Figure 6. Pcdhgc3 is the only isoform that mediates sensory neuron synapse formation in vivo and can promote postsynaptic specialization in vitro.
(A) Experiment design for expressing single Pcdhg isoforms (Pcdhga1 or Pcdhgc3) in AdvillinCre;Pcdhgfcon3/fcon3 mice.
(B) IHC images of spinal cord dorsal horn lamina III from AdvillinCre;Pcdhgfcon3/fcon3;cA1 and AdvillinCre;Pcdhgfcon3/fcon3;cC3 mice.
(C and D) Normalized densities of vGluT1+ (C) and Homer1+ (D) puncta. Each dot on the plot indicates a normalized value for an animal. One-way ANOVA test.
(E) Experiment design for deleting both copies of the Pcdhgc3 isoforms only in the dorsal horn neurons in the Lbx1Cre;PcdhgC3KO/fcon3 mice.
(F) IHC images of spinal cord dorsal lamina III from control and Lbx1Cre;PcdhgC3KO/fcon3 mice.
(G-J) Quantifications of the synapses in lamina III, showing that deleting Pcdhgc3 in the dorsal horn neurons led to reduced densities of vGluT1+ (G), Homer1+ (H) puncta, as well as the density of Homer1+ puncta surrounding vGluT1+ terminals (I). Similarly, the average size of Homer1+ puncta is reduced in Lbx1Cre;PcdhgC3KO/fcon3 mice (J). Each dot on the plot indicates a normalized value for an animal. Student’s unpaired t test.
(K) IHC showing the localizations of Pcdhgc3-mCherry in the spinal cord lamina III of P5 and P45 AdvillinCre;cC3 mice and P22 Lbx1Cre;cC3 mice (n = 3 animals for each genotype).
(L) Representative images of artificial synapse formation assays in which Nrxn1β, Pcdhgc3, or Pcdhgc3ΔEcto is expressed in HEK293T cells that are cocultured with neonatal spinal cord neurons.
(M) Representative images of in vitro synapse formation assays. Note the MAP2+ neuronal cell body in the upper right corner of the panel on the right (Pcdhga7 condition).
(N and O) Average number (N) and intensity (O) of PSD-95 puncta per HEK 293 cell for each condition. Data are means ± SEM from three independent replicates. One-way ANOVA with Tukey’s post hoc test.
ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.