Figure 6.

Pcdh single-isoform overexpression in dissociated neurons revealed exclusively homophilic neuron–neuron interactions in trans. (A) Schematic diagram of the highly frequent trans-homophilic interactions of the γA3 isoforms between Δαβγ neurons. To simplify the demonstration, each isoform complex is shown as a dimer. (B,C,E,F) E16.5 hippocampal neurons were nucleofected with γA3-tdTomato or γA3-Venus expression vectors and co-cultured for 11 days. Significantly more tdTomato⁺ and Venus⁺ dots were colocalized in the hippocampal neurons of Δαβγ mutants, compared to those of the +/+:TG^taf7 mice. The small numbers at the corners indicate the number of colocalized yellow dots (arrows). (D) A similar experiment with different Pcdh isoforms (γB2-tdTomato and γA3-Venus) in Δαβγ mutants showed that the homophilic interaction was an isoform-specific feature that was only detectable in the Δαβγ neurons. Immunostaining revealed microscope fields in which tdTomato⁺ and Venus⁺ neurites ran parallel within a distance of <0.5 μm of each other. Data were analyzed by one-way ANOVA and post-hoc Tukey tests. The numbers of neurite pairs analyzed were as follows: γA3-γA3, 11 control (+/+:TG^taf7) and 16 Δαβγ; γA3-γB2, 20 in mutant Δαβγ. Error bars represent SEM; ^***P < 0.001. Bars: 0.5 μm.