First published online 30 August 2006
doi: 10.1242/dev.02556
Development 133, 3929-3937 (2006)
Published by The Company of Biologists 2006
Loss of cyclin D1 impairs cerebellar development and suppresses medulloblastoma formation
Jennifer Pogoriler1,
Kathleen Millen2,
Manuel Utset3 and
Wei Du1,*
1 Ben May Institute for Cancer Research, The University of Chicago, 924 E. 57th
Street, Chicago, IL 60637, USA.
2 Department of Human Genetics, The University of Chicago, 924 E. 57th Street,
Chicago, IL 60637, USA.
3 Department of Pathology, The University of Chicago, 924 E. 57th Street,
Chicago, IL 60637, USA.
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Fig. 1. Cyclin D1 (brown) is expressed at high levels in the EGL during early
cerebellar development. (A-D) The E16.5 cerebellum (A, with
high-power views of boxed areas shown in B-D) expresses cyclin D1 strongly in
the ventricular region (B) and anterior EGL (C), with fewer positive cells in
the posterior EGL (D). (E-H) The P0 cerebellum (E, with high-power
views of boxed areas shown in F-H) shows some remaining ventricular expression
(F), and strong expression in the EGL extending much further posteriorly with
enlarged anterior (G) and posterior (H) views of the EGL, although the extreme
posterior still has fewer positive cells (H). (I,J) P6
cerebellum (I, with high-power of view of boxed area in J) shows strong
staining in the outer EGL throughout the entire AP axis. (K,L)
At P12, only background staining of blood vessels and meninges is seen
(arrows) (K, with high-power of view of boxed area in L). Brackets indicate
the width of the EGL. Nuclei are lightly stained purple with Hematoxylin and
Eosin. Scale bars: 50 µm.
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Fig. 2. Cyclin D2 (brown) is not expressed at high levels in the EGL until later
in cerebellar development. (A-H) The E16.5 (A, with high-power
views of boxed areas shown in B-D) and P0 (E, with high-power views of boxed
areas shown in F-H) EGL show little nuclear cyclin D2 staining. Enlarged views
of the ventricular region (B,F), anterior EGL (C,G) and posterior EGL (D,H)
are shown. Only a few nuclei in the posterior EGL (arrow in D) are weakly
positive, in comparison with strong staining of the ventricular zone of the
midbrain and of cells deeper within the cerebellum (arrowheads in C,G).
(I,J) Strong cyclin D2 expression is seen in the outer half to
two-thirds of the EGL at P6 (I, with high-power of view of boxed area in J).
(K,L) Scattered EGL cells are positive (arrows) at P12 (K, with
high-power of view of boxed area in L). Brackets indicate the width of the
EGL. Nuclei are lightly stained purple with Hematoxylin and Eosin. Scale bars:
50 µm.
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Fig. 3. Ccnd1-/- cerebella show a postnatal size defect.
The midline cross-sections of wild-type littermates (A-C) are shown
next to Ccnd1-/- (cycD1 on figure) pups
(D-F). At P0 there is no difference in cerebellar size (A,D), but by P6
Ccnd1-/- cerebella are markedly smaller (B,E). The
relative size difference is maintained but not increased by P12 (C,F).
(G) Quantification of the relative cerebellar midline area. (H)
Total body weight of Ccnd1-/- pups continued to fall
further behind wild-type littermates from P6 to P12. (I) The size of
the granule cell population was similarly decreased at P6 and P12. Scale bars:
500 µm. The number of littermates analyzed at each time point is indicated
in each chart.
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Fig. 4. Proliferation is decreased in Ccnd1-/- cerebella
during early development. (A-H) Representative BrdU (A-D) and H3-P
(E-H) immunohistochemistry of cerebella from P0 (A,B,E,F) and P6 (C,D,G,H)
pups sacrificed 1 hour after BrdU injection. (I) Quantification of BrdU
incorporation in the anterior EGL of wild-type and
Ccnd1-/- (cycD1 on figure) siblings at P0
(n=7) and P6 (n=4). *P 0.03 when
comparing between +/+ and -/- littermates. (J) Quantification of H3-P
positive nuclei in the EGL at P0 (n=8, P=0.07) and P6
(n=4, P=0.22) shows a trend of decreased mitotic cells in P0
but not in P6 GNPs in the mutant.
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Fig. 5. Purkinje cell dendritic development is abnormal in
Ccnd1-/- mice at P9. Comparison of wild-type
(A,C) and Ccnd1-/- (B,D)
Purkinje cells stained with anti-calbindin antibody shows that at P9 (A,B),
Ccnd1-/- Purkinje cell dendrites are abnormally oriented and
stunted, but by 4 weeks (C,D) they look normal. Scale bars: 50 µm in A,B;
100 µm in C,D.
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Fig. 6. Medulloblastoma development is significantly decreased in
Ptch1+/-;Ccnd1-/- mice
compared with Ptch1+/- mice, despite upregulation of both
cyclins D1 and D2 in tumors. Tumors were identified by staining with X-gal
(A,B): in the normal cerebellum (A), the staining is strongest
in the Purkinje cell layer and inner granule layer, but medulloblastomas (B)
were easily identified by their disruption of this pattern resulting in
staining throughout the tumor, including at the surface of the brain.
(C) Medulloblastomas were also identified histologically. (D-G)
Immunohistochemistry of Ptch1+/- medulloblastomas. (D) In
contrast to mature granule cells of the IGL, tumor cells are only weakly
positive for the postmitotic marker NeuN. Expression of both cyclins D1 (E)
and D2 (G), as well as BrdU incorporation (F) in the tumor but not in adjacent
IGL or molecular layers. (H)
Ptch1+/-;Ccnd1-/- (cycD1 on figure)
mice develop significantly fewer medulloblastomas than do
Ptch1+/- mice P=0.0105. (I,J) The
medulloblastoma that developed in
Ptch1+/-;Ccnd1-/- background showed
similar patterns of BrdU incorporation (I) and cyclin D2 (J) expression. Mb,
medulloblastoma; ML, molecular layer; IGL, inner granule layer. Scale bars:
500 µm in C; 100 µm in D-G,I,J.
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Fig. 7. Ptch1+/-;Ccnd1-/- mice develop
fewer preneoplastic lesions. (A-E) preneoplastic lesions (indicated
by broken lines) express both cyclins D1 (D) and D2 (E) and incorporate BrdU
(C). (A,B) Some cells in these lesions, particularly in BrdU-negative regions,
are weakly NeuN positive. The density of NeuN positive cells is increased in
the molecular layer below the lesion (A,B) when compared with below the mature
cluster (F,G). (F-J) Clusters of mature ectopic granule cells
(indicated by broken lines) do not incorporate BrdU (H) or express cyclins D1
(I) or D2 (J), and all cells are strongly positive for the post-mitotic neural
marker NeuN (F,G). Scale bars: 100 µm. (K) There is a significant
decrease in the number of preneoplastic lesions in
Ptch1+/-;Ccnd1-/- (cycD1 on figure)
mice when compared with Ptch1+/- mice
(*P=0.021), and a smaller total volume of preneoplastic
lesions (**P=0.020), but no significant difference in the
number of mature ectopic clusters seen in older adults. ML, molecular layer;
IGL, inner granule layer.
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