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. 2009 Sep 24;461(7263):524-8.
doi: 10.1038/nature08362. Epub 2009 Sep 16.

Integration of neuronal clones in the radial cortical columns by EphA and ephrin-A signalling

Masaaki Torii  1 , Kazue Hashimoto-Torii, Pat Levitt, Pasko Rakic
Affiliations

Integration of neuronal clones in the radial cortical columns by EphA and ephrin-A signalling

Masaaki Torii et al. Nature. .

Abstract

The cerebral cortex is a laminated sheet of neurons composed of the arrays of intersecting radial columns. During development, excitatory projection neurons originating from the proliferative units at the ventricular surface of the embryonic cerebral vesicles migrate along elongated radial glial fibres to form a cellular infrastructure of radial (vertical) ontogenetic columns in the overlaying cortical plate. However, a subpopulation of these clonally related neurons also undergoes a short lateral shift and transfers from their parental to the neighbouring radial glial fibres, and intermixes with neurons originating from neighbouring proliferative units. This columnar organization acts as the primary information processing unit in the cortex. The molecular mechanisms, role and significance of this lateral dispersion for cortical development are not understood. Here we show that an Eph receptor A (EphA) and ephrin A (Efna) signalling-dependent shift in the allocation of clonally related neurons is essential for the proper assembly of cortical columns. In contrast to the relatively uniform labelling of the developing cortical plate by various molecular markers and retrograde tracers in wild-type mice, we found alternating labelling of columnar compartments in Efna knockout mice that are caused by impaired lateral dispersion of migrating neurons rather than by altered cell production or death. Furthermore, in utero electroporation showed that lateral dispersion depends on the expression levels of EphAs and ephrin-As during neuronal migration. This so far unrecognized mechanism for lateral neuronal dispersion seems to be essential for the proper intermixing of neuronal types in the cortical columns, which, when disrupted, might contribute to neuropsychiatric disorders associated with abnormal columnar organization.

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Figures

Figure 1
Figure 1
Abnormal tangential organization of the cortical plate in the TKO neocortex. a, CTIP2 immunohistochemistry on wild-type (WT) and TKO brains. b, Higher magnification images of the boxed area in a. The arrow and arrowhead indicate the domains where abnormally more and less neurons are labelled, respectively. c, Quantification of the grey values of fluorescent CTIP2 labelling from the left panels in a along the medial-lateral axis (yellow arrows). Total labelling was similar in wild-type andTKO(39,361 and 39,036 in pixels, respectively). d, Irregular tangential distribution of Sox51 neurons, associated with abnormal thickening and thinning of the cortical plate ofTKO(arrow and arrowheads). e, Cutl1 and CTIP2 labelling shows an increase and decrease of labelled neuronal subpopulations (white arrows and arrowhead, respectively) in adjacent cortical domains in TKO. The increase and decrease was not always synchronized (yellow arrowhead). f, Lmo4 and CTIP2 staining shows distinct patterns of increased/decreased labelled neurons in TKO (arrow and arrowheads). g, The numbers of labelled CTPN and CPN were both highly variable among neighbouring columnar domains in TKO (arrow and arrowhead). Scale bars, 500 mm (a) and 200 mm (b–g).
Figure 2
Figure 2
Impaired lateral dispersion of cortical neurons in TKO. a, GFPlabelled (green) clonal neurons. The column width of the clonal neurons (brackets) was smaller in TKO. Nuclei were counterstained (blue). b, Magnified view of clonally related neurons. The right panel for wild-type shows the most extreme columnar case in wild-type. In the TKO cortex, straight alignments of GFP-labelled neurons (between arrows) were found. c, Left: representative image of GFP-labelled neurons along their parental radial glial fibre (RGF; arrowheads). CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Middle: box-plot of the distance between labelled migrating neurons and the parental RGF. The box represents the median and the 25th and 75th percentiles. The top and bottom whiskers denote the 5th and 95th percentiles. Plus signs denote the mean distances. The distance was significantly shorter in TKO. P50.0014 (Mann–Whitney U test, n532 from 14 TKO brains, and 21 from 10 wild-type brains). Right: cumulative percentage plot from the same data. P,0.001 (Kolmogorov–Smirnov test). d, Discontinuity of the band of NeuroD-labelled migrating neurons in the TKO cortex (arrowheads) by DTA electroporation. Similar levels of apoptotic cell death were observed in wild-type and TKO cortices as shown with adjacent slices. Note that a few surviving EYFP1 neurons can demarcate the electroporated fields (between dotted lines). Fibrous labelling in the upper intermediate zone in the NeuroD staining of TKO is a nonspecific background. e, Interruption of the NeuroD-labelled domain (arrows) in the TKO cortex, which received DTA electroporation only in a very narrow region. f, The thickness of Cutl11 cortical layers at the DTA electroporated domains (brackets) was decreased in TKO. Relative thickness of Cutl11 layers at the electroporated domains against the control hemisphere was significantly decreased in TKO. Data are mean6s.e.m. (n520 sections from five brains per genotype), P50.0006 (Student’s t-test). Scale bars, 100 mm (a), 50 mm (b, c) and 200 mm (d–f).
Figure 3
Figure 3
EphA overexpression leads to tangential sorting of cortical neurons. a, Distribution of EYFP-labelled neurons (green or black) during development in the cortex in which control or EphA7 expression plasmid was delivered using electroporation (EP) with EYFP plasmid. Scale bars, 600 mm (for E18.8), 50 mm (for E15.5) and 200 mm (for P4). b, E15.5 cortex electroporated with control or EphA7 expression plasmid at E12.5. Right panels are higher magnification views around the ventricular–intermediate zone, showing multipolar shaped migrating neurons. EYFP1 EphA7- electroporated neurons start to segregate from EYFP2 neurons in the intermediate zone (arrowheads). Scale bars, 100 mm (left panels) and 50 mm (right panels). c, Fluorescence grey values were quantified from the images in b from left to right (medial to lateral in the cortex) in the ventricular zone and the intermediate zone as indicated by blue and red arrows in b. Plot profiles indicate the clustering of labelled EphA7-electroporated neurons in the intermediate zone (arrowheads in b and c).
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