FIGURE 1

Generation and Primary Characterization of the Gjd2b ^−/− /Cx35.1^−/− Line. (A) General overview of the gjd2b ^−/− /Cx35.1^−/− gene structure and partial DNA sequence. The position of forward and reverse primers targeting exon one flanked the region containing CRISPR mutations. The sgRNA target with PAM sequence is outlined by the purple box; The predicted CRISPR-Cas9 binding region targeting a XhoI restriction site is indicated by the purple arrow. The CRISPR-Cas9 genome engineering strategy produced a 1bp substitution at position 12 of exon 1, resulting in the substitution of G to A in Cx35.1^−/− animals. The mutation is outlined with a green box. (B) Cx35.1 partial protein sequence. The mutation resulted in an early stop codon, as indicated by the purple asterisk (*). (C) Immunohistochemistry of gjd2b ^−/−/Cx35.1^−/− larvae. (C1-left) A monoclonal anti-Cx36 primary antibody (Invitrogen) was applied to 6dpf wild type (TL) to determine Cx35 immunoreactivity in a frontal section of a zebrafish larva. The retina of gjd2b ^−/−/Cx35.1^−/− fish showed fluorescence in the inner plexiform layer (IPL) and photoreceptor cell layer (PRL). The optic nerve (ON), optic chiasm (OC), and arborization fields of the retinotectal tract (AF) were immunoreactive. Scale bar 100 µm. (D, E) The immunoreactivity in the IPL and PRL of wild type TL (top) was reduced in gjd2b ^−/− /Cx35.1^−/− larvae (bottom). Arrows indicate the IPL. Scale bars 50 µm.