Fig. 3

aff3 knocked down zebrafish larvae display altered behavior and morphological anomalies. The conditions analyzed are the following: Uninjected (Un), Mock-injected (M), and aff3 knockdown (KD). A Proportions of normal and developmentally defective 5 dpf embryos. In 10% of aff3 KD zebrafish, we identified several morphological anomalies such as head malformations, belly and heart edema, skeleton-muscular dysmorphologies, and alteration of eye pigmentation. B Alcian blue staining at 5 dpf revealed jaw malformation in 57% of aff3 KD zebrafish. C Visualization of morphological inter-ocular distance (IOD) and head width (HW) measurements from dorsoventral zebrafish image. Quantification of IOD (D) and HW (E) indicates a significative decrease in IOD and HW in aff3 KD larvae; p* < 0.04; p** < 0.0049. F Touch test response assay at 3 dpf. Upon a touch stimulus, we classified the larvae swimming behavior in «normal swimming», «pause», «looping swimming», «pinwheel swimming», or «motionless» due to malformations. G Swimming global velocity analysis at 5dpf in the dark of Un, M, and aff3 KD and aff3 KD co-injected with human AFF3 (hAFF3) mRNA wild-type (Wt), which recovered 57% of the locomotion function compared to aff3 KD, or harboring the indicated missense variant, i.e., the KINSSHIP variants Ala233Thr and Val235Gly or the biallelic variants identified in this report in a healthy (Gln179Glu) or affected individuals (Lys528Arg and Thr592Ser). H Proportions of normal and developmentally defective 5 dpf embryos uninjected (Un), injected with water as control (H₂0) or with 360 ng of hAFF3 mRNA Wt or the indicated missense variant. Larvae were cataloged as described: (i) normal phenotype, (ii) Class 1 with skeletomuscular dysmorphology and/or small dimension, (iii) Class 2 with a more severe phenotype including at least three of the following characteristics: skeletomuscular dysmorphology, small dimensions, head malformations, eyes’ alteration, pericardial edema, and lateral belly edema or (iv) dead. Injections of 180 and 720 ng of hAFF3 mRNA showed similar results