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Layering defect in p35 deficiency is linked to improper neuronal-glial interaction in radial migration

Nature Neuroscience volume 6, pages 1284–1291 (2003)Cite this article

Abstract

Several genes essential for neocortical layering have been identified in recent years, but their precise roles in this process remain to be elucidated. Mice deficient in p35—an activator of cyclin-dependent kinase 5 (Cdk5)—are characterized by a neocortex that has inverted layering. To decipher the physiological mechanisms that underlie this defect, we compared time-lapse recordings between p35−/− and wild-type cortical slices. In the p35−/− neocortex, the classic modes of radial migration—somal translocation and locomotion—were largely replaced by a distinct mode of migration: branched migration. Branched migration is cell-autonomous, associated with impaired neuronal-glial interaction and rare in neurons of scrambler mice, which are deficient in Dab1. Hence, our findings suggest that inside-out layering requires distinct functions of Reelin and p35/Cdk5 signaling, with the latter being important for proper glia-guided migration.

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Figure 1: Migration behavior at E13.
Figure 3: Relationship between migrating neurons and radial glia.
Figure 4: Cell-autonomous functions of p35.
Figure 2: Migration behavior at E15.
Figure 5: Migration behavior of scrambler neurons.

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Acknowledgements

The authors thank S. Tobet at the Shriver Center for technical help with the slice procedure, F. Gertler at Massachusetts Institute of Technology, where the quantitative and trajectory analysis was performed with DIAS software, and H. Patzke, M.D. Nguyen, J. Ko and B. Samuels for comments on the manuscript. K.S. is supported by a fellowship from the Naito Foundation. This work was supported by National Institutes of Health grants to L-H.T. and by research grants from the National Eye Institute (EY00621 to A.L.P.). L.-H.T. is an associate investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Amitabh Gupta and Kamon Sanada: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Harvard Medical School and Howard Hughes Medical Institute, Boston, 02115, Massachusetts, USA

    Amitabh Gupta, Kamon Sanada & Li-Huei Tsai

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    David T Miyamoto

  3. Departments of Neurology and Cell Biology, Washington University School of Medicine, St. Louis, 63110, Missouri, USA

    Susan Rovelstad, Alan L Pearlman & Jan Brunstrom

  4. Division of Neuroscience, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK

    Bagirathy Nadarajah

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Correspondence to Li-Huei Tsai.

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Supplementary information

Supplementary Fig. 1.

(a) Examples of branched p35 deficient neurons selected from the videomicroscopic data. Branch points and branches are designated by thick and thin arrows, respectively. White and blue colors indicate two separate branching cells in the same mage field. (b) Examples of branched neurons, compiled from p35 deficient neocortices that were infected with a GFP-carrying retrovirus at E12 and subjected to GFP-immunohistochemistry 3 days later. (JPG 37 kb)

Supplementary Fig. 2.

(a) Quantification of dynamic branching of a resting p35-/- neuron with complex branching morphology. (Top) Depicted is a resting neuron at two different time points (left, right) that shows dynamic changes of its branching morphology over time. The branching morphology consists of a main branch point (white arrows), a left branch (blue arrows), a right main branch (red arrows, left: growth cone is outlined) and a smaller right side branch (asterisk), which creates a side branch point. (Bottom) For the depicted neuron, the length of the branches (in μm) and the angles of the branch points (in degrees) are plotted against time (in hours). The dynamic nature of branching is evident not only in the changes of branch lengths, but also in the changes of the main branch point angle. (b) Quantification of dynamic branching of a resting p35-/- neuron with simple branching morphology. This neuron possesses a branching morphology that comprises of one branch point from which a left and right branch extend. The lengths of the branches (in μm) and the angle of the branch point (in degrees) are plotted against time (in hours). In this case, the dynamic changes in branching can be followed from branch point formation to branch point termination. Somal movement commences only after a second branch point is established at farther distance. (c) Dynamic branching of a p35-/- neuron that moves by branch-to-branch migration. The neuron analyzed here is pictured in figure 1c. Top: Depicted are the changes in the length of branches over time at two successive branch points (black, red). Bottom: Angles of both branch points and cumulative somal movement are followed over the same time period. It appears that somal movement in this case is associated with a fairly constant branch point angle, while branch length is subject to change (note the decrease in the length of the left branch at the second branch point). (JPG 109 kb)

Supplementary Fig. 3.

Trajectory tracings of a representative wildtype (WT) and p35 deficient neuron from E15 videos. Each dot along the tracings represents the position of the cell soma at a distinct frame (time point) of the corresponding video. The tracings illustrate that while the WT and p35-/- neurons both advance in an overall radial direction, the p35-/- neuron moves in a "zig-zag"-like fashion, with significant changes in the direction of the trajectory that suggest glia-independent migration. The WT neuron, in contrast, does not deviate from its strict radial path, indicating migration along radial glia. VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. (GIF 4 kb)

Supplementary Fig. 4.

An example of a p35 deficient neuron utilizing branch-to-branch migration. The cell soma of the depicted neuron is marked by an arrowhead, whereas branch points and branches are indicated by white and blue arrows, respectively. As the cell soma moves across the intermediate zone (IZ) to approach the branch point (I-II), the left branch retracts, while the right branch extends towards the subplate (II-III), becoming the new leading process (yellow arrows in II-III). A new branch point with two new branches is established (IV), to which the cell soma continues to move (IV-V), thereby advancing the neuron farther towards the cortical plate (CP). In panel VI, a second neuron with branched morphology can also be appreciated. Branch-to-branch movement in this case is associated with a 32 degrees change in the direction of the trajectory. (JPG 42 kb)

Supplementary Video 1.

Branched migration at E13 in the p35 ko neocortex (time between frames 20 min).Quicktime movie illustrating several examples of neurons that have branched morphology and advance their cell somata by moving towards stably positioned branch points. (MOV 1182 kb)

Supplementary Video 2.

Locomotion at E15 in the wildtype neocortex (time between frames,10 min).Quicktime movie of the two cells outlined in fig 2a, which move in tandem along the same radial path,presumably along the same radial glial fiber (glial fibers cannot be labeled with the Oregon Green calcium indicator used in the imaging studies). (MOV 846 kb)

Supplementary Video 3.

Branched migration at E15 in the p35 ko neocortex (time between frames,10 min).Quicktime movie of the two cells shown in fig. 2b,which demonstrate dynamic branching and movement towards fixed branch points. (MOV 3014 kb)

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Gupta, A., Sanada, K., Miyamoto, D. et al. Layering defect in p35 deficiency is linked to improper neuronal-glial interaction in radial migration. Nat Neurosci 6, 1284–1291 (2003). https://doi.org/10.1038/nn1151

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