Figure 2. aff3 knocked-down zebrafish larvae display altered behavior and morphological anomalies.

The conditions analyzed are the following: Untreated (Un), Mock-injected (M), and aff3 Knock-Down (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) 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); p**<0.0025; p**** < 0.0001. (H) Proportions of normal and developmentally defective 5 dpf embryos uninjected (Un), injected with water as control (H₂0) or with 360ng 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 180ng and 720ng of hAFF3 mRNA showed similar results.