Expression of S100B during embryonic development of the mouse cerebellum

Abstract

Background: In the cerebellum of newborn S100B-EGFP mice, we had previously noted the presence of a large population of S100B-expressing cells, which we assumed to be immature Bergmann glial cells. In the present study, we have drawn on this observation to establish the precise spatio-temporal pattern of S100B gene expression in the embryonic cerebellum.

Results: From E12.5 until E17.5, S100B was expressed in the primary radial glial scaffold involved in Purkinje progenitor exit from the ventricular zone and in the Sox9+ glial progenitors derived from it. During the same period coinciding with the primary phase of granule neuron precursor genesis, transient EGFP expression tagged the Pax6+ forerunners of granule precursors born in the cerebellar rhombic lip.

Conclusion: This study provides the first characterization of S100B-expressing cell types of the embryonic mouse cerebellum in a high-resolution map. The transient activation of the S100B gene distinguishes granule neuron precursors from all other types of precursors so far identified in the rhombic lip, whereas its activation in radial glial precursors is a feature of Bergmann cell gliogenesis.

Figure 1

S100B gene driven expression of EGFP in S100B+ cells of the ventricular and cortical transitory zones of the cerebellum at E17.5. A: confocal fluorescent image of a parasagittal section of the E17.5 S100B-EGFP cerebellar vermis. In addition to neural cells, the EGFP reporter is strongly expressed in the mesenchyme underlying the CPe. The staining patterns for S100B and EGFP are overlapping near the MHB (B), in the VZ (C), CTZ (D), and RL (E). The white dashed lines mark the ventricular and pial limits of the Cb. In this and the following figures, numbers above bars indicate the scale in microns.

Figure 2

pattern of S100B gene expression in the cerebellum and inferior colliculus before midline fusion of the cerebellar plates (E13.5). A: lower power confocal image of the Cb primordium, illustrating the strong EGFP signal present near the MHB (red box) in a parasagittal section. B: zooming on the boxed area in A reveals the high level of S100B/EGFP colocalisation at the single cell level. C: near the MHB, and on coronal sections, EGFP tags a stretch of neuroepithelial cells approx. 300 μM in width, emitting thin processes towards the pial surface (arrowheads). The red dotted lines represent the approximate planes of sections D and F. The glial scaffold (boxed area) is entirely (D, E) or only partially visible (F), depending on how close to the midline is the plane of section. E: higher magnification of the boxed area in (D) illustrating the pattern of EGFP expression near the midline: both the S-shaped radial glial scaffold of the Cb, and the abutting IC neuroepithelium, are strongly labeled.

Figure 3

S100B and EGFP are co-expressed in the radial glial scaffold of the cerebellar plates (E14.5 and E16.5). A: lower power view of the lateral portion (future hemisphere) of the E14.5 cerebellum plate. B: higher power view of the boxed area in (A), illustrating localization of cell somata in the VZ and long radial processes traversing a CTZ filled with post-mitotic β3-tubulin+ neuron precursors (inset). C: zooming on the VZ area reveals a high level of S100B/EGFP colocalisation at the single cell level. D: As expected for radial glial cells, EGFP+ cells present in the VZ express BLBP. E: at E16.5, and in addition to radial glial cells and CPe cells, Sox9 expression is maintained in isolated cells emigrating from the VZ (arrowheads).

Figure 4

EGFP is not expressed in post-mitotic calbindin+ Purkinje progenitors (E13.5–E16.5). A: lower power view of the lateral portion (future hemisphere) of the E13.5 Cb plate illustrating the broad subcortical distribution of calbindin+ PCs. B: higher power view of the boxed area in (A), illustrating the contacts between post-mitotic calbindin+ EGFP-negative PC precursors (white dots) and EGFP+ radial glial processes. Arrowheads point to the non specific red fluorescence of small capillaries. C: at E16.5, the radial glia-derived EGFP+ cell population is greatly increased. D-F: no matter which region of the Cb primordium is examined, the EGFP+ and calbindin+ populations are clearly separate entities. Most EGFP+ cells are connected to the pial surface via their apical process, constituting the so-called secondary radial glial scaffold used by the EGL population of GPs during their postnatal phase of radial migration.

Figure 5

EGFP is not expressed in the cerebellar rhombiclip before E12.5. A lower power view of a future Cb hemisphere at E11.5 illustrates the absence of EGFP expression in the rhombic lip, contrasting with its presence in the neighbouring mesenchyme (arrowhead), posterior semicircular canal (SCC) of the otic vesicle, and glial cells of the trigeminal (V) and facial (VII) ganglions.

Figure 6

The RL generates EGFP+ cells that are not precursors of DCN neurons (E12.5–E13.5). A: lower power view of a future Cb hemisphere at E12.5 illustrating the pattern of EGFP expression in two seemingly related cell populations: tightly packed bipolar cells in the RL, and isolated cells with a unipolar shape and a leading process contacting the pial surface in the nascent EGL, which is characteristic of migrating GPs (arrows). B, C: higher power views of the E12.5 RL, illustrating the localisation of EGFP+ Pax6+ cells in the medial (B), and lateral (C), portion of the RL, and the presence of Pax6+ EGFP- cells in the nascent EGL (arrowheads). Nuclei of the cells in which EGFP/Pax6 co-expression is obvious are marked by colored dots, EGFP+ Pax6- cells by asterisks. The long branching processes emitted by the RL EGFP+ cell population are better visualized in C. D: at E13.5 and on coronal sections, the Olig2+ NTZ (1) and EGFP+ EGL (2) constitute clearly distinct populations.

Figure 7

EGFP+ Pax6+ granule precursors in the RL and EGLat E14.5. A: lower power view of the lateral portion of the future Cb hemisphere, illustrating the pattern of EGFP/Pax6 co expression in the RL and EGL at E14.5. B: higher power view of the EGL showing clusters of migrating Pax6+ EGFP+ neuron precursors with their intensely Pax6+ nuclei (upper panel) and EGFP+ cytoplasm (lower panel). C: higher power view of the RL illustrating the predominant population of EGFP+ Pax6+ cells (colored dots) and the presence of EGFP+ Pax6- cells (asterisks).