NB-3 interacts with CHL1. (A) CHL1 was co-immunoprecipitated with NB-3 from mouse brains. P7 mouse brain membrane fractions immunoprecipitated (IP) with either rabbit anti-NB-3 or nonspecific rabbit IgG were probed for the presence of CHL1 or L1 as indicated. Input, lysates before immunoprecipitation; IgG, nonspecific rabbit IgG. (B) Association of NB-3-Myc and CHL1-HA in HEK293T cells. Transfected HEK293T cells were precipitated with anti-Myc or anti-HA antibodies and were probed with anti-HA or anti-Myc antibodies, respectively. (C) Purity of recombinant mouse NB-3/Fc and CHL1/Fc proteins used in ELISA as determined by SDS–PAGE and Coomassie blue stain. (D) Binding of NB-3/Fc (50 μg/ml) to immobilized CHL1/Fc (▪), L1/Fc (▴), and F3/Fc (•) (100 μg/ml) in ELISA plates. Background was estimated in BSA-coated (1 mg/ml) wells. Readings for wells coated with NB-3/Fc (5 μg/ml) followed by ELISA were given the value as 1.0. Relative binding values for each well were calculated accordingly. Values are from two independent experiments performed in triplicate. (E) Colocalization of endogenous NB-3 and CHL1 on soma and neurites of cultured cortical neurons. E17 cortical culture (5–10 DIV) were fixed and stained with rabbit anti-NB-3 and mouse anti-CHL1 antibodies. Scale bars, 30 μm.

Expression of NB-3 in the developing mouse neocortex. (A) Localization of cells expressing NB-3 monitored by LacZ expression in the medial sagittal (a, c, and e) and coronal (d and f) sections of the Nb-3^+/− brains at various developmental stages. (b) Higher magnification of the selected area in (a). Scale bars, 1 mm. (B) Immunostaining of the medial sagittal sections of P7 wild-type mouse neocortex using rabbit anti-NB-3 antibody. Arrowheads point to NB-3-positive cells. (d–f) Higher magnification of the selected areas in (a–c), respectively. (g) Control staining of Nb-3^−/− layer V visual cortex (P7) with the same NB-3 antibody. Motor, motor cortex; SS, somatosensory cortex; Visual, visual cortex. Scale bars, 100 μm. (C) E17 cortical culture (a–f) or P7 mouse brain (g–i) were stained with rabbit anti-NB-3 antibody and monoclonal antibodies against MAP2 or GFAP. NB-3 is expressed in dendrites and soma of neurons in cortical culture (a–c) and brain tissues (g–i; arrowheads points to proximal apical dendrites) but not in astrocytes (d–f). Scale bars, 25 μm. (D) Specificity of the rabbit anti-NB-3 antibody. Whole brain homogenates from wild-type and Nb-3^−/− mice were resolved by SDS–PAGE, followed by immunoblotting with the anti-NB-3 antibody.

More severe loss-of-function phenotype of the compound heterozygous (Nb-3^+/−; Chl1^+/−) mice in apical dendrite orientation in the visual cortex. (A) Reduced expression of NB-3 and CHL1 proteins in the brains of Nb-3^+/−; Chl1^+/− mice compared to the wild-type littermates (P7). (B) Golgi-impregnated layer V visual cortex of wild-type, Nb-3^+/−, Chl1^+/−, and Nb-3^+/−; Chl1^+/− mice (1-month-old littermates). Arrows point to pyramidal neurons with misoriented apical dendrites. Scale bars, 50 μm. (C) Proportion of pyramidal neurons with misoriented apical dendrites (∣θ∣>8°) in the layer V visual cortex. Four groups of littermates (1-month-old) were analyzed (n=4). The number of neurons ranged from 238 to 462 for each genotype. Results are presented as mean±s.e.m. ^***P<0.001; ^**P<0.01; ^*P<0.05; one-way ANOVA with repeated measures followed by Tukey's post-test.

CHL1 and NB-3 regulate PTPα signaling to p59^fyn. (A, B) Transfected HEK293T cells were incubated with nonspecific mouse IgG or clustering antibodies at 37°C for 30 min as indicated. Cells were lysed, and levels of p59^fyn dephosphorylated at Tyr-531 (non-P-Y531) were measured and normalized to total p59^fyn protein. b-fyn, brain isoform of p59^fyn. Results from four independent experiments (n=4) are presented as mean±s.e.m. ^*P<0.05; ^**P<0.01; one-way ANOVA with repeated measures followed by Tukey's post-test. (C–F) Non-P-Y531 p59^fyn is reduced in the Chl1^−/− and the Nb-3^−/− brains. Brains of Chl1^−/− (P2-7) and Nb-3^−/− (P5-7) mice and the wild-type littermates were homogenized and immunoprecipitated using monoclonal anti-p59^fyn antibody (α-Fyn(15)), followed by immunoblotting as indicated. (D, F) Levels of non-P-Y531 p59^fyn were normalized to total p59^fyn levels. Results from four independent experiments (n=4) are presented as mean±s.e.m. for each panel. ^*P<0.05; one-sample t-test.

CHL1 enhances cell surface expression of NB-3. (A) Transfected COS1a cells were fixed and stained with anti-Myc and/or anti-HA antibodies to reveal the localization of NB-3-Myc and CHL1-HA proteins. When expressed alone, NB-3-Myc was mainly located inside the cells. When coexpressed with CHL1-HA, some NB-3-Myc protein moved to the cell periphery and colocalized with CHL1-HA (arrowheads). Scale bars, 50 μm. (B) Increase of cell surface NB-3-Myc when coexpressed with CHL1-HA in COS1a cells. After transfected with NB-3-Myc alone (a) or with both NB-3-Myc and CHL1-HA cDNAs (b–e), live COS1a cells were incubated with anti-Myc antibody followed by fixation and staining for cell surface NB-3-Myc protein alone (a and b), or double staining with anti-HA for CHL1-HA proteins (c–e). Note the cells with high surface NB-3-Myc level also expressed high level of CHL1-HA; CHL1-negative cells (arrowheads) had low levels of surface NB-3-Myc. Scale bars, 50 μm. (C) Analysis of surface expression of NB-3-Myc and CHL1-HA in single- and double-transfected COS1a cells. Cell surface proteins were biotinylated and precipitated using NeutrAvidin Gel. Pull-down samples were blotted with anti-Myc or anti-HA antibodies to assess the level of cell surface NB-3-Myc and CHL1-HA proteins. (D, E) Analysis of surface expression of NB-3 protein in cortical neurons from Chl1^−/− mice (D) and CHL1 protein in neurons from Nb-3^−/− mice (E). Cell surface proteins of E17 cortical cultures (7 DIV) were biotinylated. Pull-down samples using NeutrAvidin Gel were blotted with antibodies as indicated. For quantification in panels C–E, levels of cell surface proteins were normalized to corresponding input proteins. Results from four independent experiments (n=4) are presented as mean±s.e.m. for each panel. ^*P<0.05; one-sample t-test. α-α-Tub, anti-α-tubulin antibody.

Misorientation of apical dendrites of layer V pyramidal neurons in the visual cortex of Nb-3^−/− mice. (A) Morphology of layer V pyramidal neurons in the motor, somatosensory (SS), visual and auditory cortices of Nb-3^+/+ (a–d, i) and Nb-3^−/− (e–h, j) littermates. To visualize the morphology of layer V neurons in neocortex, NB-3 mutant mice were crossed to the Thy1-YFPH transgenic mice, which express YFP in layer V cells in the neocortex. (i) and (j) are higher magnification of the selected areas in (c) and (g), respectively. Arrowheads point to neurons displaying misoriented apical dendrites. Scale bar, 100 μm. (B) Golgi-impregnated visual cortex of wild-type (wt) and Nb-3^−/− mice (1-month-old littermates). Arrows point to neurons with misoriented apical dendrites. Scale bars, 30 μm. (C) Scoring method for apical dendrite orientation (see Supplementary data). (D, E) Average ∣θ∣ of layer III (D) and layer V (E) pyramidal neurons of motor, somatosensory, visual and auditory cortices from wild-type and Nb-3^−/− littermates. (F, G) Proportion of pyramidal neurons with misoriented apical dendrites was significantly increased in layer V but not in the layer III visual cortex of Nb-3^−/− mice. Misoriented apical dendrites were defined as those with ∣θ∣>8°. For the above quantification, three to six pairs of wild-type and Nb-3^−/− littermates (1-month-old) were analyzed. The number for layer V neurons ranged from 80 to 445 for each cortical region of respective genotypes, and ranged from 40 to 145 for layer III neurons. Results are presented as mean±s.e.m. ^***P<0.001; two-way ANOVA with repeated measures followed by Bonferroni post-tests.

CHL1 interacts with PTPα. (A) Association of ectopically expressed CHL1-HA and VSVG-PTPα in HEK293T cells. Transfected HEK293T cells were lysed and immunoprecipitated (IP) with anti-VSVG or anti-HA antibodies and were blotted with anti-HA or anti-PTPα antibodies, respectively. (B) PTPα associates with CHL1 in the mouse brain. Wild-type or Ptpα^−/− mouse (P0) brain membrane fractions were immunoprecipitated with either rabbit anti-PTPα or nonspecific rabbit IgG, and were blotted for the presence of CHL1 protein. (C) Live cortical neurons were treated with antibodies at 37°C for 2 h as indicated, followed by fixation and immunostaining for PTPα and CHL1. Note that CHL1 antibody induced clustering of CHL1 at the cell surface and redistribution of PTPα to the CHL1 clusters (arrowheads in h). (d and h) Higher magnification of selected areas in (c) and (g), respectively. Scale bars, 15 μm.

Aberrant apical dendrite projection of layer V pyramidal neurons in the Ptpα^−/− cortex. (A) Golgi-impregnated somatosensory cortex of wild-type (wt) and Ptpα^−/− mice (1-month-old littermates). Arrows point to pyramidal neurons with misoriented apical dendrites. Scale bars, 50 μm. (B, C) Proportion of layer V pyramidal neurons with misoriented (∣θ∣>8°; B) or inverted (∣θ∣>90°; C) apical dendrites. Four pairs of wt and Ptpα^−/− littermates (1-month-old) were analyzed (n=4). The number of layer V neurons ranged from 204 to 652 for each cortical region in respective genotypes. Results are presented as mean±s.e.m. ^***P<0.001; ^*P<0.05; two-way ANOVA with repeated measures followed by Bonferroni post-tests. (D) A model depicting molecular pathway of CHL1- and NB-3-regulated apical dendrite orientation. CHL1 enhances the surface expression of NB-3. Upon activation, CHL1 and NB-3 recruit PTPα into this complex and result in subsequent activation of p59^fyn to mediate oriented growth of apical dendrites of layer V pyramidal neurons in the caudal cortex.