Extended Data Figure 1: Ten3 staining details and controls.

a, Diagram of Ten3 protein showing location of antibody epitopes, specific domains, and region deleted in the Ten3^Δ4 mutant. In Ten3^Δ4, a neomycin resistance cassette (Neo) replaces 110 base pairs of sequence directly N-terminal to the transmembrane domain. The Ten3IC antibody was used for all Ten3 stainings in the paper except d and e of this figure. Scale bar, 200 amino acids. b, c, Ten3IC staining (red) on P9 horizontal sections of Ten3^WT (b) and mutant Ten3^Δ4/Δ4 (c) brains, showing loss of staining in Ten3 mutants. Note that the Ten3IC epitope is located N-terminal to Δ4, suggesting that in Ten3^Δ4/Δ4 either the mRNA undergoes nonsense-mediated decay or the truncated protein is not stable. d, e, Ten3EC staining on P10 horizontal sections of Ten3^WT (d) and mutant Ten3^Δ4/Δ4 (e) brains. Staining has a higher background than Ten3IC antibody, but signal is present in proximal CA1 (arrow) and distal subiculum (arrowheads), similar to Ten3IC, which is absent in the knockout (open arrow/arrowheads). f, Ten3 staining (red) on P10 horizontal section with boxes around regions magnified in g–i. g, Ten3 staining in dentate gyrus from f, CA3, and CA1. Intensity was increased to highlight Ten3 signal in axons and dendrites. Ten3 in stratum radiatum of proximal CA1 (arrowhead) is most probably from CA1 dendrites, since CA3 cells, the major source of axons in this layer, did not express Ten3 mRNA (Fig. 1b). Ten3 in the molecular layers of dentate gyrus and CA3 (arrows) is probably contributed by the axons of MEC layer II neurons⁵⁶, since dentate gyrus and CA3 neurons did not express Ten3 mRNA (Fig. 1b). h, Proximal CA1 pyramidal cell layer from f, showing Ten3 signal in cell bodies. i, Proximal CA1 stratum lacunosum-moleculare from f, showing Ten3 signal in the region where MEC axons synapse onto CA1 pyramidal neuron dendrites. j, In situ hybridization on P9 horizontal section for Ten3 mRNA (magenta) combined with immunostaining for PCP4 (green), a marker of CA2 neurons⁵⁷. No overlap between Ten3 and PCP4 was observed. Scale bars, 200 μm in b–g, j, and 100 μm in h, i.