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doi: 10.1242/10.1242/dev.00553

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Rb and p107 are required for normal cerebellar development and granule cell survival but not for Purkinje cell persistence

¹ Institute of Clinical Pathology, University Hospital, Schmelzbergstrasse 12, 8091 Zürich, Switzerland
² Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands
³ Institute of Neurology, Queen Square, London WC1N 3BG, UK

View larger version (70K): [in a new window]   Fig. 5. Proliferation and apoptosis in cerebella lacking Rb or Rb/p107 at P15. Proliferation (assessed by incorporation of BrdU) in the EGL (A) and IGL (C) is increased in the Rb- and p107-deficient vermis, but is counterweighted by an increased apoptosis rate in the vermis deficient of Rb and Rb/p107 (B,D). Proliferation and apoptosis were most pronounced if both Rb and p107 alleles were lost. Asterisks indicate that the value is significantly different from control value (P<0.01 for all values). (D) There is no statistically significant difference between En2cre; RbLoxP/LoxP; p107+/- (purple bar) and En2cre; RbLoxP/LoxP (red bar). Standard deviations (±1 s.d.) are indicated by error bars. (E,H) BrdU labeling of P15 wild-type (E; arrows indicate proliferating cells in the EGL) and Rb/p107 double mutant (H) illustrated the markedly increased proliferation in the broadened EGL and in the IGL. The cell layers in the double mutant (H) are indicated (EGL, external granular layer; PCL, Purkinje cell layer; IGL internal granular layer). Red circles indicate the inner border of the presumed IGL, which would normally form the cerebellar white matter. The cell cycle inhibitor p27 is expressed in cells of the inner EGL, in most migrating and IGL neurons (F). The outer p27-negative EGL region is broadened in Rb/p107 double mutant cells (red arrow in F indicates the layer that is one or two cells thick; arrows in I indicate the broadening of the p27-negative outer EGL). Likewise, a substantial proportion of migrating and IGL neurons are p27 negative (arrows, I). As indicated by the graphs (B,D), there is also significantly increased apoptosis in double mutant (J) compared with wild-type (G) vermis. Arrow in G indicates a single TUNEL-positive cell. Scale bar: 100 µm.

View larger version (48K): [in a new window]   Fig. 1. Cerebellar phenotype of Rb and Rb/p107 compound mutant mice at P15. Gross appearance of the cerebellum of wild type (A) and mutant (B-D) mice at postnatal day 15. Note the reduced size of the double mutant vermis. (E-H) Hematoxylin and Eosin stained median sagittal sections through the cerebellum of En2cre; RbLoxP/LoxP mice (n=3) (F) show preserved foliation, but slight reduction of the vermis size and retarded migration of granule cells (inset, arrows indicate EGL thickness), when compared with a wild-type littermate (n=3) (E). A similar, but more pronounced phenotype was observed in the cerebella of Rb/p107 double mutant mice (p107+/-; n=9, p107-/-; n=3) (G,H). Scale bar: 1 mm (100 µm in insets).

View larger version (139K): [in a new window]   Fig. 2. Analysis of the cerebellar phenotype of Rb and Rb/p107 compound mutant mice. Sagittal sections of wild-type (A-E) (n=3), Rb (F-J) (n=3) and Rb/p107 compound mutant (K-T) (p107+/-; n=3, p107-/-; n=4) cerebella at P20. NeuN immunostaining (A,F,K,P) reveals marked loss of granule cells in the Rb-deficient vermis (F), while additional loss of one or both p107 alleles results in impaired terminal differentiation and in almost complete granule cells loss (K,P). Aberrantly migrating neurons can still be detected by immunostaining for TuJ1 (B,G,L,Q). Inset in B shows expression of TuJ1 in the inner postmitotic, pre-migratory cell population (arrowheads), but not in the outer layers of the EGL in a P8 control mouse. Circles indicate the outer margin of the cerebellar folium. In L,Q, there is still a TuJ1-expressing cell population in the EGL present. Inset in Q shows a higher magnification of TuJ1-expressing cells above the level of dispersed Purkinje cells. Calbindin-positive Purkinje cells (C,H,M,R) are increasingly disarranged with dystrophic dendrites in Rb-deficient (H) and in Rb/p107 double mutants (M,R). Likewise, parvalbumin antiserum stains Purkinje cells (D,I,N,S) but also reveals a loss of interneurons of the molecular layer (arrowheads in D,I). Dystrophic Bergmann glia and an increasing astrogliosis are highlighted by GFAP immunostaining (E,J,O,T). (U-X) Representative drawings of midsagittally cut cerebella with indication of the areas (red square) shown at high magnification in A-T. Scale bar: 100 µm in A-T.

View larger version (114K): [in a new window]   Fig. 3. Recombination analysis in the vermis of En2cre; RbLoxP/LoxP mice. (A,B) Whole-mount ß-galactosidase staining of the vermis of En2cre, ROSA26LoxP indicator mice shows the area of recombination from outside (A) and after sagittal sectioning (B). (C) Histology of an area corresponding to the red square in B (Nuclear Red counterstaining; EGL, external granular layer; PC, Purkinje cells; IGL, internal granular layer), demonstrating the lacZ expression in all areas of the cerebellum. Adjacent sections were immunostained for calbindin (D) or NeuN (E) to confirm the En2cre-mediated recombination in these cell populations. (F-I) PCR analysis of cell fractions obtained from Percoll gradients. Fraction 1 contains larger cells (astrocytes, GFAP immunostaining in F; Purkinje cells, Hematoxylin and Eosin staining in G), while Fraction 2 contains small cells, such as granule cells and granule cell precursors (Hematoxylin and Eosin staining in H). (I) PCR recombination analysis of both fractions shows a 283 bp product representing the floxed allele (primers Rb19E and Rb18) and a 260 bp product of recombined Rb allele (primers Rb212 and Rb18). Genomic DNA extracted from Percoll-separated cerebellar fractions of RbLoxP/LoxP and En2cre;RbLoxP/LoxP mice were used in lanes 3,4 and 5,6, respectively. Lanes 3 and 5, larger size fraction 1. Lanes 4 and 6, lower size fraction 2. Two controls with partial recombination are shown in lanes 1 and 2.

View larger version (132K): [in a new window]   Fig. 4. Purkinje cell-specific inactivation of Rb and p107 at P30. Purkinje cells are enlarged, show bizarre nuclei, and are occasionally displaced into the molecular layer (D,G) when compared with control (A). Calbindin staining shows a largely intact dendritic arborization in the molecular layer (E,H), similar to wild-type cerebella (B). The cell cycle inhibitor p27 is accumulated in the nucleus of most Rb- or Rb/p107-deficient Purkinje cells (F,I), when compared with wild-type cells (C). Controls, n=2; L7cre;RbLoxP/LoxP, n=3; L7cre;Rb LoxP/LoxP;p107-/-, n=3. Scale bar: 100 µm in A,B,D,E,G,H; 200 µm in C,F,I.

View larger version (91K): [in a new window]   Fig. 6. Lack of p53 does not rescue cerebellar neurons deficient for Rb and p107. TUNEL staining of control (A), En2cre; RbLoxP/LoxP; p107+/- (B) and En2cre; RbLoxP/LoxP; p107+/-; p53-/- (C) cerebella at postnatal day 15 shows no difference in the extent of apoptosis upon loss of p53. The cell layers in the double mutant (B,C) are indicated (EGL, external granular layer; PCL, Purkinje cell layer; IGL internal granular layer). Broken red lines indicate the inner border of the presumed IGL, which would normally form the cerebellar white matter. The cerebellum of a wild-type littermate is shown on the left as a control (A). Scale bar: 50 µm.