Activation of Wnt/β-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of ex vivo cerebellar slice culture and in vivo genetic manipulation to dysregulate its activity during late embryonic development. Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis

Wnt/β-catenin Signalling Is Active in a Highly Dynamic Pattern during Development of the Mouse Cerebellum

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

Effects of <i>in vivo</i> activation of Wnt/β-catenin signalling on development of VZ-derived cells.

<p><i>Apc^lox/+</i> and <i>Apc^lox/lox</i> embryos were electroporated with a <i>Cre-GFP</i> plasmid at E13.5 and analysed at E18.5. Electroporated cells and their progeny, marked by expression of GFP, were examined for expression of Sox9 (A–C), Pax2 (D–F) and PCNA (G–I). Immunohistochemical analyses were quantitated by counting the number of GFP+ cells expressing each marker (white arrows) as a proportion of total GFP+ cell numbers per section and comparing by Student's T-test between the two genotypes for each marker. For each test, n = 4, error bars = SEM, * = p<0.05, ** = p<0.01, *** = p<0.001. Scale bar = 25 µm.</p

BAT-gal expression in the P5 cerebellum.

<p>(A) DAB immunohistochemistry for β-gal and (B) <i>LacZ</i> 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).</p

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

<p>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 <i>LacZ</i> 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).</p

Wnt/β-catenin pathway activation <i>ex vivo</i> is mitogenic but has no affect on apoptosis.

<p>E18.5 cerebellum slices were cultured in the presence of DMSO (A,D), 20 µM BIO (B,E) or 50 µM CHIR (C,F) and analysed by immunohistochemistry for the retention of a BrdU label administered two hours prior to fixation (A–C) or apoptotic marker Caspase3 (D–F). (G) Graph showing proportion of BrdU+ cells in specific areas of cultured slices. (H) Graph showing density of Caspase3 labelled cells per unit area in specific regions of cultured slices. In all cases statistical comparisons were made by comparing each treatment group with the appropriate DMSO control using a Student's T-test. For each test, n = 3, error bars = SEM, * = p<0.05, ** = p<0.01, *** = p<0.001. All sections counterstained with TOPRO3. Scale bar = 100 µm.</p

Genetic activation of Wnt/β-catenin signalling in VZ-derived cells by <i>in utero</i> electroporation.

<p>(A) Schematic illustration showing <i>in utero</i> electroporation technique. E13.5 <i>Apc^lox/lox</i> or <i>Apc^lox/+</i> embryos were injected <i>in utero</i> with a <i>Cre-GFP</i> plasmid delivered to the fourth ventricle and electroporated. (B) GFP expression in an electroporated embryo at E14.5, revealing extensive expression along the VZ. (C, D) By E18.5 GFP expression was widespread throughout the developing cerebellum of both <i>Apc^lox/+</i>(C) and <i>Apc^lox/lox</i> (D) embryos. (E–G) High magnification analysis of GFP+ cells revealed that majority of them also displayed ectopic nuclear accumulation of β-catenin (white arrowheads) compared to non-GFP cells (empty arrowheads). All sections were counterstained with TOPRO3. CP = choroid plexus, DHB = dorsal hindbrain, EGL = external granule layer, IV = fourth ventricle, URL = upper rhombic lip, VZ = ventricular zone. Scale bar = 100 µm (A–C), 10 µm (D–F).</p

Wnt/β-catenin pathway activation <i>ex vivo</i> affects expression of VZ-derived lineage markers.

<p>(A–I) E18.5 cerebellum slices were cultured in the presence of DMSO (A,D,G), 20 µM BIO (B,E,H) OR 50 µM CHIR (C,F,I) and analysed by immunohistochemistry for the expression of Sox9 (A–C), GFAP (D–F), Pax2 (G–I) and Calbindin (J–L). (M) Graph showing the proportion of Sox9+ cells in specific regions of cultured slices. (N,O) qRT-PCR analysis of expression of <i>Sox9</i> (N) and <i>Gfap</i> (O) expression, relative to EIF4A2 control. (P) Graph showing the proportion of Pax2+ cells in specific regions of cultured slices. (Q) Graph showing the proportion of Calbindin+ cells in the dorsal region of each slice. In all cases, direct comparisons were made by comparing each treatment group with the relevant DMSO control with a Student's T-test. For each test, n = 3, error bars = SEM, * = p<0.05, ** = p<0.01, *** = p<0.001. All sections counterstained with TOPRO3. Scale bar = 100 µm.</p

BAT-gal expression in the P10 cerebellum.

<p>(A) DAB β-gal immunohistochemistry and (B) <i>LacZ</i> 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).</p

Summary of Wntβ-catenin signalling during cerebellum development.

<p>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.</p