicting littermate WT, tm9sf3+/-, and tm9sf3−/− zebrafish at 1 month post-fertilization (mpf). Dashed lines indicate the body length used for quantification. Lateral view. k Quantitative analysis of body length from (j) (WT, n = 54; tm9sf3+/-, n = 96; tm9sf3−/−, n = 7). P-values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. The P values for the comparison are as follows: WT vs tm9sf3+/-, 0.6064; WT vs tm9sf3−/−, 0.0011; and tm9sf3+/- vs tm9sf3−/−, 0.0274. l Survival rate of zebrafish shown in (j). h, k Data are presented as mean values ± SD. All source data are provided as a Source Data file. SE superecliptic, EVL enveloping layer, WT wild-type. Scale bars, 100 µm in (b, c), 200 µm (d), 50 µm (f), 20 µm (g, i) and 1 mm (j).
