Reelin-induced tyrosine [corrected] phosphorylation of disabled 1 during neuronal positioning

Abstract

The reelin (reln) and disabled 1 (dab1) genes both ensure correct neuronal positioning during brain development. We have found that the intracellular Dab1 protein receives a tyrosine phosphorylation signal from extracellular Reln protein. Genetic analysis shows that reln function depends on dab1, and vice versa, as expected if both genes are in the same pathway. Dab1 is expressed at a higher level, yet phosphorylated at a lower level, in reln mutant embryo brains. In primary neuronal cultures, Dab1 tyrosine phosphorylation is stimulated by exogenous Reln. These results suggest that Reln regulates neuronal positioning by stimulating Dab1 tyrosine phosphorylation.

Figure 1

Histology of wild-type, single, and compound mutant brains. (A,B) Histology (hematoxylin and eosin stain) at postnatal day 23 (P23). The wild-type (wt) mouse was inbred strain 129/Sv. The single and compound homozygous mutant mice were siblings. (A) Coronal sections of cerebral cortex. The marginal zone (MZ) is cell poor in wild-type mice and densely packed in the mutants. Scale bar is 50 μm. (B) Sagittal sections of cerebella. (M) Molecular layer; (G) granule cell layer; (arrowheads), Purkinje cells. Scale bar is 50 μm. Note that the wild-type cerebellum is foliated, and the lower half of the section shown is a mirror image of the upper half. The mutant cerebella are not foliated. (C) Nuclei in the marginal zone (0.09 mm²) were counted and expressed as a ratio to the number of nuclei in layer 6 (0.13 mm²). Counts were within 15% on duplicate section. (D) PCR genotypes of dab1 reln, dab1, and reln samples.

Figure 2

Comparison of Dab1 protein and phosphotyrosine levels between wild-type and reln mutant brains. Brains were collected either at E16 or at P21 from wild-type or mutant animals, weighed, and homogenized in RIPA buffer. Lysates were normalized for total protein concentration, then immunoprecipitated with either anti-Dab1 (+), or preimmune (−) antibodies. Immunoprecipitates were analyzed by SDS-PAGE, followed by Western blotting with either anti-phosphotyrosine (top) or anti-Dab1 antibody (middle). Relative levels of Dab1 protein were estimated by dilution of reln mutant samples (right). High Dab1 tyrosine phosphorylation was observed only in the embryonic samples. Equivalent protein concentration in cell lysates was confirmed by Coomassie stain (not shown) and blotting with anti-enolase antibody (bottom). Enolase is more highly expressed in P21 brain. (wt) Wild-type; (rl) reln; (arrow) position of Dab1.

Figure 3

Tyrosine phosphorylation of Dab1 p80 is induced by the addition of Reln to dissociated embryonic brain cells. (A) Detection of Reln protein (triangle) by Western blot in conditioned media used to treat brain cells. (C) pCDNA3 vector; (R) Reln-expressing pCrl plasmid. (B) Cells isolated from E16 reln forebrains were treated for 0 or 10 min with fresh medium (−) or control (C) or Reln (R) conditioned media. Total cell lysates were analyzed using antiphosphotyrosine antibody. A Reln-induced 80-kD tyrosine phosphorylated protein is indicated by the arrow. (C) Cell lysates were immunoprecipitated with either anti-Dab1 (+) or preimmune (−) antibodies, and analyzed by Western blotting with anti-phosphotyrosine (top) or anti-Dab1 (bottom) antibodies. The Dab1 p80 doublet (arrow) may represent differential serine-threonine phosphorylation.

Figure 4

Cultured neurons respond to Reln: effect of genotype, time of exposure, and inhibitors. (A) E16 wild-type (wt) and reln (rl) mutant forebrain cells were dissociated and cultured for 3 days before treatment with fresh (−), control (C) or Reln (R) conditioned media for 30 min. Lysates were incubated with anti-Dab1 (+) or preimmune (−) antibodies, and immunoprecipitated proteins were Western blotted with anti-phosphotyrosine (top) and anti-Dab1 (bottom) antibodies. Anti-phosphotyrosine signal was quantitated by dilution of Reln-stimulated samples compared to undiluted control sample (right). (B) Reln mutant cells were treated for 0, 10, 30, or 240 min with fresh, control, or Reln media. (C) Reln mutant cells were treated for 30 min with control or Reln media in the presence or absence of EDTA (5 mm) or Na₃VO₄ (0.2 or 2 mm). (D) Cultured cells were fixed and stained with neuronal specific anti β-tubulin antibody Tuj1 (green, left), anti-Dab1 (red, right) and Dapi (blue) to determine the percentage of cells that were neurons and express Dab1.

Figure 4

Cultured neurons respond to Reln: effect of genotype, time of exposure, and inhibitors. (A) E16 wild-type (wt) and reln (rl) mutant forebrain cells were dissociated and cultured for 3 days before treatment with fresh (−), control (C) or Reln (R) conditioned media for 30 min. Lysates were incubated with anti-Dab1 (+) or preimmune (−) antibodies, and immunoprecipitated proteins were Western blotted with anti-phosphotyrosine (top) and anti-Dab1 (bottom) antibodies. Anti-phosphotyrosine signal was quantitated by dilution of Reln-stimulated samples compared to undiluted control sample (right). (B) Reln mutant cells were treated for 0, 10, 30, or 240 min with fresh, control, or Reln media. (C) Reln mutant cells were treated for 30 min with control or Reln media in the presence or absence of EDTA (5 mm) or Na₃VO₄ (0.2 or 2 mm). (D) Cultured cells were fixed and stained with neuronal specific anti β-tubulin antibody Tuj1 (green, left), anti-Dab1 (red, right) and Dapi (blue) to determine the percentage of cells that were neurons and express Dab1.

Figure 4

Cultured neurons respond to Reln: effect of genotype, time of exposure, and inhibitors. (A) E16 wild-type (wt) and reln (rl) mutant forebrain cells were dissociated and cultured for 3 days before treatment with fresh (−), control (C) or Reln (R) conditioned media for 30 min. Lysates were incubated with anti-Dab1 (+) or preimmune (−) antibodies, and immunoprecipitated proteins were Western blotted with anti-phosphotyrosine (top) and anti-Dab1 (bottom) antibodies. Anti-phosphotyrosine signal was quantitated by dilution of Reln-stimulated samples compared to undiluted control sample (right). (B) Reln mutant cells were treated for 0, 10, 30, or 240 min with fresh, control, or Reln media. (C) Reln mutant cells were treated for 30 min with control or Reln media in the presence or absence of EDTA (5 mm) or Na₃VO₄ (0.2 or 2 mm). (D) Cultured cells were fixed and stained with neuronal specific anti β-tubulin antibody Tuj1 (green, left), anti-Dab1 (red, right) and Dapi (blue) to determine the percentage of cells that were neurons and express Dab1.

Figure 4

Cultured neurons respond to Reln: effect of genotype, time of exposure, and inhibitors. (A) E16 wild-type (wt) and reln (rl) mutant forebrain cells were dissociated and cultured for 3 days before treatment with fresh (−), control (C) or Reln (R) conditioned media for 30 min. Lysates were incubated with anti-Dab1 (+) or preimmune (−) antibodies, and immunoprecipitated proteins were Western blotted with anti-phosphotyrosine (top) and anti-Dab1 (bottom) antibodies. Anti-phosphotyrosine signal was quantitated by dilution of Reln-stimulated samples compared to undiluted control sample (right). (B) Reln mutant cells were treated for 0, 10, 30, or 240 min with fresh, control, or Reln media. (C) Reln mutant cells were treated for 30 min with control or Reln media in the presence or absence of EDTA (5 mm) or Na₃VO₄ (0.2 or 2 mm). (D) Cultured cells were fixed and stained with neuronal specific anti β-tubulin antibody Tuj1 (green, left), anti-Dab1 (red, right) and Dapi (blue) to determine the percentage of cells that were neurons and express Dab1.