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. 2017 Mar 9;7:42895.
doi: 10.1038/srep42895.

Ascl1 Promotes Tangential Migration and Confines Migratory Routes by Induction of Ephb2 in the Telencephalon

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Free PMC article

Ascl1 Promotes Tangential Migration and Confines Migratory Routes by Induction of Ephb2 in the Telencephalon

Yuan-Hsuan Liu et al. Sci Rep. .
Free PMC article

Abstract

During development, cortical interneurons generated from the ventral telencephalon migrate tangentially into the dorsal telencephalon. Although Achaete-scute family bHLH transcription factor 1 (Ascl1) plays important roles in the developing telencephalon, whether Ascl1 regulates tangential migration remains unclear. Here, we found that Ascl1 promoted tangential migration along the ventricular zone/subventricular zone (VZ/SVZ) and intermediate zone (IZ) of the dorsal telencephalon. Distal-less homeobox 2 (Dlx2) acted downstream of Ascl1 in promoting tangential migration along the VZ/SVZ but not IZ. We further identified Eph receptor B2 (Ephb2) as a direct target of Ascl1. Knockdown of EphB2 disrupted the separation of the VZ/SVZ and IZ migratory routes. Ephrin-A5, a ligand of EphB2, was sufficient to repel both Ascl1-expressing cells in vitro and tangentially migrating cortical interneurons in vivo. Together, our results demonstrate that Ascl1 induces expression of Dlx2 and Ephb2 to maintain distinct tangential migratory routes in the dorsal telencephalon.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Overexpression of Ascl1 and Dlx2 in the ventral telencephalon promotes tangential migration.
Four days after electroporation, brains of E19.5 rats were dissected and sectioned in the coronal plane. Electroporated cells were labeled with anti-GFP in green; nuclear DNA was stained with DAPI in blue. Red dashed lines indicate the electroporation region in (AG). (A) A confocal image of brain section; red dashed lines mark the electroporated region and a red square indicate the area for (BD). ST: striatum, LV: lateral ventricle. (BD) GFP-positive cells were distributed in the ST adjacent to the LV. (E) In the control group, some GFP-positive cells were distributed in the VZ/SVZ (white arrows) of the dorsal telencephalon, while most remained in the ventral telencephalon. (F) In Ascl1 group, GFP-positive cells were distributed in both the VZ/SVZ (white arrows) and IZ (white arrowheads) of the dorsal telencephalon. (G) In Dlx2 group, many GFP-positive cells were distributed in the VZ/SVZ (white arrow). Red squares indicate the zoom-in areas for 1E’ to G’. Length of the scale bar is 40 μm in (BD) and (E’–G’), 250 μm in (EG). (H) Quantification of GFP-positive cell density in the dorsal telencephalon. (I) Quantification of GFP-positive cells in the VZ/SVZ, IZ, or non-tangentially migrating cells in the dorsal telencephalon. Data were presented as mean ± standard error of the mean (SEM) with all data points and analyzed by Student’s t-test, n = 3. *p < 0.05; **p < 0.01 compared to the control group.
Figure 2
Figure 2. Overexpression of Ascl1 in the dorsal telencephalon induces ectopically tangential migration.
Four days after electroporation to the dorsal telencephalon, brains of E19.5 rats were dissected and sectioned in the coronal plane. Electroporated cells were labeled with anti-GFP in green; nuclear DNA was stained with DAPI in blue. (A) A confocal image with a red square indicating the electroporated site. (B’–G’) are zoomed regions of (B–G) indicated by red squares. (B) In the control group. GFP-positive cells were distributed near the electroporated area and many of them extended processes radially. GFP-positive axons toward the contralateral side were observed (yellow dashed lines). (C) In Neurog2 group, most GFP-positive cells were distributed in the cortical plate (CP). GFP-positive axons toward the contralateral side were observed (yellow dashed lines). (D) In Ascl1 group, many GFP-positive cells were distributed in the VZ/SVZ (white arrows) and IZ (white arrowheads) dorsomedially to the electroporated site. (E) In Dlx2 group, many GFP-positive cells were distributed in the VZ/SVZ (white arrows) dorsomedially to the electroporated site. (F) In Ascl1 + shLacZ#1 group, GFP-positive cells were distributed in a similar pattern as Ascl1 group in (D). (G) In Ascl1+shDlx2#1 group, many GFP-positive cells were distributed in the IZ (white arrowheads) dorsomedially to the electroporated site. Few GFP-positive cells were distributed in the VZ/SVZ (white arrows). Length of the scale bar is 120 μm in (BG), and 100 μm in (B’ to G’). (H) GFP-positive cells were categorized into radial, tangential, or other types according to the orientation of their leading processes. (I) Quantification of GFP-positive cells in the VZ/SVZ and IZ. Data are presented as box and whisker plots with all data points. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentiles, and the whiskers indicate the maximum and minimum values of the results. Data are analyzed by using Student’s t-test, n = 6 in all groups. *p < 0.05; **p < 0.01.
Figure 3
Figure 3. Overexpression of Ascl1 changes the migratory behavior of neurons in the dorsal telencephalon.
Ascl1 or US2 control expression construct were co-electroporated with a GFP expression plasmid. Two days after electroporation to the dorsal telencephalon, brains of E17.5 rats were dissected and sectioned in the coronal plane for slice culture and live imaging recording. The video from 5 to 11 hours after recording was used for tracking the migratory behavior. We set 0° to 180° axis in parallel to the lateral ventricle and the dorsomedial side as 180°. The line connecting the cell body location in the first frame (starting point) and the last frame (end point) of the video was used for measuring migratory angle. The average migratory rate was calculated as accumulated distance of every six-minute interval divided by recording time. 50 GFP-positive cells were counted for the control and 32 were counted for Ascl1 group. All counts were plotted into histograms according to their migratory angle or rate. (A) In the control group, most GFP-positive cells migrated radially in a migratory angle between 90° to 130°. (B) In Ascl1 group, GFP-positive cells were categorized into radially (90°–130°) and tangentially (140°–180°) migrating cells. (C) In the control group, GFP-positive cells migrated in the rate of 12.9 ± 3.6 μm/hour (mean ± SEM). (D) In Ascl1 group, radially migrating cells migrated in the rate of 30.2 ± 8.7 μm/hour and tangentially migrating cells migrated in the rate of 40.8 ± 9.3 μm/hour.
Figure 4
Figure 4. Identification of Ascl1 target genes of the Eph and Ephrin (Efn) family.
P19 cells were transfected with Neurog2 , Ascl1, or Dlx2 expression constructs and total RNA was extracted two days after transfection. The expression of Ephs (A,B) and Efns (C) were normalized to the expression of TATA-box binding protein (Tbp). Ephb1 and Ephb2 were expressed at higher levels in Ascl1-expressing cells than those in Neurog2 and Dlx2-expressing cells. Data are presented as box and whisker plots with all data points. Data are analyzed by using Student’s t-test, n = 4. *p < 0.05; **p < 0.01.
Figure 5
Figure 5. Knockdown of Ephb1 or Ephb2 disrupted tangential migration promoted by Ascl1.
Four days after electroporation to the dorsal telencephalon, brains of E19.5 rats were dissected and sectioned in the coronal plane. Electroporated cells were labeled with anti-GFP in green. (A’–C’) are zoomed regions of (AC) indicated by red squares, which were dorsomedial to the electroporated site. The cortex was divided equally into 10 bins. (A) In Ascl1 + shLacZ#1 group, many GFP positive cells were distributed in the VZ/SVZ (bins 1 and 2) and IZ (bin 5). (B,C) In Ascl1 + shEphB1#1 and Ascl1 + shEphB2#1 groups, GFP-positive cells were distributed in the VZ/SVZ (bins 1 and 2) and IZ (bin 5) were reduced. Length of the scale bar is 120 μm in (AC), and 40 μm in (A’–C’). (D) Distribution of GFP-positive in the cortex. Only GFP-positive cells dorsomedial to the electroporated site were counted. Data were presented as mean ± SEM with all data points and analyzed by one-way ANOVA with Tukey’s-HSD post hoc test, n = 4. *p < 0.05; **p < 0.01 compared to Ascl1 + shLacZ#1 group.
Figure 6
Figure 6. Ephb2 is a direct target of Ascl1.
(A,B) Putative E-box sequences were identified in the 3 Kb upstream of the transcription starting site (TSS) for Ephb1 and Ephb2. Four fragments (B1R1 to B1R4) of Ephb1 and five fragments (B2R1 to B2R5) of Ephb2 containing E-box sequences were designed for ChIP. Two fragments (B1NE and B2NE) without E-box sequences were selected as negative controls. (C,D) DNA from P19 cells transfected with Ascl1 expression vectors was extracted for ChIP. A fragment in the promoter region of Dll1 that has been demonstrated to interact with Ascl1 was used as a positive control. After ChIP, enrichment of the DNA fragments was quantified by qPCR (C,D) and regular PCR (E). (C) The promoter region of Dll1 was enriched by ChIP with anti-Ascl1. No fragment in Ephb1 was enriched. (D) The promoter region of Dll1, as well as B2R1 and B2R2 sites of Ephb2 were enriched by ChIP with anti-Ascl1. (E) Regular PCR result. The number in the parentheses indicated PCR cycle. Data were presented as mean ± SEM with all data points and analyzed by Student’s t-test, n = 3; *p < 0.05 compared to the IgG control.
Figure 7
Figure 7. Ephrin-A5 has a repulsive effect on Ascl1-expressing cortical neurons.
Control (US2) or Ascl1 expression constructs were electroporated into the dorsal telencephalon of E15.5 rats. The dorsal telencephalon was dissected two days after electroporation and dissociated into individual cells. These cells were cultured on coverslips coated with Fc-control or Ephrin-A5-Fc stripes. Cells were fixed 16–18 hours after plating and cells on strips or between strips were counted. Electroporated cells were labeled with anti-GFP in green, nuclear DNA was stained with DAPI in blue. GFP-positive cells on stripes are indicated by white arrowheads; GFP-positive cells between stripes are indicated by white arrows. (A) On a coverslip coated with Fc-control, GFP-positive Ascl1-expressing cells and GFP-negative cells were distributed evenly on stripes and between stripes. (B) On a coverslip coated with Ephrin-A5-Fc, GFP-positive Ascl1-expressing cells were preferentially distributed between stripes, while GFP-negative cells were evenly distributed on stripes and between stripes. Length of the scale bar is 50 μm. (C,D) Distribution of GFP-positive and GFP-negative cells. 150 cells from each group were counted in each experiment. Data are presented as mean ± SEM with all data points and analyzed by using Chi-square test, n = 3. *p < 0.05 compared to the Fc-control.
Figure 8
Figure 8. Ephrin-A5 repulses cortical interneurons.
Control (US2) or EfnA5 expression constructs were electroporated into the dorsal telencephalon together with a DsRed expression construct at E14.5 of Gad67-GFP mice. Brains were dissected four days after electroporation and sectioned coronally. (A,B) Confocal image of E18.5 mouse telencephalon. The cortex was divided into three regions: (A”,B”) the “in” region that contained DsRed-positive cells; (A”’,B”’) the “pre” region was ventrolateral to the “in” region; (A’,B’) the “post” region was dorsomedial to the “in” region. (A,B) In Ephrin-A5 group, fewer GFP-positive cells were present in “post” regions than those of the control group. Length of the scale bar is 100 μm. (C) Quantification of GFP fluorescent intensity in the MZ. (D) Quantification of GFP-positive cells in the CP, IZ and VZ/SVZ. A 200 μm wide cortical area in each region was selected for quantification. For the pre-DsRed, we selected the 200 μm wide cortical area 50 to 100 μm from the pre-/in-DsRed boundary. For the in-DsRed, we selected 50 to 100 μm from the in-/post-DsRed boundary. For the post-DsRed, we selected 50 to 100 μm from the in-/post-DsRed boundary. Data are presented as box and whisker plots and analyzed by using Student’s t-test, n = 4. *p < 0.05.

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