Dendritic planarity of Purkinje cells is independent of Reelin signaling

Abstract

The dendritic planarity of Purkinje cells is critical for cerebellar circuit formation. In the absence of Crk and CrkL, the Reelin pathway does not function resulting in partial Purkinje cell migration and defective dendritogenesis. However, the relationships among Purkinje cell migration, dendritic development and Reelin signaling have not been clearly delineated. Here, we use synchrotron X-ray microscopy to obtain 3-D images of Golgi-stained Purkinje cell dendrites. Purkinje cells that failed to migrate completely exhibited conical dendrites with abnormal 3-D arborization and reduced dendritic complexity. Furthermore, their spines were fewer in number with a distorted morphology. In contrast, Purkinje cells that migrated successfully displayed planar dendritic and spine morphologies similar to normal cells, despite reduced dendritic complexity. These results indicate that, during cerebellar formation, Purkinje cells migrate into an environment that supports development of dendritic planarity and spine formation. While Reelin signaling is important for the migration process, it does not make a direct major contribution to dendrite formation.

Fig. 1

Expression of Crk and CrkL in normal and mutant cerebellum. Calbindin immunostaining of normal (a, c) and Crk/CrkL mutant (b, d) cerebellum. Panels c and d are high-magnification images of boxes in panels a and b, respectively. Crk (22) immunostaining of low (e, f) and high (g, h) resolution images of normal and Crk/CrkL mutant. Crk (1154) immunostaining of low (i, j) and high (k, l) resolution images of normal and Crk/CrkL mutant cerebellum. CrkL (C-20) immunostaining of low (m, n) and high (o, p) resolution images of normal and Crk/CrkL mutant cerebellum. Crk/L (1157) immunostaining of low (q, r) and high (s, t) resolution images of normal and Crk/CrkL mutant cerebellum. ml molecular layer, pcl PC layer, igl internal granular cell layer

Fig. 2

3-D tomographic volume-rendered images of cerebellar tissues. a 3-D image of an entire cerebellum in a normal mouse (Movie S1). The lobular and PC layer structures are marked by the white and light green dashed lines, respectively. b 3-D magnified image of the white box region in panel a (Movie S2). PC and granule cells are marked by blue and magenta asterisks, respectively. The cerebellar layer arrangement is described by the white dashed lines. ml molecular layer; pcl PC layer, igl internal granular cell layer. c 3-D image of an entire cerebellum in a Crk/CrkL knockout mouse (Movie S3). Migrated and non-migrated PC are marked by the white and cyan blue boxes, respectively. d 3-D magnified image of the white box region in panel c (Movie S4). Migrated PC are marked by blue asterisks. ml molecular layer, pcl PC layer. e 3-D magnified image of the cyan blue box region in panel c (Movie S5). Non-migrated PC are marked by red asterisks

Fig. 3

Quantitative characterization of planarity of PC dendrites. a–c 3-D tomographic volume-rendered image of a normal, a migrated and a non-migrated Crk/CrkL knockout PC (Movie S6–8). Here, y–z defines the sagittal plane, x–z, the coronal plane, and x–y, the transverse plane. d–f Projections on the x–y plane of the dendritic branch (green dots) and the end (blue dots) points from the images of panels a–c, respectively. The soma is marked by a red circle at the origin. F is the formula for ‘flattening’ ratio, F = (a − b)/a (a is the length of the semi-major axis in the projections; b is its semi-minor axis). g Angular distribution in the x–y plane of the dendritic branch and the end points for 5 normal (black), 5 migrated mutant (blue), and 10 non-migrated mutant (red) PC. The plot shows the percentage of the points found in each 10-degree angular interval. The error bars correspond to the SEM. h Flattening ratio of PC projections with the distance from the cerebellar surface (DCS) for 5 normal, 5 migrated mutant (the light blue region), and 10 non-migrated mutant (the pink region: n = 3 for DCS = 200–400 μm; n = 3 for DCS = 400–600 μm; n = 4 for DCS = 600–800 μm) PC. The error bars correspond to the SEM

Fig. 4

3-D quantitative analysis of the branching rules and the fractal dimension of PC. a Branch angles. b Branch segment length. c Branch numbers, as determined by 3-D Sholl analysis. d 3-D Fractal dimension. 5 normal, 5 migrated and 10 non-migrated mutant PC were tested. The error bars correspond to the SEM

Fig. 5

Nano-radiographic images of PC spines and quantitative analysis of their densities. a–c Entire PC nano-images of a normal, a migrated and a non-migrated Crk/CrkL knockout PC. d–f Magnified images of the box regions in panels a–c, respectively. g Spine numbers per 10 μm of dendrites. Ten mice were analyzed for each group. The error bars correspond to the SEM. NS not significant; ***p < 0.001 compared to normal