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CXCL12-Mediated Murine Neural Progenitor Cell Movement Requires PI3Kβ Activation

Abstract

The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.

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Acknowledgments

We thank L. Gómez for animal handling, and C. Bastos and C. Mark for secretarial and editorial assistance, respectively. BLH received an FPI predoctoral fellowship (BES-2006-12965) from the Spanish Ministry of Science and Innovation. This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2011-27370), the RETICS Program (RD08/0075/0010, RD12/0009/0009; RIER), the Madrid regional government (S2010/BMD-2350; RAPHYME), and the European Union (FP7-integrated project Masterswitch 223404).

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Correspondence to Mario Mellado.

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Online Resource 1. NPC migration on fibronectin-coated coverslips. Videomicroscopy analysis of NPC motility. Fluorescent-labeled NPC (1 μM CFSE) were injected into a fibronectin-coated chamber alone (left) or with CXCL12 (right). After 10 min, confocal fluorescence and transmitted light images were acquired every 20 s for 90 min. Time indicated in seconds. (MOV 3356 kb)

figure8

Online Resource 2. NPC motility on laminin-coated surfaces. a Effect of fibronectin- (20 μg/mL) and laminin-coated (20 μg/mL) surfaces on average NPC speed in the absence of stimulus (fibronectin, n = 35 cells; laminin, n = 44 cells). b Effect of fibronectin- and laminin-coated surfaces on NPC total track length in the absence of stimulus (fibronectin, n = 35 cells; laminin, n = 44 cells). c Analysis of average speed of CXCL12-induced NPC migration on laminin-coated surface (none, n = 35 cells; CXCL12, n = 44 cells). d Analysis of total track length of CXCL12-induced NPC migration on laminin-coated surfaces (none, n = 35 cells; CXCL12, n = 44 cells). Data correspond to three independent experiments. Student’s t test; ***P < 0.001; n.s. not significant. (JPEG 3134 kb)

figure9

Online Resource 3. Inhibitor treatment and cell viability. a NPC migration assay in response to different CXCL12 concentrations. Figure shows the percentage of migrated cells expressed as mean ± SEM (n = 3). b Cell cycle analysis of NPC treated with PTx (0.1 μg/mL), AMD31000 (10 μM) or glycerol (0.025 %) by PI staining and flow cytometry. c Cell cycle phase quantitation of NPC in (b). Data show mean ± SEM (n = 3). d Cell cycle analysis by PI staining and flow cytometry of NPC treated with 0.1 % DMSO (control), JAK2 Inhibitor II (10 μM), LY294002 (10 μM), or Src Inhibitor I (10 μM). e Cell cycle phase quantitation of NPC in (d). Data show mean ± SEM (n = 3). (JPEG 3059 kb)

figure10

Online Resource 4. Class I PI3K isoform expression and role of p110γ and p110δ in CXCL12-triggered NPC migration. a Q-PCR analysis of the expression of distinct catalytic and regulatory class I PI3K subunits mRNA in Wt NPC. b Western blot analysis of PI3K catalytic subunit expression in Wt NPC. c Wt and p110γ−/− mouse NPC were allowed to migrate toward CXCL12 (50 nM). Figure shows the percentage of migrated cells expressed as mean ± SEM (n = 3). d Wt and p110δ−/− mouse NPC were allowed to migrate as in (c). Figure shows the percentage of migrated cells expressed as mean ± SEM (n = 3). e shRNA control or shRNA p110β nucleofection efficiency of NPC was controlled by flow cytometry using GFP detection. Data are expressed as mean ± SEM (n = 3). n.s. not significant. (JPEG 3286 kb)

figure11

Online Resource 5. CXCL12-mediated JAK and PI3K β are independent signaling pathways. a NPC lysates were immunoprecipitated using an anti-p110β antibody, and kinase activities were assayed in vitro (top) in the presence of TGX-221 (0.5 and 0.05 μM) or JAK2 Inhibitor II (10, 0.5, and 0.001 μM). As controls, we show kinase activity in the presence of DMSO (0.1 %) and in the absence of the immunoprecipitating anti-p110β antibody (Neg). Arrow indicates migrated PtdIns-3-P. One representative assay is shown of three performed. As a protein loading control (bottom), aliquots were immunoblotted with anti-p110β antibody in the same experiment. b Lysates of CXCL12-, EGF-, and LPA-activated NPC cells pretreated with JAK2 Inhibitor II or DMSO (control) were analyzed by Western blot with anti-p-Akt Ab. As control, the membrane was reprobed with anti-Akt Ab. c JAK2−/− mouse NPC were nucleofected with control or JAK1 siRNA; after 24 h, cells were allowed to migrate in response to 50 nM CXCL12 in the presence of TGX-221 or DMSO (control). Data shown as mean ± SEM for one representative experiment of three performed. (JPEG 2820 kb)

Online Resource 1. NPC migration on fibronectin-coated coverslips. Videomicroscopy analysis of NPC motility. Fluorescent-labeled NPC (1 μM CFSE) were injected into a fibronectin-coated chamber alone (left) or with CXCL12 (right). After 10 min, confocal fluorescence and transmitted light images were acquired every 20 s for 90 min. Time indicated in seconds. (MOV 3356 kb)

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Holgado, B.L., Martínez-Muñoz, L., Sánchez-Alcañiz, J.A. et al. CXCL12-Mediated Murine Neural Progenitor Cell Movement Requires PI3Kβ Activation. Mol Neurobiol 48, 217–231 (2013) doi:10.1007/s12035-013-8451-5

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Keywords

  • CXCR4
  • Cell migration
  • p110β
  • Neural precursor
  • Interneuron
  • JAK