Wnt/β-catenin signalling is active in a highly dynamic pattern during development of the mouse cerebellum

Abstract

The adult cerebellum is composed of several distinct cell types with well defined developmental origins. However, the molecular mechanisms that govern the generation of these cell types are only partially resolved. Wnt/β-catenin signalling has a wide variety of roles in generation of the central nervous system, though the specific activity of this pathway during cerebellum development is not well understood. Here, we present data that delineate the spatio-temporal specific pattern of Wnt/β-catenin signaling during mouse cerebellum development between E12.5 and P21. Using the BAT-gal Wnt/β-catenin reporter mouse, we found that Wnt/β-catenin activity is present transiently at the embryonic rhombic lip but not at later stages during the expansion of cell populations that arise from there. At late embryonic and early postnatal stages, Wnt/β-catenin activity shifts to the cerebellar ventricular zone and to cells arising from this germinal centre. Subsequently, the expression pattern becomes progressively restricted to Bergmann glial cells, which show expression of the reporter at P21. These results indicate a variety of potential functions for Wnt/β-catenin activity during cerebellum development.

Figure 1. BAT-gal expression in the E12.5 and E14.5 cerebellum.

(A) DAB Immunohistochemistry for β-galactosidase (β-gal) on sagittal sections of E12.5 cerebellum revealed two key expression domains: the isthmus (Is) and the cerebellar rhombic lip (RL). At E14.5 (B) expression was also found in the early external granule layer (EGL, black arrowheads) but was notably absent from the ventricular zone (VZ) lining the fourth ventricle (IV). (C) Double immunofluorescence for β-gal and PCNA confirms the expression of β-gal in the RL and EGL (white arrowheads). β-gal protein was validated as a Wnt/β-catenin reporter by in situ hybridisation for LacZ (D, E) and Wnt target Axin2 (F, G)) mRNA. At both time points β-gal protein and LacZ mRNA were expressed in the same domains as Axin2, although the LacZ mRNA expression appeared less diffuse than that of Axin2. (A–B counterstained with hematoxylin. Scale bars: A, B, D–E  = 100 µm, B  = 50 µM).

Figure 2. BAT-gal expression in the E18.5 and P1 cerebellum.

DAB immunohistochemistry for β-gal in sagittal sections of the E18.5 cerebellum (A) reveals widespread expression, including predominant staining in the VZ (black arrowheads). A similar pattern is observed at P1 (B). These expression patterns are mirrored by those observed for LacZ mRNA visualised with in situ hybridisation (C–D). Double immunofluorescence for β-gal and PCNA reveals an almost complete lack of BAT-gal reporter expression in the EGL at both E18.5 (E) and P1 (F), though β-gal+ cells can be observed within the developing cerebellum at both time points, in some cases colocalised with PCNA (white arrows). At the VZ, BAT-gal expression can be observed colocalised with PCNA (white arrowheads) at both E18.5 (G) and P1 (H). (A–B counterstained with hematoxylin. Scale bars: A, B = 100 µm, E–H = 50 µm).

Figure 3. BAT-gal expression in the P5 cerebellum.

(A) DAB immunohistochemistry for β-gal and (B) LacZ in situ hybridisation in the P5 cerebellum. (C) Higher magnification of the region boxed in (A) reveals expression spread through all layers except the EGL. The Purkinje cell layer (PCL) and the white matter (WM) in particular contained many β-gal+ cells (black and white arrowheads respectively). Double immunofluorescence for β-gal and PCNA (D) revealed the presence of β-gal+ cells within the PCL and white matter (white arrowheads). Although β-gal+ cells were observed in close proximity to proliferating cells (unfilled arrowheads) very few β-gal+/PCNA+ cells were observed. Double immunofluorescence for β-gal and Pax2 (E) showed the close proximity of β-gal+ cells (white arrowhead) to Pax2+ interneurons (unfilled arrowhead) but no double-labelled cells were observed. Double immunofluorescence for β-gal and NeuN showed β-gal+ cells (white arrows) located outwith the IGL. (A,C counterstained with hematoxylin and D–F with Topro3. Scale bars: A = 500 µm, B–C = 100 µm, D–E = 50 µm).

Figure 4. BAT-gal expression in the P10 cerebellum.

(A) DAB β-gal immunohistochemistry and (B) LacZ in situ hybridisation in P10 cerebellum. (C) Higher magnification of the region boxed in (A) reveals a more restricted pattern than that seen at P5, with strongest staining observed within the PCL (black arrowheads – also in B). At higher magnification, β-gal+ cells within the PCL (white arrowheads) were observed in close proximity to both PCNA+ (D) and Pax2+ (E) cells (unfilled arrowheads), though no colocalisation was observed between β-gal and PCNA or Pax2. (F) Double immunofluorescence for β-gal and NeuN confirms the presence of β-gal+ cells at the PCL on the edge of the IGL, while double immunofluorescence for β-gal and calbindin (G) confirms the lack of BAT-gal reporter expression in Purkinje cells (PC). (H) Colocalisation with glial marker s100β confirms the identity of β-gal+ cells within the PCL as Bergmann glia (white arrowheads), though not all Bergmann glia express β-galactosidase (unfilled arrowhead). (A, C are counterstained with hematoxylin and D–H with Topro3 Scale bars: A = 500 µm, B–C = 100 µm, D–H = 50 µm).

Figure 5. BAT-gal expression in the P21 cerebellum.

(A) DAB immunohistochemistry for β-gal in the P21 cerebellum. (B) Higher magnification of the region boxed in (A) reveals that BAT-gal reporter expression is largely restricted to the PCL (black arrowheads), with few β-gal+ cells observed in other layers. Double immunofluorescence experiments confirmed this localisation of β-gal+ cells (C–F). Double immunofluorescence for Pax2 (C), NeuN (D) and Calbindin (E) show the β-gal+ cells (white arrows) located in the PCL do not express markers of interneurons, granule neurons or PCs respectively. (F) Colocalisation with glial marker s100β confirms the identity of these cells as Bergmann glia (white arrows). (A–B are counterstained with hematoxylin and C–F with Topro3. Scale bars: A = 500 µm, B = 100 µm, C–F = 50 µm).

Figure 6. Summary of Wntβ-catenin signalling during cerebellum development.

Wnt/β-catenin signalling is present in a dynamic spatio-temporal specific pattern in the developing cerebellum. Initially it is observed at the cerebellar rhombic lip but by E18.5 its expression expands into a more widespread pattern with particularly strong expression at the VZ during the birth of glia and interneurons. During postnatal development it is largely restricted to the PCL, consistent with a subpopulation of Bergmann glia.