Collapsin response mediator protein 1 mediates reelin signaling in cortical neuronal migration

Abstract

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1-/- mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A-/- and crmp1+/-;sema3A+/- cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

Figure 1.

Abnormally positioned neurons in the crmp1^−/− cerebral cortex. A, Distribution of cells in the P10–BrdU–E14.5 crmp1^+/− or crmp1^−/− coronal cortical sections. The rate of the BrdU-labeled nuclei of each bin (see Materials and Methods) is shown in b. B, Distribution of cells in the P10–BrdU–E16.5 crmp1^+/− and crmp1^−/− coronal cortical sections. The rate of the BrdU-labeled nuclei of each bin is shown in b. In all cases, three littermates of each genotype were used for quantification. C, E16.5 crmp1^+/− or crmp1^−/− coronal cortical sections immunostained with anti-MAP-2 (2a + 2b) (a) and anti-Nestin (b) antibodies. D, Expression levels of MAP-2 (2a + 2b) and Nestin at E16.5 crmp1^+/− or crmp1^−/− mouse cortex lysates from two individual embryos of each genotype. Equal amounts of protein were analyzed, as indicated by the loading control (β-actin). Cortical layers are shown on the left. Scale bars: A, B, 100 μm; C, 10 μm. MZ, Marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. *p < 0.05, **p < 0.01 compared with the corresponding value in crmp1^+/−.

Figure 2.

Observation of BrdU birth-dating analysis in sema3A^−/− and sema3A^+/−;crmp1^+/− cortices. A, Distribution of cells in the E18.5–BrdU–E14.5 wt, sema3A^+/−, and sema3A^−/− coronal cortical sections. MZ, Marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. B, Distribution of cells in the P10–BrdU–E14.5 crmp1^+/− and crmp1^+/−;sema3A^+/− coronal cortical sections. C, Distribution of cells in the P10–BrdU–E16.5 crmp1^+/− and crmp1^+/−;sema3A^+/− coronal cortical sections. Cortical layers are shown on the left. Scale bar, 100 μm.

Figure 3.

Expression and tyrosine phosphorylation of CRMP1 in Reln^rl/rl cortex and the genetic interaction between crmp1 and dab1 in the hippocampus. A, Tyrosine phosphorylation of CRMP1 by Fyn. CRMP1-Myc was introduced with or without the wild-type (WT), constitutive-active (CA), or dominant-negative (DN) form of Fyn in HEK293T cells. Immunoprecipitation with anti-Myc antibody was performed and was thereafter immunoblotted with anti-p-Tyr and anti-Myc antibodies. B, Tyrosine phosphorylation of CRMP1 in Reln^rl/+ and Reln^rl/rl lysates from mouse cortex at E16.5. Immunoprecipitation with anti-CRMP1 antibody was performed and was thereafter immunoblotted with anti-p-Tyr and anti-CRMP1 antibodies. C, Expression levels of CRMP1 at E16.5 Reln^rl/+ and Reln^rl/rl brain lysates. Immunoblot analysis of anti-CRMP1 and anti-Dab1 antibodies of three individual embryos of each genotype was performed. Equal amounts of protein were analyzed, as indicated by the loading control (β-actin). D, Immunohistochemistry with anti-CRMP1 (magenta) and anti-Dab1 (green) antibodies in E16.5 Reln^rl/+ (a–c) and Reln^rl/rl (d–f) coronal cortical sections. Cortical layers are shown on the left. MZ, Marginal zone; CP, cortical plate; SPP, superplate. E, Nissl staining of the brains at P10 of crmp1^−/− (a, d), crmp1^−/−;dab1^yot/+ (b, e), and crmp1^−/−;dab1^yot/yot (c, f) mice. Coronal sections of the hippocampus region are presented. Magnified images of the CA1 region in a, b, and c are shown in d, e, and f, respectively. Scale bars: D, 10 μm; E, 100 μm.