EphA4 Regulates Neuroblast and Astrocyte Organization in a Neurogenic Niche

Abstract

Significant migration cues are required to guide and contain newly generated rodent subventricular zone (SVZ) neuroblasts as they transit along the lateral ventricles and then through the anterior forebrain to their ultimate site of differentiation in the olfactory bulbs (OBs). These cues enforce strict neuroblast spatial boundaries within the dense astroglial meshwork of the SVZ and rostral migratory stream (RMS), yet are permissive to large-scale neuroblast migration. Therefore, the molecular mechanisms that define these cues and control dynamic interactions between migratory neuroblasts and surrounding astrocytes are of particular interest. We found that deletion of EphA4 and specifically ablation of EphA4 kinase activity resulted in misaligned neuroblasts and disorganized astrocytes in the RMS/SVZ, linking EphA4 forward signaling to SVZ and RMS spatial organization, orientation, and regulation. In addition, within a 3 week period, there was a significant reduction in the number of neuroblasts that reached the OB and integrated into the periglomerular layer, revealing a crucial role for EphA4 in facilitating efficient neuroblast migration to the OB. Single-cell analysis revealed that EPHA4 and its EFN binding partners are expressed by subpopulations of neuroblasts and astrocytes within the SVZ/RMS/OB system resulting in a cell-specific mosaic, suggesting complex EphA4 signaling involving both homotypic and heterotypic cell-cell interactions. Together, our studies reveal a novel molecular mechanism involving EphA4 signaling that functions in stem cell niche organization and ultimately neuroblast migration in the anterior forebrain.SIGNIFICANCE STATEMENT The subventricular zone neurogenic stem cell niche generates highly migratory neuroblasts that transit the anterior forebrain along a defined pathway to the olfactory bulb. Postnatal and adult brain organization dictates strict adherence to a narrow migration corridor. Subventricular zone neuroblasts are aligned in tightly bundled chains within a meshwork of astrocytes; however, the cell-cell cues that organize this unique, cell-dense migration pathway are largely unknown. Our studies show that forward signaling through the EphA4 tyrosine kinase receptor, mediated by ephrins expressed by subpopulations of neuroblasts and astrocytes, is required for compact, directional organization of neuroblasts and astrocytes within the pathway and efficient transit of neuroblasts through the anterior forebrain to the olfactory bulb.

Keywords: astrocyte; migration; neuroblast; olfactory bulb; rostral migratory stream; subventricular zone.

Figure 1.

Disorganized SVZ neuroblasts breach standard boundaries in EphA4^−/− mice. A, En face view of lateral ventricle wall whole mounts from EphA4^+/+ and EphA4^−/− adult mice. Top, Region of whole-mount preparations. Black arrowheads indicate neuroblast chains. B, TEM montages of the SVZ and adjacent corpus callosum (cc) of EphA4^+/+ and EphA4^−/− adult mice. Orange line indicates boundary between cc and SVZ. Black arrowheads indicate neuroblasts that have breached standard SVZ boundaries. C, D, Immunohistochemistry confirms the presence of Dcx⁺ (red) neuroblasts (white arrows) in neighboring striatum (St) and cc. Green represents GFAP⁺ astrocytes. Schematics indicate regions depicted in C, D. V, Ventricular space. Scale bars: A, 100 μm; C, D, 50 μm.

Figure 2.

EphA4 forward signaling is required to organize migrating neuroblasts into a compact, cell-dense adult RMS. A, Schematic of EphA4 forward, reverse, and bidirectional signaling in EphA4^+/+, EphA4^−/−, and KD-EphA4^eGFP/eGFP adult mice. B, Representative coronal sections of mature RMS neuroblasts (Dcx-immunolabeled) proximal to the lateral ventricle from EphA4^+/+, EphA4^−/−, and KD-EphA4^eGFP/eGFP mice. Bottom left, Region where coronal sections were isolated. C, Representative sagittal sections of mature RMS neuroblasts from EphA4^+/+, EphA4^−/−, and KD-EphA4^eGFP/eGFP mice. Yellow arrowheads indicate neuroblasts migrating outside of the standard RMS trajectory. D, Quantification of RMS cross-sectional areas from EphA4^+/+, EphA4^−/−, and KD-EphA4^eGFP/eGFP adult mice. *p < 0.05. **p < 0.01. Error bars indicate SEM. Scale bars: B, 100 μm; C, 20 μm.

Figure 3.

EphA4 acts during postnatal development to condense neuroblasts within what is to become the mature RMS. A, Representative sagittal sections of the RMS showing organization of astrocytes (GFAP; green), neuroblasts (Dcx; red), and vasculature (PECAM-1; blue) in EphA4^+/+ and EphA4^−/− mice at P6 and P12. B, Representative coronal sections of RMS neuroblasts (Dcx immunolabeled) from EphA4^+/+ and EphA4^−/− mice at postnatal day (P) 6 and P12. Scale bar, 50 μm. C, Quantification of RMS areas in P6 and P12 EphA4^+/+ and EphA4^−/− mice. *p < 0.05. Error bars indicate SEM. Scale bar, 50 μm.

Figure 4.

Astroglial meshwork morphology is disorganized in the RMS of adult EphA4^−/− and KD-EphA^eGFP/eGFP mice. Representative sagittal sections of RMS astrocytes (GFAP-immunolabeled) from EphA4^+/+, EphA4^−/−, and KD-EphA4^eGFP/eGFP mice. Right, To better portray cell morphology, red boxed regions represent “zoomed-in” images. Red arrowheads indicate astrocytes exhibiting hypertrophy and/or abnormal organization. Scale bar, 200 μm.

Figure 5.

EphA4 is needed for directed neuroblast migration to the OB and integration into the PGL. A, Representative images of EdU⁺ cells in the PGL of OB from EphA4^+/+ and EphA4^−/− mice. B, Total number of EdU⁺ cells in the SVZ, RMS, OB, and PGL of EphA4^+/+ and EphA4^−/− mice. C, Percentage BrdU⁺/Ki67⁺ cells in the SVZ of EphA4^+/+ and EphA4^−/− mice. D, Number of caspase-3⁺ cells in the SVZ, RMS, and OB of EphA4^+/+ and EphA4^−/− mice. *p < 0.05. ***p < 0.001. Error bars indicate SEM. Scale bar, 25 μm.

Figure 6.

RMS neuroblasts and astrocytes display a mosaic expression of EPHA4 and EFN genes. A, qPCR of total RMS lysates. All expression values are displayed as inverse C_t normalized to Gapdh expression. Bottom, Statistical analysis of relative expression. *p < 0.05. **p < 0.01. ***p < 0.001. Error bars indicate SEM. B, C, Heatmaps of single-cell gene expression (hierarchical clustering) for EPHA4, EFNA2, EFNA3, EFNA5, EFNB1, EFNB2, and EFNB3 in RMS neuroblasts (B) and RMS astrocytes (C). All expression values are displayed as inverse C_t. Euler diagrams below each heat map represent gene coexpression in the respective cell type. For ease of interpretation, the Euler diagrams are shown with (top) and without (bottom) EPHA4.

Figure 7.

RMS neuroblasts and astrocytes express distinct patterns of EphA4 and ephrin proteins. Coronal sections of the RMS of adult EphA4^+/+ mice show a unique pattern of EphA4, ephrinA2, and ephrinB expression on neuroblasts and astrocytes. Far right, To better portray Eph/ephrin expression in astrocytes, red boxed regions represent “zoomed-in” images. Green represents GFAP. Blue represents Dcx. Red represents specific EphA4/ephrin immunolabeling. Scale bar, 50 μm.

Figure 8.

Schematic of EphA4/ephrin expression in the adult EphA4^+/+ and EphA4^−/− mice. Illustration represents the compact, organized EphA4^−/− RMS in contrast to the disorganized and dispersed RMS of EphA4^−/− mice. EphA4 and ephrin expression is represented as percentages determined via single-cell transcriptional analysis.