Figure 1. Generation and characterization of Snx6 CNS-specific knockout mice.

(A) Domain structure of SNX6. (B) Schematic diagram of the Snx6 gene locus, the targeting vector, and the mutant alleles after homologous recombination. FRTtF/FRTtR and loxtF/loxtR: primer pairs used for genotyping. The XhoI and HpaI probes used for Southern blotting analysis are shown. Neo: the neomycin resistance cassette. (C) Southern blotting analysis of wild-type (WT) and two independent clones of targeted ES cells (9 hr and 5D). (D) Immunoblots of tissue lysates from mouse littermates, probed with antibodies to SNX6. (E) Comparison of brain weight of Snx6^fl/fl (15) and Nestin-Cre; Snx6^fl/fl mice (12). Data represent mean ± SEM for each group. (F) Nissl staining of sagittal sections of whole brain from Snx6^fl/fl and Nestin-Cre; Snx6^fl/fl mice. Also shown are magnification of the cerebellum (middle panel) and the hippocampus/cortex area (right panel). Scale bar: 1 mm.

DOI: http://dx.doi.org/10.7554/eLife.20991.003

Figure 1—figure supplement 1. Expression and subcellular distribution of SNX6 in the CNS.

(A) Western blotting of wild-type mouse tissue lysates. (B) A schematic showing the relative positions of coronal sections in (D) and (E) in sagittal view. (C–E) Immunohistochemical analysis of SNX6 expression in mouse brain. Coronal sections of wild-type mouse were fixed and stained with antibodies to SNX6 and counterstained with hematoxylin. (F) Mouse hippocampal neurons were cultured in vitro for 18 days, fixed and immunostained with antibodies to SNX6 and Tau1 or MAP2. Shown are representative confocal microscopy images. (G) Background-subtracted mean intensity of SNX6 fluorescence in primary axon and dendrites. Measurement of fluorescence intensity is expressed in arbitrary units per square area in both axons and dendrites. All images (1024 × 1024 pixels, 16 bit) were obtained in the same settings (mean ± SEM, n = 15). (H) Mouse hippocampal neurons were cultured in vitro for 18 days, fixed and immunostained with antibodies to SNX6 and synaptophysin (SYP) or PSD95. Shown are representative confocal microscopy images. OB, olfactory bulb. Fi, fimbria. LD, lateral dorsal nucleum of thalamus. Sm, stria medullaris. MH, medial habenula. LH, lateral habenula. Bars: 100 μm in (C), (D), (E) and (D-1), 20 μm in (C-1 – 4 ), (D-2 – 3) and (E-1 – 8 ), 2 μm in (F) and (H).

DOI: http://dx.doi.org/10.7554/eLife.20991.004