Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

  1. Ayumu Mubuchi
  2. Mina Takechi
  3. Shunsuke Nishio
  4. Tsukasa Matsuda
  5. Yoshifumi Itoh
  6. Chihiro Sato
  7. Ken Kitajima
  8. Hiroshi Kitagawa
  9. Shinji Miyata  Is a corresponding author
  1. Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Japan
  2. Graduate School of Bioagricultural Sciences, Nagoya University, Japan
  3. Faculty of Food and Agricultural Sciences, Fukushima University, Japan
  4. Kennedy Institute of Rheumatology, University of Oxford, United Kingdom
  5. Bioscience and Biotechnology Center, Nagoya University, Japan
  6. Institute for Glyco-core Research, Nagoya University, Japan
  7. Laboratory of Biochemistry, Kobe Pharmaceutical University, Japan
11 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
NCAN is a major CSPG produced by developing cortical neurons.

(a) Detection of the neoepitope of CSPGs after digestion with chondroitinase ABC (Chase). (b) Immunoblotting of the neoepitope from undigested (Chase -) and digested (Chase +) cerebral cortex lysates prepared at E18.5. The arrowhead indicates the major CSPG at 130 kDa. (c) List of peptide fragments identified from the 130 kDa band. Positions indicate the amino acid number in full-length NCAN. (d) Domain structure of mouse NCAN. Ig: immunoglobulin-like domain, Link: hyaluronan-binding link module, EGF: epidermal growth factor-like repeat, CLD: C-type lectin domain, CS: chondroitin sulfate chain. (e, f) NCAN expression in the developing cerebral cortex from E13.5 to postnatal day (P) 42. The arrowheads indicate the full-length and N-terminal fragment of NCAN. Values in (f) are normalized to GAPDH and represented relative to P2. N=3 for each point. Mean ± SD. (g) Quantitative RT-PCR analysis of Ncan mRNA in the developing cerebral cortex. Values are normalized to Gapdh and represented relative to E13.5. N=3 for each point. Mean ± SD. (h) Immunoblot analysis of NCAN in the cultured medium of primary cultured cortical neurons 1, 3, and 5 days after plating. (i) Experimental model for in utero labeling. Radial glial cells in the VZ were labeled with GFP on E14.5. GFP-positive cells were isolated 0.6–4 days later. (j) Expression of Ncan mRNA in GFP-labeled cells isolated on the indicated days after in utero electroporation. Values are normalized to Gapdh and represented relative to 0.6 days. N=3 for each point. Mean ± SD.

Figure 1—figure supplement 1
Location of GFP-positive cells 0.6, 2, and 4days after in utero electroporation.

Most GFP-positive cells are in the VZ on day 0.6. GFP-positive cells begin to migrate radially through the SP/IZ on day 2 and reach the CP on day 4. Scale bar represents 100 μm.

Figure 2 with 1 supplement
Neuron-derived NCAN forms a pericellular matrix with HA.

(a) In situ hybridization analysis of Ncan mRNA on the E16.5 cerebral cortex. (b) Localization of NCAN protein (green) in the E16.5 cerebral cortex. Nuclei were counterstained with DAPI (blue). (c) Distribution of HA (green) visualized by the biotinylated HA-binding protein (b-HABP) in the E16.5 cerebral cortex. (d) High-magnification views of a Tuj-1-positive primary cultured cortical neuron (green) 5 days after plating. White arrows indicate the co-localization of NCAN (magenta) and HA (cyan). Orthogonal projections in the X-Z and Y-Z planes taken along the white lines showed the localization of NCAN and HA at the adhesion sites between the neuron and culture substrate. (e) Schema of the pull-down assay for analyzing binding between endogenous HA and its interactors. (f) Immunoblotting of the input, precipitate (P), and supernatant (S) with an anti-NCAN antibody. NCAN was precipitated with HA by adding b-HABP (b-HABP +). NCAN was not precipitated without b-HABP (b-HABP -) or after digestion with hyaluronidase (HAase +). (g) Co-precipitation of NCAN with exogenously added biotinylated HA (b-HA +) or endogenous HA (b-HABP +). Scale bars represent 200 μm (a–c) and 5 μm (d).

Figure 2—figure supplement 1
Pull-down assay of HA with recombinant GFP-fused NCAN.

(a) Recombinant expression of GFP-fused full-length, N-terminal, and C-terminal half of NCAN. The culture medium of transfected HEK293 cells was digested with chondroitinase ABC (Chase) and analyzed by immunoblotting with an anti-GFP antibody. (b) Quantification of precipitated HA by the full-length, N-terminal, and C-terminal half of NCAN. A negative control experiment (NC) was conducted without NCAN. N=3 for each point. Mean ± SD.

Screening of the interacting partners of NCAN.

(a) Schematic of the pull-down assay to identify NCAN-interacting partners. (b) Co-precipitation of TNC with the full-length and C-terminal half of NCAN. Interactions between NCAN and TNC disappeared following the addition of EDTA but were not affected by Chase digestion. The N-terminal half of NCAN did not bind to TNC. (c, e) TNC expression in the developing cerebral cortex from E13.5 to postnatal day (P) 42. Values in (e) are normalized to GAPDH and represented relative to E18.5. The same brain lysate as in Figure 1e were analyzed. The values for GAPDH were calculated based on the data presented in Figure 1e. N=3 for each point. Mean ± SD. (d) Quantitative RT-PCR analysis of Tnc mRNA in the developing cerebral cortex. Values are normalized to Gapdh and represented relative to E13.5. N=3 for each point. Mean ± SD. (f, g) In situ hybridization analysis of Tnc mRNA (f) and immunohistochemical localization of TNC protein (g) in the E16.5 cerebral cortex. Scale bar represents 200 μm.

Figure 4 with 1 supplement
Alphafold2 prediction of the NCAN-TNC complex.

(a) Domain structure of mouse (m) TNC. EGF: epidermal growth factor-like repeat, FNIII: fibronectin type-III domain. (b) Five predicted complex models of the mNCAN and mTNC were generated using AlphaFold2 multimer implemented in ColabFold, and the best-predicted complex was shown. (c, d). Sequence alignments of CLD of mNCAN and rat (r) ACAN (c) and FNIII-3–5 of mTNC and rTNR (d). Circles under alignments indicate the key residues of the rACAN-rTNR complex. Triangles over alignments indicate the residues in the interface of mNCAN-mTNC. Green and magenta circles/triangles are the residues involved in the interaction between L4 loop of CLD and βC, F, G strands of FNIII-4 and β6, 7 strands of CLD and CC’ loop of FNIII-4, respectively. Black circles/triangles indicate residues involved in the interaction between CLD and FNIII-4–5 linker region/FG loop of FNIII-5. (e) Model for forming the ternary complex of NCAN, HA, and TNC.

Figure 4—figure supplement 1
Alphafold2 prediction of NCAN-TNC complex.

(a) Model confidence of NCAN-TNC complex. Each residue is colored by pLDDT score, and a high pLDDT score indicates high accuracy. (b) Crystal structure of rat (r) ACAN-TNR complex (PDB ID: 1TDQ). Each domain is colored the same as in Figure 4c. (c) Sequence alignments of fourteen FNIII of mTNC. Green letters indicate residues involved in the interaction between FNIII-4 and CLD of NCAN.

HA, NCAN, and TNC form the ternary complex in the developing cerebral cortex.

(a) Triple staining of the E17.5 mouse cerebral cortex with HA (cyan), NCAN (magenta), and TNC (green). (b) High-magnification images show the co-localization of the three components in the upper part of the SP/IZ but not in the CP or VZ. (c, d) Localization of TNC (c, magenta), NCAN (c, d, cyan), and HA (d, magenta) around GFP-labeled bipolar neurons (green) in the upper SP/IZ at E17.5, 3 days after in utero labeling. Orthogonal views taken along the white dashed lines showed the contact between the ternary complex and the surface of bipolar neurons, as indicated by the arrows. Scale bars represent 100 μm (a), 5 μm (b), and 2 μm (c, d).

Figure 6 with 4 supplements
Defects in NCAN and TNC retards neuronal migration.

(a) Low-magnification images of FT-labeled cells (black or green) in WT and DKO mouse coronal sections at E16.5. Nuclei were counterstained with DAPI (blue). (b, c) Radial distribution of FT-labeled cells (green) in the lateral (b) and medial (c) cortices of WT and DKO mice at E16.5. A quantitative analysis of migration profiles across the cortex is shown on the right. The cerebral cortex is divided into five equal areas (bins 1–5) from the pia to the ventricle, and the proportion of FT-labeled cells in each bin was calculated. The nuclei staining images (blue) on the left illustrate the boundaries of the cortical layers. N=19–20 mice per group. Mean ± SD; *p<0.05; Student’s t-test. Scale bars represent 200 μm (a) and 50 μm (b, c).

Figure 6—figure supplement 1
Characterization of DKO mice.

(a) Immunoblot analysis of TNC and NCAN from WT and DKO mouse cerebral cortex lysates. (b) Immunostaining of NCAN and TNC on coronal sections of WT and DKO mouse brains at E17.5. Scale bars represent 200 µm. (c) No significant difference in cerebrum weights between WT and DKO mice at E16.5. N=5 mice per group. Mean ± SD; ns > 0.05; Student’s t-test.

Figure 6—figure supplement 2
Restored neuronal migration in DKO mice after 3 days of labeling.

(a, b) Radial distribution of FT-labeled cells (green) in the lateral (a) and medial (b) cortices of WT and DKO mice at E17.5. A quantitative analysis of migration profiles across the cortex is shown on the right. The nuclei staining images (blue) on the left illustrate the boundaries of the cortical layers. N=4 mice per group. Mean ± SD; ns > 0.05; Student’s t-test. Scale bars represent 50 µm.

Figure 6—figure supplement 3
The laminar organization of the postnatal cortex in WT and DKO mice.

(a) Immunostaining of NeuN-positive (green) and Ctip2-positive (magenta) neurons in the cerebral cortices of WT and DKO at 2 weeks of age. Nuclei were counterstained with DAPI (blue). (b, c) Distribution of NeuN-positive (b) or Ctip2-positive (c) neurons in the cerebral cortices of WT and DKO mice. N=3 mice per group. Mean ± SD; ns >0.05; Student’s t-test. Scale bars represent 200 µm (a).

Figure 6—figure supplement 4
Histochemical analysis of radial glial cells, intermediate progenitor cells, and the morphology of radial fibers in DKO mice.

(a) Immunostaining of Pax6-positive radial glial cells (green) and Tbr2-positive intermediate progenitor cells (red) in the VZ of WT and DKO cerebral cortices at E16.5. Bar graphs show the numbers of Pax6- and Tbr2-positive cells per 150×150 µm2. N=5 mice per group. Mean ± SD; ns > 0.05; Student’s t-test. (b) Immunostaining of nestin-positive radial fibers in WT and DKO cerebral cortices at E16.5. (c) High-magnification images of mCherry-positive bipolar neurons and radial fibers in the WT and DKO at E16.5, 2 days after in utero labeling. Bipolar neurons in the SP/IZ-attached radial fiber, irrespective of genotypes. Scale bars represent 20 µm (a), 50 µm (b), and 5 µm (c).

Figure 7 with 1 supplement
Single deletion of NCAN or TNC results in mild abnormalities in neuronal migration.

(a) Radial distribution of FT-labeled cells (green) in the lateral cortices of WT, NCAN KO, and TNC KO mice at E16.5. A quantitative analysis of migration profiles across the cortex is shown on the right. The nuclei staining images (blue) on the left illustrate the boundaries of the cortical layers. N=20–21 mice per group. Mean ± SD; *p<0.05 vs. WT; Dunnett’s test. (b, c) Localization of TNC in WT and NCAN KO mice at E16.5 (b). Localization of NCAN in WT and TNC KO mice at E16.5 (c). The normalized fluorescence intensity profiles of TNC and NCAN are shown on the right of the images. The maximum intensity value for WT mice was set to 1. N=8–13 mice for each group. Mean ± SD (shaded area). (d) In utero electroporation of Turbo-RFP alone or with GFP-fused full-length NCAN (GFP-NCAN) into the NCAN KO brain at E16.5. (e) Immunostaining of TNC (magenta) around Turbo-RFP-positive neurons (cyan) 2 days after electroporation. (f) Staining intensity of TNC around the control and GFP-NCAN-expressing neuron. N=20 images per group. Mean ± SD; *p<0.05; Student’s t-test. (g) High magnification of the boxed region in (e). The arrows indicate the juxtaposed localization of TNC (magenta) and GFP-NCAN (green). Scale bars represent 50 µm (a, b, c), 5 µm (e), and 2 µm (g).

Figure 7—figure supplement 1
Localization of HA in WT, DKO, NCAN KO, and TNC KO mice.

(a) Distribution patterns of HA in the E16.5 cerebral cortices of WT and DKO mice. The fluorescence intensity profile is shown on the right. The maximum intensity value for WT mice was set to 1. N=11 mice for each group. Mean ± SD (shaded area). (b) Comparison of HA staining in the E16.5 cerebral cortices of WT, NCAN KO, and TNC KO mice. The normalized fluorescence intensity profiles of HA are shown on the right of the image. The maximum intensity value for WT mice was set to 1. N=7–9 mice for each group. Mean ± SD (shaded area). Scale bars represent 50 µm.

Transient disruption of the ternary complex by hyaluronidase injection.

(a) Localization of HA, NCAN, and TNC in the cerebral cortex 2 days after intraventricular injection of PBS or hyaluronidase (HAase) at E14.5. The normalized fluorescence intensity profiles of HA, NCAN, and TNC are shown on the right of each image. The maximum intensity value for PBS-injected mice was set to 1. N=5 mice for each group. Mean ± SD (shaded area). (b) Immunoblot analysis of NCAN and TNC in cerebral cortex lysates 2 days after intraventricular injection of PBS or HAase. The broadening of the NCAN band is due to the absence of chondroitinase ABC digestion. (c) The NCAN (left) and TNC (right) amounts were represented relative to the PBS-injected group. N=4 mice per group. Mean ± SD; *p<0.01; Student’s t-test. (d, e) Distribution of FT-labeled cells (green) in the lateral (d) and medial (e) cortices 2 days after injection of PBS or HAase at E14.5. N=9–10 mice per group. Mean ± SD; *p<0.05; Student’s t-test. Scale bars represent 50 µm.

Delayed multipolar-to-bipolar transition in DKO mice.

(a) Schematic of the morphological analysis. (b, c) Migration of mCherry-labeled cells in the WT and DKO cerebral cortices 53 hr after in utero labeling (b). High-magnification images of mCherry-labeled cells in the IZ (c). The images below show the morphology of neurons in the upper IZ (U–IZ) and lower IZ (L–IZ). (d, e) The length-to-width ratio (e) and the major neurite angle (f) of mCherry-positive neurons in WT and DKO mice. N=128–134 cells from 6 mice per group. *p<0.05; Student’s t-test. (f) The proportion of bipolar neurons among mCherry-labeled neurons in the WT and DKO cortices. N=6 mice per group. Mean ± SD; *p<0.05; Student’s t-test. Scale bars represent 50 μm (b).

Figure 10 with 3 supplements
Morphological maturation of cortical neurons by TNC and NCAN.

(a) Experimental model for in utero cell labeling and the primary neuronal culture. (b) Morphological stages of primary cultured cortical neurons. (c) Representative images of GFP-labeled neurons cultured for 2 days on cover glasses coated with the control substrate, poly-L-ornithine (POL), and 10 μg/mL of TNC, HA, and NCAN. (d-f) The length of the longest neurite (d), total length of neurites (e), and number of neurites (f) of neurons cultured on the indicated substrate for 2 days. N=55–80 cells per condition. *p<0.05 vs Control (Con); Dunnett’s test. (g) The percentage of neurons with each morphological stage after culturing on the indicated substrate for 2 days. N=4 wells per condition. Mean ± SD. *p<0.05 vs Con for stage 1; Dunnett’s test. (h) Representative images of GFP-labeled neurons derived from WT and NCAN KO mice cultured for 2 days on cover glasses coated with POL. (i–k) The length of the longest neurite (i), total length of neurites (j), and number of neurites (k) of WT and NCAN KO neurons cultured for 2 days. N=96–101 cells per condition. *p<0.05; Student’s t-test. Scale bars represent 20 μm (c, h).

Figure 10—figure supplement 1
Morphological analysis of cortical neurons cultured for 3 days on cover glasses coated with HA, NCAN, and TNC.

(a) Representative images of GFP-labeled neurons after culturing on the indicated substrate for 3 days. (b–d) The length of the longest neurite (b), total length of neurites (c), and number of neurites (d) of neurons cultured on the indicated substrate for 3 days. N=55–78 cells per condition. *p<0.05 vs Control (Con); Dunnett’s test. Scale bars represent 20 μm.

Figure 10—figure supplement 2
Neurite outgrowth analysis with anti-Tuj-1 antibody staining.

(a) Representative images of neurons stained with anti-Tuj-1 antibody after 3 days of culture on the indicated substrate. (b–d) The length of the longest neurite (b), total length of neurites (c), and number of neurites (d) of neurons. Neurites were analyzed using AutoNeuriteJ. N=30–76 cells per condition. *p<0.05 vs Control (Con); Dunnett’s test. Scale bars represent 200 μm.

Figure 10—figure supplement 3
Neurite outgrowth analysis of WT and NCAN KO neurons with anti-Tuj-1 antibody staining.

(a) Representative images of WT and NCAN KO neurons stained with anti-Tuj-1 antibody after 3 days of culture. (b–d) The length of the longest neurite (b), total length of neurites (c), and number of neurites (d) of neurons. Neurites were analyzed using AutoNeuriteJ. N=70–80 cells per condition. *p<0.05; Student’s t-test. Scale bars represent 200 μm.

Author response image 1
In situ hybridization analysis of Has2 and 3 mRNA on the E16.

5 cerebral cortex. Upper images show results of in situ hybridization using antisense against Has2 and 3. Lower images are in situ hybridization using sense probes as negative controls.

Tables

Table 1
List of proteins identified from GFP-NCAN and negative control resins.
GFP-NCAN resinNegative control resin
Protein nameNo. of peptideProtein nameNo. of peptide
Neurocan core protein34Tubulin beta-2B chain21
Tenascin22Tubulin beta-5 chain17
Tubulin alpha-1A chain18Tubulin beta-3 chain15
Tubulin beta-2B chain17Tubulin beta-6 chain15
Tubulin beta-5 chain16Tubulin alpha-1A chain13
Actin, cytoplasmic 211Actin, cytoplasmic 210
Actin, cytoplasmic 19Elongation factor 1-alpha 15
Tubulin beta-6 chain8Macrophage migration inhibitory factor5
Elongation factor 1-alpha 16Crk-like protein4
Crk-like protein4Keratin, type II cytoskeletal 14
Profilin-24Peroxiredoxin-13
Eukaryotic translation initiation factor 3 subunit L4L-lactate dehydrogenase A chain3
Histone deacetylase 64Keratin, type I cytoskeletal 103
40 S ribosomal protein SA3Hemoglobin subunit alpha3
Macrophage migration inhibitory factor3Hemoglobin subunit beta-13
Eukaryotic translation initiation factor 3 subunit E3Keratin, type II cytoskeletal 1b3
Keratin, type II cytoskeletal 1b3
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)ICR miceJapan SLCRRID:MGI:5462094
Strain, strain background (M. musculus)C57BL/6 N miceJapan SLCRRID:MGI:5295404
Strain, strain background (M. musculus)B6.Cg-Tnc<tm1Sia>/RbrcRIKEN Bioresource CenterRBRC00169
Strain, strain background (M. musculus)DKO mice for TNC and NCANThis paperN/AMice deficient for TNC and NCAN
Cell line (Homo sapiens)HEK293Riken Cell BankRCB1637
RRID:CVCL_0045
Verified by the Riken Cell Bank and tested negative for mycoplasma
Cell line (H. sapiens)HEK293TRiken Cell BankRCB2202
RRID:CVCL_0063
Verified by the Riken Cell Bank and tested negative for mycoplasma
AntibodyAnti-tenascin C Rat IgG2A (Clone # 578)R&DMAB2138,
RRID:AB_2203818
IF(1:400), WB (1:3000)
AntibodyAnti-β-Tubulin (Tuj-1) Mouse IgG1(clone TUB 2.1)SigmaT4026
RRID:AB_477577
IF(1:1000)
AntibodyAnti-Neurocan Sheep IgGR&DAF5800
RRID:AB_2149717
IF(1:400), WB (1:3000)
AntibodyAnti-GFP Alexa Fluor 488 conjugate Rabbit IgGInvitrogenA21311
RRID:AB_221477
IF(1:1000)
AntibodyAnti-Nestin Mouse IgGMilliporeMAB5326
RRID:AB_94911
IF(1:400)
AntibodyAnti-Pax6 Rabbit IgGFujifilm015–27293IF(1:400)
AntibodyAnti-EOMES (Tbr2) Rat IgG2AInvitrogen14-4875-82
RRID:AB_11042577
IF(1:400)
AntibodyAnti-NeuN Rabbit IgGProteintech26975–1-AP
RRID:AB_2880708
IF(1:1000)
AntibodyAnti-Ctip2 Rat IgG2AabcamAb18465
RRID:AB_2064130
IF(1:400)
AntibodyAnti-GAPDH Mouse IgG1(Clone # 5A12)Wako016–25523
RRID:AB_2814991
WB (1:10000)
AntibodyAnti-Chondroitine-4-Sulfate Mouse IgG1 (Clone # BE-123)MilliporeMAB2030
RRID:AB_11213679
WB (1:50000)
AntibodyAlexa Fluor 488 donkey anti-Mouse (H+L)Wako715-545-151
RRID:AB_2341099
IF(1:400)
AntibodyAlexa Fluor 594 goat anti-Rat IgG (H+L)Thermo FisherA-11007
RRID:AB_10561522
IF(1:400)
AntibodyAlexa Fluor 647 goat anti-Rat IgG (H+L)Thermo FisherA-21247
RRID:AB_141778
IF(1:400)
AntibodyAlexa Fluor 647 donkey anti-Sheep IgG (H+L)Thermo FisherA-21448
RRID:AB_2535865
IF(1:400)
AntibodyMouse IgG HRP-conjugated AntibodyMBLPM009-7WB (1:2500)
AntibodySheep IgG HRP-conjugated AntibodyR&DHAF016
RRID:AB_562591
WB (1:2500)
AntibodyRat IgG HRP-conjugated AntibodyCell Signaling Technology7077 S
RRID:AB_10694715
WB (1:2500)
AntibodyRabbit IgG HRP-conjugated AntibodyCell Signaling Technology7074 S
RRID:AB_2099233
WB (1:2500)
Recombinant DNA reagentpCAG-GFP
(plasmid)
AddgenePlasmid #11150
Recombinant DNA reagentpCAG-mGFP
(plasmid)
AddgenePlasmid #14757
Recombinant DNA reagentpCAG-mCherry
(plasmid)
This paperN/AVector backbone: pCAG
Gene/Insert name: mCherry
Recombinant DNA reagentpCAGGS-TurboRFP
(plasmid)
This paperN/AVector backbone: pCAGGS
Gene/Insert name: TurboRFP
Recombinant DNA reagentpCAG-Full length NCAN-GFP
(plasmid)
This paperN/AVector backbone: pCAG
Gene/Insert name: GFP-fused full length NCAN
Recombinant DNA reagentpCAG-N half NCAN-GFP
(plasmid)
This paperN/AVector backbone: pCAG
Gene/Insert name: GFP-fused N half of NCAN
Recombinant DNA reagentpCAG-C half NCAN-GFP
(plasmid)
This paperN/AVector backbone: pCAG
Gene/Insert name: GFP-fused C half of NCAN
Sequence-based reagentISH TNC probe fThis paperN/ACGGAATTCATCTTTGCAGAGAAAGGACAGC
Sequence-based reagentISH TNC probe rThis paperN/AGCTCTAGACTGTGTCCTTGTCATAGGTGGA
Sequence-based reagentISH NCAN probe fThis paperN/AGCGAATTCAGAATGCCTCTCTTGTTGGTG
Sequence-based reagentISH NCAN probe rThis paperN/AGCTCTAGACTACAATAGTGAGTTCGAGGCC
Sequence-based reagentcrRNA, Mm.Cas9.NCAN.1.AAIDTREF #101658344ACCUUAGUCCACUUGAU
CCGGUUUUAGAGCUAUGCU
Sequence-based reagentqPCR for Ncan, forwardThis paperN/ACCCTGCTTCTTTACCCTGCA
Sequence-based reagentqPCR for Ncan, reverse,This paperN/ACGTTGTCTTTGGCCACCAAG-3'
Sequence-based reagentqPCR for Tnc, forwardThis paperN/AACCATGGGTACAGGCTGTTG
Sequence-based reagentqPCR for Tnc, reverseThis paperN/ACCTTTCCAGCCTGGTTCACA
Sequence-based reagentqPCR for Gapdh, forwardThis paperN/AGACTTCAACAGCAACTCCCAC
Sequence-based reagentqPCR for Gapdh, reverseThis paperN/ATCCACCACCCTGTTGCTGTA
Peptide, recombinant proteinAlt-R S.p. Cas9 Nuclease V3IDTCatalog #1081058
Peptide, recombinant proteinTrypsin (Sequencing Grade)PromegaV511C
Peptide, recombinant proteinHRP-conjugated streptavidinWako190–17441
Peptide, recombinant proteinHyaluronidase from streptomyces hyalurolyticusSigmaH1136
Peptide, recombinant proteinRecombinant Human Tenascin C ProteinR&D3358-TC
Peptide, recombinant proteinRecombinant Human Neurocan ProteinR&D6508-NC-050
Peptide, recombinant proteinChondroitinase ABC Protease FreeSeikagaku Corporation100332
Commercial assay or kitBCA assay kitThermo Fisher23227
Commercial assay or kitRNeasy Mini KitQiagen74104
Commercial assay or kitPowerUp SYBR Green Master MixThermo FisherA25741
Commercial assay or kitDIG RNA Labeling Kit (SP6/T7)Roche11175025910
Commercial assay or kitNEBuilder HiFi DNA Assembly Master MixNew England BiolabsE2621
Commercial assay or kitIn-Fusion HD Cloning KitTakara639648
Commercial assay or kitKOD -Plus- Mutagenesis KitToyoboSMK-101
Software, algorithmMASCOT serverMatrix sciencehttps://www.matrixscience.com/server.html
RRID:SCR_014322
Software, algorithmImageJSchneider et al., 2012https://imagej.nih.gov/ij/
OtherDAPI solutionDOJINDOLot.PF082 340–07971
OtherN-2 SupplementThermo Fisher17502–048
OtherB-27 SupplementThermo Fisher17504–044
OtherNeurobasal MediumThermo Fisher21103–049
OtherPenicillin-StreptomycinThermo Fisher15070–063
OtherHBSS (X10)Thermo Fisher14185–052
OtherHEPES (1 M)Thermo Fisher15630–080
OtherPapainWorthington Biochemical CorporationLK003178
OtherFast GreenWako061–00031
OtherProtease Inhibitor CocktailSigmaP8340-1ML
OtherStreptavidin Magnetic BeadsThermo Fisher88817
OtherPoly-L-Ornithine SolutionWako163–27421
OtherSkim Milk PowderWako190–12865
OtherImmobilon Transfer Membrane PVDFMilliporeIPVH00010
OtherImmobilon Western ChemiluminescentMilliporeWBKLS0500
OtherSodium Hyaluronate (M2) (Mw:600,000~1,120,000)PG ResearchNaHA-M2
OtherBiotinylated Sodium Hyaluronate (M1) (Mw:600,000~1,120,000)PG ResearchBHHA-M1
OtherCarboxyfluorescein diacetate succinimidyl esterDOJINDO341–07401
OtherGFP-Trap-AgaroseChromotekgta-20
OtherELISA 96 well plateIWAKI3801–096
OtherBLOCK ACE PowderKACUKB80
OtherELISA POD Substrate TMB SolutionNacalai05299–54
OtherHyaluronan Binding Protein (HABP)Cosmo-bioBC40
OtherHyaluronic Acid Sodium SaltWako083–10341
OtherStreptavidin, Alexa Fluor 488 conjugateThermo FisherS-11223
OtherBiotinylated Hyaluronan Binding ProteinHokudoBC41
OtherSuperScript III reverse transcriptaseThermo Fisher18080044
OtherOCT CompoundSakura Finetek45833
OtherAnion exchange chromatography resinTosohTOYOPEARL DEAE-650M
OtherHeparin-agaroseSigmaH6508
OtherUltrafiltration filterAmiconYM-10
Otherα-cyano-4-hydroxycinnamic acidShimadzu70990
OtherLipofectamine 3000 ReagentThermo FisherL3000008
OtherElectroporatorNEPA GENENEPA21
Other5 mm-diameter tweezers-type disc electrodesNEPA GENECUY650P5
Other2.5 mm x 4 mm tweezers-type disc electrodesNEPA GENECUY652P2.5X4
OtherVibratomeLeicaVT1200S
OtherLuminoGraphATTOWSE-6100H-ACP
OtherConfocal laser scanning microscopeNikonAX
OtherConfocal laser scanning microscopeCarl ZeissLSM 710 NLO
OtherFluorescence stereomicroscopeLeicaM165FC
OtherAnimal anesthetizerMuromachiMK-AT210
OtherStepOne Real-Time PCR SystemThermo Fisher4376374
OtherCryostatLeicaCM3050 S
OtherDirect nanoLC/MALDI fraction systemKYA technologiesDiNa-MaP
OtherMALDI mass spectrometerSCIEXTOF/TOF 5800
OtherLC-MS/MS SystemSCIEXQTRAP 5500
OtherFluorescence-activated cell sorterBay bioscienceJSAN

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  1. Ayumu Mubuchi
  2. Mina Takechi
  3. Shunsuke Nishio
  4. Tsukasa Matsuda
  5. Yoshifumi Itoh
  6. Chihiro Sato
  7. Ken Kitajima
  8. Hiroshi Kitagawa
  9. Shinji Miyata
(2024)
Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons
eLife 12:RP92342.
https://doi.org/10.7554/eLife.92342.3