Figure 2: Generation and basic characterization of Salm4^−/− mice.

(a) Strategy used to generate Salm4^−/− mice. LacZ, β-galactosidase gene. Arrows flanking the neomycin gene indicate loxP sites. Arrows at the start of Exon 2 indicate primer sites for PCR genotyping. Note that the lacZ and neo are two separate markers. (b) PCR genotyping of Salm4^−/− (KO), Salm4^+/− (HT) and WT mice. (c) Lack of in situ hybridization signals in the Salm4^−/− brain (P56). (d) Salm4^−/− brain lacks detectable SALM4 proteins, as shown by two different SALM4 antibodies (1820 and 2026 guinea pig). (e) Normal gross morphology of Salm4^−/− brain regions (left, forebrain coronal section; right, cerebellar coronal section). Scale bar, 500 μm. (f) Normal numbers of neurons in hippocampal CA1 and CA3 regions. Scale bar, 20 μm. (g) Normal morphology of the apical and basal dendrites of Salm4^−/− hippocampal CA1 pyramidal neurons. Neuronal infusion of biocytin was followed by the analysis of apical (middle) and basal (right) dendritic complexity by Sholl analysis. Scale bar, 50 μm. (h) Distribution patterns of SALM4 proteins in the mouse brain, as revealed by X-gal staining of sagittal Salm4^+/− brain slices at 5 weeks. Scale bars, 1 mm. (i) SALM4 deletion does not induce compensatory changes in other SALMs, as determined by immunoblot analysis of Salm4^−/− whole brain (WB) and hippocampal lysates (3 weeks). n=3 mice, ns, Student’s t-test. (j) SALM4 deletion does not cause significant changes in the levels of synaptic proteins (scaffolds, receptors and signalling molecules). Salm4^−/− WB and hippocampal (HC) lysates (3 weeks) were immunoblotted by the indicated antibodies. n=3 mice, ns, Student’s t-test.