Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin α5β1

Abstract

Birthdate-dependent neuronal layering is fundamental to neocortical functions. The extracellular protein Reelin is essential for the establishment of the eventual neuronal alignments. Although this Reelin-dependent neuronal layering is mainly established by the final neuronal migration step called "terminal translocation" beneath the marginal zone (MZ), the molecular mechanism underlying the control by Reelin of terminal translocation and layer formation is largely unknown. Here, we show that after Reelin binds to its receptors, it activates integrin α5β1 through the intracellular Dab1-Crk/CrkL-C3G-Rap1 pathway. This intracellular pathway is required for terminal translocation and the activation of Reelin signaling promotes neuronal adhesion to fibronectin through integrin α5β1. Since fibronectin is localized in the MZ, the activated integrin α5β1 then controls terminal translocation, which mediates proper neuronal alignments in the mature cortex. These data indicate that Reelin-dependent activation of neuronal adhesion to the extracellular matrix is crucial for the eventual birth-date-dependent layering of the neocortex.

Figure 1. Involvement of the Dab1-Crk/CrkL-C3G pathway in terminal translocation

Cerebral cortices at P0.5 electroporated with the indicated plasmids plus pCAGGS-EGFP at E14.5 (A–E). Red denotes NeuN-positive neurons. Blue denotes Cux1-positive upper CP (uCP: layer II–IV) neurons. PCZ denotes the NeuN-negative region (arrows). Layer V and VI neurons are equally divided into deep CP 1 (dCP1) and dCP2. (A′–E′) Graphs show the estimation of cell migration. Note that all of Dab1-KD, Crk-KD, CrkL-KD and DN-C3G affected the neuronal entry into the PCZ. n=5–7 brains. ** p<0.01. Scale bars, 100 μm. See also Figure S1.

Figure 2. Rap1 has dual functions for neuronal migration, and the Rap1/N-cadherin pathway is not sufficient for the neuronal entry into the PCZ

Cerebral cortices at P0.5 (A–C) and at E16.5 (D) electroporated at E14.5. Note that Tα1-Spa1 affected the neuronal entry into the PCZ (arrows) whereas CAG-Spa1 affected the neuronal entry into the CP. (A′, C′) Graphs show the estimation of cell migration. (B′) Statistical analyses of terminal translocation failure of (B). GFP-positive cells within the uCP were counted. **, p=0.00194. n=6 (control), n=8 (Tα1-Spa1), and n=7 brains (CAG-Spa1). (D) Immunohistochemisty of neuronal marker Hu. The CAG-Spa1 expressed cell is positive for Hu. (E, F) Cerebral cortices (E16.5) electroporated at E14.5. CAG-Spa1 positive cells were observed from the VZ (arrows) to the IMZ (arrowheads) (E), whereas Tα1-Spa1 positive cells were mainly observed in the IMZ (arrowheads) (F). (G–K) Cerebral cortices (P0.5) electroporated at E14.5. Note that neither CAG-Spa1+N-cadherin nor DN-C3G+N-cadherin could rescue the terminal translocation failure. (L) Statistical analyses of GFP-positive cells within the NeuN-negative PCZ (arrows) shown in (G–K). **, p<0.01. n=6 or 7 brains. Scale bars, 100 μm (A, C), 50 μm (B, E–K), 10 μm (D). See also Figure S2.

Figure 3. Integrin β1 is activated in the leading process of the translocating neurons

(A, B, B′) Immunohistochemistry for fibronectin, Reelin, and activated integrin β1. (A) PFA-fixed P0.5 cortex. Note the positive staining for fibronectin (green) in the Reelin (red)-positive MZ as well as in the deep part of the CP and IMZ. (B) Fresh frozen sections of the P0.5 cortex. Activated integrin β1 was detected in the MZ by 9EG7 antibody (green). (B′) Higher magnification view of (B). (C) PFA-fixed sections of heterozygous mutant of Reelin (P1). Activated integrin β1 was detected in the MZ (green). (D) Cerebral cortices (E18.5) electroporated at E14.5. Note that integrin β1 is activated in the leading process which attached to the MZ. (D′) is a higher magnification of the white square of (D). (E) PFA-fixed sections of reeler mutant which is a littermate of (C) (P1). Note that there is no obvious accumulation of the activated integrin β1. Scale bars, 100 μm (A, C, E), 200 μm (B), 50 μm (B′), 10 μm (D), 2.5 μm (D′). See also Figure S3.

Figure 4. Reelin regulates the inside-out activation of integrin α5β1

(A–A″) in vitro integrin activation assay using primary cortical neurons at E14. The amount of activated integrin β1 was quantified by the amounts of bound 9EG7 antibody. n=6, ** p=0.00209. (B, B′) Cell adhesion assay using the primary cortical neurons at E16. Note that the Reelin-dependent promotion of neuronal adhesion to fibronectin was blocked by co-treatment with a functional blocking antibody for integrin α5 (MFR5). n=4. **, p<0.01. No adhesion to the BSA-coated dishes was observed, irrespective of the presence/absence of Reelin (data not shown). (C) Reelin-dependent cell adhesion was blocked by co-treatment with receptor associated proteins (RAP). ** p<0.01. (n=3) (D) 2A-Reelin mutant could not promote neuronal adhesion to fibronectin. (n=3) (E) Reelin-dependent cell adhesion was not observed in the primary cortical neurons obtained from Dab1-deficient mice (yotari). ** p<0.01 (n=3) (F) Reelin-dependent cell adhesion was blocked by co-treatment of function-blocking antibody to Reelin (CR-50). * p<0.05, ** p<0.01 (n=3) (G) Cell adhesion assay using the migrating neurons at E17.5 electoporated at E14.5. Note that Reelin-dependent promotion of adhesion to fibronectin was significantly impaired in the Reelin-receptor KD neurons, Dab1-KD neurons, or Spa1-overexpressing neurons. n=4. *, p<0.05. (H) Cell adhesion assay using reconstructed HEK-293T cells. Cells were transfected with the indicated plasmids. Note that Reelin promoted cell adhesion to fibronectin only when both ApoER2 and Dab1 were transfected. n=3. **, p<0.01. (I) PFA-fixed cerebral cortices (P0.5) electroporated with pCAGGS-Reelin at E14.5. Neuronal aggregates were observed in the IMZ (white square). Magenta signal shows activated integrin β1 detected using 9EG7 antibody. Right four panels are the higher magnification view of this aggregate. Note that integrin β1 is highly activated in the cell-sparse center of this aggregate. Scale bars, 100 μm (I, left panel), 50 μm (I, right panels). See also Figure S4.

Figure 5. Involvement of integrin α5β1 in terminal translocation

(A) Effects of integrin β1 KD for terminal translocation. Cerebral cortices (P0.5) electroporated at E14.5. Note that integrin β1-KD neurons could not enter the PCZ. (A′) Graphs show the estimation of cell migration of (A). (B) Schemes of plasmids used in the rescue experiments. (C–I) Integrin β1, talin1, and integrin α5 in neurons were required for terminal translocation. Cerebral cortices (P0.5) electroporated at E14.5. Arrows show the PCZ. Note that each resistant vector expressed under the control of a Tα1 promoter rescued the effects of each KD vector. (J) Statistical analyses of terminal translocation failure. **, p<0.01, *, p<0.05. n=6–9 brains. (K) Morphological analyses of neurons (E18.5) electroporated at E14.5. Scale bars, 100 μm (A), 50 μm (C–I), 25 μm (K). See also Figure S5.

Figure 6. Reelin activates integrin α5β1 during terminal translocationin vivo

(A–E) Rescue of the terminal translocation failure caused by ApoER2/VLDLR double KD (DKD). Cells were transfected with the indicated plasmids. Note that co-transfection of CA-integrin α5 and Akt rescued the terminal translocation failure. Arrows show the PCZ. (F) Statistical analyses of (A–E). * p<0.05, ** p<0.01. n=8–12 brains. Scale bar, 50 μm (A).

Figure 7. Integrin α5β1 is required for the eventual neuronal positioning in the mature cortex

(A–C) Sequential in utero electroporation examined at P7 electroporated with the indicated plasmids. Note that KD of integrin α5 or integrin β1 in later-born neurons affected the inside-out arrangement of neurons in the mature cortex. (A′–C′) Bin analyses of (A–C). Layer II–IV was divided into 10 bins. (D) Overlapping index of sequential electroporation. ** p<0.01, * p<0.05. n=9 (control-control case), n=7 (control-integrin β1 KD case), n=8 (control-integrin α5 KD case). Scale bar, 50 μm (A).

Figure 8. Reelin dependent switch of adhesion molecules controls terminal translocation and inside-out lamination

(A) Scheme of the migratory mode changes. Multipolar migrating neurons change their migratory behavior to locomotion through Rap1, and adhere to the radial glial fibers in a N-cadherin-dependent manner. When the locomoting neurons reach beneath the PCZ, at which site a dense accumulation of immature neurons is observed, Reelin triggers the C3G-dependent Rap1 pathway for integrin activation. Then, the leading processes of the locomoting neurons adhere to the ECM, such as fibronectin in the MZ through integrin α5β1 and change their migratory mode to terminal translocation. The interaction between migrating neurons and ECM is fundamental for the eventual neuronal layering in the mature cortex. (B) Scheme of Reelin-dependent integrin α5β1 inside-out activation. 1. Reelin (input) phosphorylates Dab1 through ApoER2/VLDLR, recruits Crk/CrkL, and phosphorylates C3G. 2. C3G activates Rap1. 3. Activated Rap1 recruits some effectors, and 4. the conformation of the integrin subunits change. 5. Finally, activated integrin α5β1 adheres to fibronectin in the MZ to mediate terminal translocation and layer formation (output).