Figure 3. mafba and mafbb are required for valve formation and function in the facial lymphatics (A) Confocal projection of facial lymphatics and valve (arrowheads) labeled with Tg(fli1a:nEGFP) (cyan) and Tg(prox1a:RFP) (magenta) at 5 dpf, masked with surface rendering of both channels. Scale bars: 50 and 20 μm. Dashed box: magnified images. (B) Valve-length measure from (A). Embryos per genotype; n = 15. Kruskal-Wallis test: ∗∗p ≤ 0.009 for double mutants versus sibling or mafbb; ∗p = 0.044 mafba versus double mutants; other comparisons: ns p > 0.9999. (C) (Top) Projections of the average phenotype of siblings (n = 12), mafba mutants (n = 11), and double mutants (n = 12) in Tg(prox1a:RFP) embryos at 5dpf. Arrow: lymphatic valve; ∗: its absence. (Bottom) Overlay of siblings (green) with siblings and mafba or double mutants (magenta). Arrow: valve; ∗: absent valve. Scale bar, 50 μm. (D) Surface rendering of Tg(prox1a:RFP) showing valve structure in siblings, mafba mutants, and double mutants at 5 dpf. Fontal and lateral views. Scale bar, 10 μm. (E) Confocal imaging of Tg(fli1a:nEGFP) (yellow, endothelium), Tg(prox1a:RFP) (green), and Qtracker 665 (magenta) in the facial lymphatics at 5 dpf, in siblings and mafbb, mafba, and double mutants, 5 min post-injection. FCLV, facial collecting lymphatic valve; FLV, facial lymphatic vessel. ∗: absence of dye in FCLV. Scale bar, 50 μm. (F) Percentage of embryos with dye leakage into FCLV at 5 min post-injection in siblings (n = 17), mafbb mutants (n = 8), mafba mutants (n = 15), and double mutants (n = 6). Chi-squared; p = 0.0159. Single comparisons with Fisher’s exact test: ∗∗ p ≤ p = 0.009 for double mutants versus siblings or mafbb; ∗p = 0.0456 for mafba versus double mutants; other comparisons: ns with p ≥ 0.399. ns, non-significant; stars are significant; error bars are mean ±SD. See also Figure S2 and Video S2.
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