Glyphosate exposure impairs visual function in zebrafish larvae through retinoic acid-dependent regulation of six3b expression

Glyphosate (GLY) is a widely used, broad-spectrum organophosphorus herbicide. Epidemiological studies have raised concerns about its potential risks due to environmental residues and occupational exposure, yet the molecular mechanisms underlying its toxicity, particularly in the visual system, remain poorly understood. This study investigates the toxic effects and mechanisms of GLY on the visual system using zebrafish. Wild-type and six homeobox 3b ( six3b )-overexpression zebrafish embryos at 5 h post-fertilization (hpf) were exposed to environmentally relevant concentrations of GLY (0.14, 0.7, 1.4, and 2.8 mg/L) until 144 hpf. Results indicated that exposure to varying concentrations of GLY did not significantly affect basic developmental parameters, such as hatchability, mortality, malformation rates, or larval eye diameter. However, it markedly impaired visual motor response and optomotor response in zebrafish larvae. At 1.4 mg/L, GLY exposure reduced optic nerve size, suppressed six3b gene transcription, decreased Six3 protein expression, and lowered retinoic acid (RA) concentration in larvae. Both six3b overexpression and RA supplementation rescued GLY-induced optomotor response deficits. Moreover, RA supplementation restored six3b expression and normalized optic nerve morphology in GLY-exposed larvae. Taken together, these findings demonstrated that exposure to environmentally relevant concentrations of GLY impairs visual behavior in zebrafish larvae through optic nerve hypoplasia, mediated by RA-dependent downregulation of six3b expression.