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 …

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) …

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 …

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 …

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 …

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). …

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 …

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 …

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 …

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 …

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) …

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 …

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 …

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. …

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, …

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 …

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 …

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 …

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), …

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 …

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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