Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons - PubMed (original) (raw)

Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons

Elaine M Pinheiro et al. Nat Cell Biol. 2011.

Abstract

During corticogenesis, pyramidal neurons (∼80% of cortical neurons) arise from the ventricular zone, pass through a multipolar stage to become bipolar and attach to radial glia, and then migrate to their proper position within the cortex. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular and intermediate zones, regions rich in tangentially migrating interneurons and axon fibre tracts. We examined the role of lamellipodin (Lpd), a homologue of a key regulator of neuronal migration and polarization in Caenorhabditis elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing pyramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode.

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Figures

Figure 1

Figure 1. Lpd silencing impairs neuronal positioning

(a–d) Mouse embryos were electroporated in utero at E14.5 and harvested at E18.5 (a, b, c) or P3 (d). (a) Quantification of cell distribution in cortical sections co-electroporated with mCherry and either Lpd shRNA, control (Ctrl) shRNA, or Lpd shRNA + Lpd rescue construct (Lpd*)(** p <0.01, *** p <0.001, one-way ANOVA, n = 4 brains per condition). (b) Rescue of the shRNA-mediated phenotype with an RNAi-resistant Lpd construct (Lpd*) expressed under the NeuroD1 promoter (* p <0.05, ** p <0.01, Student’s t-test, n = 4–5 brains per condition). (c) Images of a sequential electroporation of an embryo at E13.5 with Lpd shRNA plus Venus and subsequently at E14.5 with mCherry. (d) Distribution of cells at P3 after electroporation at E 14.5 (* p <0.05, **p <0.01, *** p <0.001, Student’s t-test, n = 3 brains per condition). Scale bars: 50 μm (a,b,c). Bar graphs are plotted as mean ± SEM.

Figure 2

Figure 2. Suppression of Lpd increases the number of tangentially oriented bipolar pyramidal neurons in the IZ/SVZ

(a–e) Mouse embryos were electroporated in utero at E14.5 and harvested at E18.5. (a) Percentage of multipolar versus bipolar cells in the SVZ and lower IZ of brains electroporated with either control (Ctrl) or Lpd. (b) Orientation of control (orange arrowhead) and Lpd shRNA (blue arrowhead) bipolar cells that co-express Venus in the SVZ/IZ. The graph represents the distribution of bipolar cells based upon the angle of their leading process with respect to the pial surface (90°) or ventricular surface (−90°). Cells were counterstained with Hoechst 33342 dye to visualize nuclei (blue). (c) Percentage of Lpd shRNA or control bipolar cells that are tangentially oriented in the IZ/SVZ (leading process angles between 30° and −30°). (d) Image of a tangentially oriented bipolar cell expressing Lpd shRNA identified by mCherry expression (red) in brain sections immunostained with axon fiber tract marker Neurofilament (green). (e) Tangential cells expressing Lpd shRNA do not align with radial glial fibers. The angle of the leading process of the tangential (as determined in (c)) Lpd shRNA-expressing cells was measured with respect to the radial glial fibers (identified by the expression of Nestin) and compared to that of bipolar control cells. Scale bars: 5 μm in (b), (d) and (e). Bar graphs are plotted as mean ± SEM (**p <0.01, *** p <0.001, Student’s t-test, n = 3 brains per condition).

Figure 3

Figure 3. Lpd depleted bipolar pyramidal neurons migrate tangentially within the IZ/SVZ but do not exhibit a change in cell fate

(a) E18.5 cortical sections electroporated at E14.5 with Lpd shRNA and Venus. Cells were counterstained with Hoechst 33342 dye to visualize nuclei (blue). Enlargement of boxed region is shown below. The distance between the cell body of individual tangential cells and the border of the electroporated region is indicated (arrows). (b) Time-lapse of a tangentially oriented bipolar cell migrating in the IZ/SVZ (arrowhead). (c) Image of a tangentially oriented bipolar cell, co-electroporated with Lpd shRNA and Venus, that expressed the neuronal marker, Cux1 (red). Nuclei were visualized with Hoechst 33342 dye. Cortical neurons expressing Lpd shRNA and Venus (arrowheads) lack detectable (d) GABA expression (red), and (e) CXCR4 (red) expressed by migrating interneurons (arrow). (f) Orientation of the leading process of Lpd-depleted tangentially oriented bipolar cells in the medial, mediolateral, and lateral regions of the cortex. Brains were electroporated at E11.5 and harvested 37 hrs later. Bar graphs are plotted as mean ± SEM. Scale bars: 50 μm (a), 40 μm (b) 5 μm (c), and 10 μm (d) and 10μm (e).

Figure 4

Figure 4. Lpd affects the orientation of cortical bipolar cells through an SRF/MAL-dependent pathway

(a–d) Mouse embryos were electroporated in utero at E14.5 and harvested at E18.5. (a) Expression levels of rhodamine-phalloidin (red) in control and Lpd shRNA bipolar cells that co-express Venus in the SVZ/IZ. Nuclei were visualized with Hoechst 33342 dye (blue). Scale Bar: 5 μm. Quantification of F-actin levels in Lpd knock-down tangentially oriented and control bipolar neurons as measured by relative phalloidin fluorescence intensity normalized to background fluorescence intensity (***p<0.001; cells from 3 control and 3 Lpd shRNA brains were included for quantification). (b) In utero luciferase reporter assay. Tissue was from brains that were co-electroporated with SRF reporter 3D. ALuc and pRL-TK plasmids along either Lpd shRNA, control (Ctrl) shRNA, or control (empty NeuroD1 vector), DN-SRF, or R62D expression vectors and subsequently analyzed for luciferase activity. Lpd knockdown significantly decreased SRF activity, represented by the relative firefly luciferase activity normalized to Renilla luciferase activity (*** p <0.001, Student’s t-test, n = 4 brains per condition) (* p <0.05, ** p <0.01, one-way ANOVA, n = 4–6 brains per condition). (c) Percentage of control and SRF knockdown (* p <0.05, Student’s t-test, n = 3 brains per condition) or (d) control, DN-SRF, and R62D bipolar cells that are tangentially oriented (leading process angles between 30° and −30°) in the IZ/SVZ (*** p <0.001, one-way ANOVA, n = 3 brains per condition).

Figure 5

Figure 5. Expression of MAL G-actin binding motifs (RPEL) rescues the Lpd knockdown orientation and positioning defects of bipolar pyramidal neurons

Mouse embryos were electroporated in utero at E14.5 and harvested at E18.5. (a) Schematic representation of SRF/MAL activity upon RPEL-NLS rescue of the Lpd knockdown. (b) Percentage of bipolar tangentially oriented cells in the IZ/SVZ of samples co-electroporated with Lpd knockdown vector and RPEL-NLS or RPEL(*)-NLS containing RPEL motif (mutations) ** p <0.01, Student’s t-test, n = 3 brains per condition). Bar graphs are plotted as mean ± SEM. (c) Quantification of cell distribution in cortical sections co-electroporated with Venus, Lpd shRNA and either RPEL-NLS or RPEL(*)-NLS containing mutations in the RPEL motif that disrupts G-actin binding. (* p <0.05, **p <0.01, *** p <0.001, Student’s t-test, n = 3 brains per condition). Scale bars: 50 μm. Bar graphs are plotted as mean ± SEM.

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