Fig. 5. Lactate is required for eye morphogenesis by regulating eye transcriptional programs.

A, B The addition of sodium L-lactate, which does not change the pH, significantly rescued the OV phenotype (arrowheads) in the presence of 20 µM GNE-140 (LDHi). C Quantification analysis using RT-qPCR confirmed that Rax and Six3 expression were also rescued by the addition of sodium L-lactate (25 mM). D Generation and genotyping of Ldha^delta/flox;Rax-Cre conditional mouse embryos (CKO) and derivation of ESCs. The delta allele was generated by germline deletion using Ella-Cre mice. Only one flox allele remains upon conditional Cre expression. Representative micrographs are shown as similar results were obtained from three independent experiments. E Genetic deletion of LDHA in the eye field-specific region leads to defects in optic vesicle formation in Ldha mutant organoids. The addition of lactate rescued the eye defects as indicated by the formation of OVs (arrowheads). F–I Immunostaining and RT-qPCR analyses showed that Ldha-depleted organoids recovered Rax and Six3 expression in the presence of 25 mM lactate (arrowheads); however, Ldha expression in the mutant OVs is not detected (F). J RNA-sequencing was performed to compare LDH block (LDHi) vs LDHi plus sodium L-lactate. Addition of lactate resulted in significant transcriptional changes as indicated by heatmap analysis that identified changes in the expression of various genes known to regulate eye formation (arrows). Scale bars, 100 µm (B, E–H). One-way ANOVA followed by Tukey’s post-hoc test was performed (C, I). ****p < 0.0001, ***p < 0.001, **p < 0.01, n.s. not significant (C, I). Data are presented as mean values +/−SEM (C, I). Source data are provided as a Source Data file. n = 3 biologically independent experiments were performed.