SRP373654 PRJNA835105 GSE202238 Alx function in the developing eye Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. We show that zebrafish alx1; alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity. Overall design: Germline samples: RNAseq was performed on single zebrafish embryos derived from an alx1;alx3/+ mutant incross. A total of 12 samples (4 each of alx1, alx1; alx3/+ and alx1; alx3) were used for pairwise comparisons; CRISPANTs samples: Additional samples were sequenced using 72hpf alx1; alx3 CRISPANTs (multiple alx3 guides injected into alx1 embryos). A total of 12 samples (6 from alx1; alx3 CRISPANTs and 6 WT). GSE202238 pubmed 35142342