Fig. 1

Characterization of the lhx4 mutation. (A) A 5 bp deletion mutation (denoted by red dashes) at the end of exon 1 of the lhx4 gene was generated by the CRISPR-Cas9 system. (B) The cDNA sequences derived from WT and lhx4-mutant brains indicate a frameshift caused by the deletion mutation (underlined in WT sequence), leading to 13 altered amino acids (aas) (red) and an early stop codon in exon 2. (C) Gel Electrophoresis of PCR products amplified from WT and lhx4-KO cDNAs using a primer set targeting exons 1 and 3 yielded a 265 bp WT and 260 bp mutant product. The similar product lengths confirm that the splicing of the lhx4 mRNA was not altered by the mutation. (D) The lhx4 mutation resulted in a predicted 41 aa truncated protein (bottom), including altered aas (orange), as compared with the 391 aa WT protein (top). The positions of the LIM1 and LIM2 domains (yellow) and homeobox domain (green) are designated in the WT protein.