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EGFRs mediate chemotactic migration in the developing telencephalon

Department of Neurobiology and Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, W1454 Biomedical Science Tower, Pittsburgh, PA 15261, USA

View larger version (80K): [in a new window]   Fig. 1. EGFRs mediate chemotaxis after viral transduction of explants. (A) How the explants were dissected and manipulated to alter the orientation of infected cells relative to ligand. Dorsolateral cortex was used in all experiments except L, where dorsolateral and medial cortex was used. In all micrographs, the ventricular surface is down, whether explants were grown VZ up or down, and the relative location of ligand is indicated. At least three explants were analyzed per condition. Similar distributions of cells were noted in triplicates. (B) E12.5 mouse, control virus, grown VZ down, no ligand; (C) E12.5 mouse, control virus, VZ down, TGF 10 ng/ml; (D) E12.5 mouse, control virus, VZ up, no ligand; (E) E12.5 mouse, control virus, VZ up, HB-EGF 10 ng/ml; (F) E12.5 mouse, EGFR virus, VZ down, no ligand; (G) E12.5 mouse, EGFR virus, VZ down, HB-EGF 1 ng/ml; (H) E12.5 mouse, EGFR virus, VZ up, HB-EGF 1 ng/ml; (I) E10.5 mouse, EGFR virus, VZ up, HB-EGF 10 ng/ml; (J) E12 rat, EGFR virus, VZ down no ligand; (K) E12 rat, EGFR virus, VZ down, TGF 0.1 ng/ml; (L) E12 rat, EGFR virus, VZ down, TGF 1 ng/ml (arrow indicates midline between hemispheres); and (M) E12 rat, EGFR virus, VZ up, TGF 10 ng/ml.

View larger version (36K): [in a new window]   Fig. 2. Chemotaxis is independent of cell type. (A) The identity of cells that chemotax in response to TGF or HB-EGF (1 ng/ml) was determined in E12 rat cortical explants infected with EGFR virus. MAP2 was used to identify neurons, S-100ß and GFAP were used to distinguish astrocytes. EGFR-infected cells that chemotaxed (labeled with anti-ß-gal in B,D; arrows) expressed the neuronal marker MAP2 in C or the astrocyte marker GFAP in E (arrows). To determine whether any of the cells that chemotax were stem cells, explants were dissociated after 3 days in ligand and the ability of EGFR-infected cells to divide in response to EGF (0.1-1 ng/ml) to generate neurospheres was assessed after 10 days (A), as described in the Materials and Methods. Approximately 25% of the EGFR-infected cells that chemotax could generate neurospheres, a characteristic of stem cells.

View larger version (76K): [in a new window]   Fig. 3. Expression of EGFRs changes during development. At E13 (A,C) EGFR immunostaining is relatively low, but increases by E16 (B,D) in mouse telencephalon. The distribution of cells expressing a high level of EGFR immunoreactivity was graded in the E16 cortex (B),with more cells expressing a high level of staining in lateral regions (L) than in medial regions (M). Asterisk indicates corticostriatal sulcus. The distribution of EGFRs on the cell surface also changes. At E13, EGFR immunostaining was concentrated at the apical surfaces of cells lining the lateral ventricle (A,C), but at E16 it was distributed more uniformly over the cell surface (B,D). C,D are higher magnifications A,B, respectively. The cells that expressed a high level of EGFRs were seen in proliferative zones (B,D) and in migration pathways, including the intermediate zone (B,D), the lateral cortical stream (LCS) (F-I) and the rostral migratory stream (RMS) (E,J). E illustrates the RMS in a sagittal view, with the letter J indicating the approximate position of the image in J. OB, olfactory bulb. F illustrates the LCS migration route, with letters indicating the approximate positions of images in panels G-I (M and L refer to medial and lateral). G shows EGFR expression at the beginning of the pathway, near the lateral ventricle (corticostriatal sulcus), as the pathway curves around the lateral ganglionic eminence (LGE). H shows the LCS in the ventrolateral cortex, with EGFR-positive cells leaving to migrate radially to the cortical plate. I shows the LCS leading into the piriform cortex (P). J shows EGFR-positive cells in the RMS posterior to the olfactory bulb in a parasagittal section. All other sections are transverse.

View larger version (72K): [in a new window]   Fig. 4. Endogenous EGFRs mediate chemotaxis. Explants of E16 dorsolateral cortex were cultured VZ down without (A) or with (B) exogenous HB-EGF (10 ng/ml) for 3 days and the location of cells expressing a high endogenous level of EGFRs was determined immunocytochemically.

View larger version (75K): [in a new window]   Fig. 5. Temporal and spatial patterns of HB-EGF staining in E13 (A) and E16 mouse (B,C). At E13 (A), HB-EGF immunoreactivity was seen at the ventricular surface. By contrast, at E16 (B), staining was most intense in two additional layers, corresponding to the marginal zone (MZ) and subplate/white matter (SP/WM). Diffuse staining was also visible in the intermediate zone (IZ). VZ, ventricular zone; SVZ, subventricular zone. Staining was more intense laterally (L) than medially (M) (B,C). (D) Western blot of lysates of E16 mouse dorsolateral cortex probed with anti-HB-EGF. Lane 1, recombinant HB-EGF (R&D); lane 2, E16 lysate.

View larger version (34K): [in a new window]   Fig. 6. Mis-expression of EGFRs in vivo at E14.5. The laminar and regional positions of cells infected in utero with control or EGFR virus were determined 3-4 days after infection. (A) Illustration of regional divisions. M, medial; DM, dorsomedial; D, dorsal; L, lateral; VL, ventrolateral. For control virus, 2757 cells in five embryos were counted; for EGFR virus, 3103 cells in 4 embryos were counted. (B) Regional distribution of infected cells in cortex (dorsal, ventrolateral) and olfactory bulb (OB). Note that mis-expression of EGFRs does not promote migration to the olfactory bulb via the RMS or the VL cortex via the LCS. (C) Laminar positions of infected cells in dorsal cortex. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; sp/wm, subplate/white matter; cp-l, lower half of cortical plate; cp-u, upper half of cortical plate; MZ, marginal zone. More of the EGFR-infected cells were located in sp/wm and MZ than control-infected cells (28.9±7.5 versus 2.2±1.9; P=0.01). By contrast, more control-infected cells were located in the inner half of the cerebral wall (VZ+SVZ+IZ) than EGFR-infected cells (87.4±5.9 versus 35.6±9.9; P=0.01). (D) Micrograph of cells infected with control virus. Several cells in the inner half of the cerebral wall are shown (arrow points to cells in VZ). (E) Micrograph of cells infected with EGFR virus. Several cells in the sp/wm are shown (arrows). (F) The laminar positions of infected cells in the olfactory bulb indicated that within the bulb, EGFR-misexpression promoted migration out of proliferative zones and into the differentiating zone (78.9±13.5 versus 22.3±5.9; P=0.025), as in dorsal cortex.

View larger version (80K): [in a new window]   Fig. 7. Infection at E10.5 results in region-specific changes in migration. (A) Micrograph of control-infected cells 6 days after injection at E10.5. Note clusters of cells in cortical plate (CP). (B) Micrograph of EGFR-infected cells 6 days after infection at E10.5. Note cells in subplate/white matter (SP/WM) in lateral and ventrolateral cortex, but not dorsomedial cortex, where they remained in proliferative zones (VZ, ventricular zone). Insets: higher magnification images of cells in dorsomedial (left) and ventrolateral (right) cortex. In ventrolateral cortex, EGFR-infected cells were found in the lateral cortical stream (C,D), migrating toward the piriform cortex (P). (C) Brightfield micrograph of X-gal labeled cells, (D) DAPI. (E,F) Control-infected cells were also found in the lateral cortical stream (E, brightfield; F, DAPI). (G,H) EGFR-infected cells (arrows) in the lateral cortical stream were found among cells that express high endogenous EGFRs. G was stained with an antibody that recognizes both endogenous and virally transduced EGFRs; in H, the EGFR-infected cells could be distinguished by expression of ß-galactosidase.

View larger version (37K): [in a new window]   Fig. 8. Laminar and regional distributions of infected cells 6 and 4 days after infections at E10.5. For control virus, 7535 cells in four embryos were analyzed 6 days after infection, and 2757 cells in five embryos were analyzed 4 days after infection. For EGFR virus, 8431 cells in three embryos were analyzed 6 days after infection, and 2293 cells in three embryos were analyzed 4 days after infection. (A) In dorsomedial cortex, the migration of EGFR-infected cells out to the cortical plate was reduced relative to control infected cells, with more of the EGFR-infected cells located in proliferative zones (VZ, SVZ) and the IZ 6 days after infection. By contrast, in dorsal (B), lateral (C) and ventrolateral cortex (D), EGFR-infected cells were found in greater proportions in sp/wm and MZ layers 6 days after infection. (E) In dorsal cortex 4 days post-infection, more of the EGFR-infected cells were located in proliferative zones, compared with control infected cells, suggesting that their migration to the sp/wm and MZ occurred between 4 and 6 days post-infection. (F) Comparison of the proportion of infected cells in different regions 4 and 6 days after infection revealed a dorsal to ventrolateral shift in EGFR-infected cells, but not control-infected cells. This suggests that EGFR-infected cells from dorsal cortex were diverted into the LCS. Asterisk indicates that the difference between the proportion of EGFR-infected cells in dorsal cortex at 4 days versus 6 days was significant (30.5±6.1 versus 10.5±2.9; P=0.04), as was the difference in ventrolateral cortex comparing 4 and 6 days (9.6±0.7 versus 30.1±4.7; P=0.01). (G) Micrograph of a clone of EGFR-infected cells in dorsal cortex 5 days post-infection. Note the radial alignment of cells in proliferative zones and the lateral displacement in the IZ. Inset: higher magnification image of cells in the IZ displaced laterally. (G) Model summarizing migration pathways containing EGFR-positive cells, ligands and the effects of EGFR misexpression. Pink circles represent cells expressing high endogenous EGFR, blue circles represent EGFR-infected cells, stippling represents ligands (HB-EGF and/or TGF). Left, transverse view; right, sagittal view.

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