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First published online 3 October 2007
doi: 10.1242/dev.005447

Development 134, 3883-3891 (2007)
Published by The Company of Biologists 2007
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Divergent roles of ApoER2 and Vldlr in the migration of cortical neurons

Iris Hack1,*, Sabine Hellwig1,2,*, Dirk Junghans3, Bianka Brunne4, Hans H. Bock4, Shanting Zhao1 and Michael Frotscher1,4,{dagger}

1 Institut für Anatomie und Zellbiologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
2 Neurologische Universitätsklinik, Neurozentrum, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
3 Max-Planck-Institut für Immunbiologie, D-79108 Freiburg, Germany.
4 Zentrum für Neurowissenschaften, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.


Figure 1
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  		Fig. 1. Labeling of early and late born cortical neurons in wild-type, Vldlr-/- and ApoER2-/- mice at P7 using ER81 and Cux2. (A,D,G) In situ hybridization for ER81, a marker of early generated neurons, showed strongest expression in cortical layer V neurons on sagittal brain sections of wild-type animals (A) and Vldlr-/- mice (D). In ApoER2 mutants (G), labeling in the outer portion of the cortex, normally occupied by layer II-IV neurons, was found, indicating a more superficially located layer V. (B,E,H) In situ hybridization for Cux2, a marker of late generated neurons, revealed expression in the upper cortical layers (II-IV) in wild-type animals (B). In Vldlr mutants (E), the Cux2-positive population showed a similar distribution, but the marginal zone was invaded by many Cux2-positive cells. In ApoER2 mutants (H), in situ hybridization for Cux2 showed two bands, a superficial one underneath layer I, and a deep one, located in the innermost portion of the cerebral wall. A comparison of the expression patterns of ER81 (G) and Cux2 (H) in this mutant suggests that the upper Cux2-positive cells represent layer IV neurons. (C,F,I) The prominent phenotype of ApoER2 mutants when compared to Vldlr-/- mice is also seen in Nissl-stained sections. Arrow in I indicates cell accumulation in deep cortical layers. Scale bar: 200 µm.

 

Figure 2
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  		Fig. 2. Labeling of early born cortical neurons in wild-type and ApoER2-/- mice at P7 using Tbr1 and Tle4. Immunostaining for Tbr1 (A,B) and in situ hybridization for Tle4 (C,D) on sagittal brain sections revealed the strongest signal in deep cortical layers of wild-type animals (A,C). Similar to the staining for ER81, both markers show a shift in early born neurons towards more superficial regions in the ApoER2 mutant cortex (B,D). Scale bars: 200 µm in A,B; 100 µm in C,D.

 

Figure 3
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  		Fig. 3. ApoER2 controls late stages of cortical layer formation. (A,F) BrdU-labeling at E12.5 and staining of sagittal brain sections through the cerebral cortex at P0 showed the strongest signal close to the ventricle in wild-type mice (A, arrowhead). By contrast, in ApoER2 mutants (F), many heavily BrdU-labeled cells were also found in superficial portions of the cortex. (B,G) Labeling at E13.5 revealed many BrdU-stained cells in the inner to middle portion of the cortex in wild-type animals (B, arrowhead), whereas in ApoER2 mutants (G) an altered pattern was observed (arrowhead), and the formation of two separate bands became apparent. When labeling for BrdU was carried out at E14.5 (C,H) and E15.5 (D,I), the separation of these two layers was more clearly visible (H,I, arrowheads), whereas in wild-type (C,D) mice, only the upper cortical layers were strongly labeled (arrowheads). (E,J) Labeling at E16.5, a late stage of corticogenesis, resulted in a heavily stained band in the outermost portion of the cortex in wild-type animals (E, arrowhead), whereas in ApoER2 mutants (J) strongly labeled cells were located close to the ventricle (arrowhead). Scale bar: 200 µm.

 

Figure 4
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  		Fig. 4. Cux2 and RORbeta staining in wild-type animals and ApoER2 mutants at P7. (A,E) In situ hybridization for RORbeta, a marker of layer IV neurons, showed strong expression in layer IV in sections of wild-type animals (arrow in A). In ApoER2 mutants (E), strongest RORbeta expression was found in superficial portions of the cortex underneath the marginal zone and close to the ventricle (arrows). (B-D,F-H) Double fluorescence in situ hybridization for Cux2 (green) and RORbeta (red) revealed that Cux2 labels mainly layer II-III neurons and only a superficially located subpopulation of layer IV in wild-type animals (B-D). Arrowheads in D point to double-labeled cells. In ApoER2 mutants (F-H), Cux2- and RORbeta-labeled neurons were scattered throughout the cortex with cell accumulations underneath the marginal zone and close to the ventricle (arrows). Arrowheads in H point to double-labeled cells. Scale bars: 200 µm.

 

Figure 5
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  		Fig. 5. Expression pattern of ApoER2 transcripts at different developmental time points. (A-F) In situ hybridization for ApoER2 on sagittal sections of E16.5 (A), E18.5 (C) and P7 (E) wild-type cortex showed expression throughout the whole cortical wall. Higher magnification of the neocortex revealed the strongest signal in upper cortical layers (B,D,F). (G-I) Double fluorescence in situ hybridization for Cux2 (G, green) and ApoER2 (H, red) shows colocalization (I, yellow) of the two markers, indicating that late generated cortical neurons express ApoER2. Scale bars: 200 µm in A,C,F-I; 100 µm in B,D; 500 µm in E.

 

Figure 6
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  		Fig. 6. Cortical radial glial scaffold in wild-type and ApoER2-/- mice. Staining for BLBP, nestin and GLAST of frontal sections through the telencephalon of ApoER2-/- (B,D,F) mice at E16.5 showed no alteration of the radial glial scaffold when compared to wild-type animals (A,C,E). Scale bar: 100 µm.

 

Figure 7
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  		Fig. 7. Invasion of the marginal zone in Vldlr mutants. (A,B) In situ hybridization for Cux2 on sagittal brain sections through the neocortex of adult wild-type mice (A) showed only few neurons in layer I, whereas many Cux2-positive cells were found in the marginal zone of Vldlr-/- mice (B). (C-E) Sagittal brain sections through adult neocortex stained for NeuN similarly showed few neurons in wild-type animals (C) and ApoER2 mutants (D), but numerous cells in Vldlr mutants (E). Scale bars: 100 µm in A,B; 200 µm in C-E. (F) Stereological estimation of cell numbers in the marginal zone was performed in four animals of each group. Vldlr-/- mice showed significantly more neurons (mean±s.e.m.) in the marginal zone compared with wild-type animals and ApoER2 mutant mice (P=0.029). No significant difference was found between wild-type animals and ApoER2 mutants.

 

Figure 8
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  		Fig. 8. Ectopic pyramidal cells in the marginal zone of Vldlr mutants. (A,B) Calbindin-immunoreactive neurons were almost absent from the marginal zone in wild-type animals (A), but were numerous in Vldlr mutants (B). (C) Immunostaining for Tbr1, a deep layer marker, revealed labeled cells in the marginal zone of Vldlr mutant mice. (D-G) Immunolabeling for SMI32, a pyramidal cell marker, revealed strongest expression in layer V on sagittal sections through adult neocortex of wild-type mice (D). In Vldlr mutants (E), but not in wild-type animals and ApoER2 mutants, a few SMI32-positive cells were observed in the marginal zone (arrowhead; higher magnification in F). Reminiscent of the reeler phenotype, inverted pyramidal cells were occasionally observed (G). Scale bars: 50 µm in A,B,F,G; 100 µm in C,D,E.

 




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