Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum
Takao Yamasaki1,2,
Kousuke Kawaji1,2,
Katsuhiko Ono3,
Haruhiko Bito4,5,
Tomoo Hirano1,2,
Noriko Osumi2,6 and
Mineko Kengaku1,2,*
1
Department of Biophysics, Graduate School of Science, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
2
CREST, Japan Science and Technology Corporation, Kawaguchi 332-0012,
Japan
3
Department of Anatomy, Shimane Medical University, Izumo 693-8501
4
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto
606-8315
5
TOREST, Japan Science and Technology Corporation, Kawaguchi 332-0012,
Japan
6
Department of Developmental Neurobiology, Tohoku University Graduate School of
Medicine, Sendai 980-8575, Japan
*
Author for correspondence (e-mail:
kengaku{at}nb.biophys.kyoto-u.ac.jp
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Fig. 1. Disrupted formation of the EGL and the parallel fibers in Pax6
mutant (rSey2/rSey2) cerebella. (A,B)
HE-stained cross-sections of E21 cerebella. The wild-type EGL (A) is
subdivided in the outer germinal layer (g) and the inner premigratory layer
(m). Spindle-shaped cells (arrows) in the premigratory layer are presumably
migrating along the young parallel fibers. Pax6 mutant EGL (B) is
thicker and uniform accumulation of round cells is observed. (C,D) DiI-labeled
parallel fibers. Horizontal beams of parallel fibers in the deep EGL of the
wild-type cerebellum (C) are not formed in the Pax6 mutant (D). Pial
surface of the cerebella is indicated by asterisks. (E,F) Expression of TAG-1
in E21 cerebella. TAG-1 signal delineates the boundary between the EGL and the
molecular layer in the wild-type cerebellum (E). TAG-1 is diffusely expressed
throughout the EGL in the Pax6 mutant cerebellum (F). Some background
autofluorescence can be observed in the vascular cells on the pial surface of
the EGL. Scale bars: 25 µm in A-D; 40 µm in E,F.
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Fig. 2. Pax6 expression in differentiating granule cells during
development. (A-D) In situ hybridization with a Pax6 probe was performed on
sagittal cryosections of developing rat cerebella of E20 and postnatal days 5,
10 and 16. Pax6 is continuously expressed in developing granule cells
in the EGL and the IGL. (E) Higher magnification of P10 cerebellum. Expression
is stronger in the EGL than in the IGL. Some cells in the ML also express
Pax6 mRNA (arrows). (F) Immunohistochemistry of P10 cerebellum for
Pax6 protein reveals intense expression in the EGL and in migrating granule
cells with fusiform nuclei in the ML (arrows); expression in the IGL is
relatively weak. Scale bars: 250 µm in A-D; 25 µm in E,F.
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Fig. 3. Abnormal morphology and migration of granule cells in microexplant cultures
of the Pax6 mutant (rSey2/rSey2) EGL.
(A,B) Transmission electron micrographs of the EGL cells in culture (3 DIV).
Spindle-shaped granule cell has long, thick leading process associated with
preformed neurites of other cells in the wild-type culture (A). Pax6
mutant cells are round in morphology and form short thin processes attached to
(arrow) or detached from (arrowhead) neighboring cells (B). (C,D)
Phase-contrast light micrographs of EGL cells migrating from the explant (2
DIV). Colored lines are trajectories of the granule cells traced for 8 hours.
While wild-type cells extend long radial neurites and migrate along the fibers
(C), Pax6 mutant cells lack long neurites and move randomly (D).
(E,F) Pax6 expression. (G,H) TAG-1 expression. Wild-type cells extend
TAG-1-positive long neurites (G), while mutant cells express TAG-1 around
their cell bodies and in the short neurites (H). (I,J) MAP2 expression
demarcates the leading processes of migrating neurons. (K,L) Differentiation
of parallel fibers is shown by 440 kDa AnkyrinB-positive staining
in wild-type cells (K). Pax6 mutant cells express AnkyrinB
in irregularly aligned processes (L). Cultures were maintained for 40 hours
(E-J) and 72 hours (K,L). Scale bars: 10 µm in A,B; 50 µm in C-L.
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Fig. 4. Abnormal elongation and migration of Pax6 mutant
(rSey2/rSey2) EGL cells co-cultured with
wild-type cells. (A) PKH26-labeled wild-type and Pax6 mutant EGL
cells from E21 embryos were mixed with P2 wild-type EGL cells at a ratio of
1:20 and cultured in cellular reaggregates. (B,D) Wild-type cells elongate
long axons (B), which occasionally display T-shaped morphology (D). (C,E)
Mutant cells rarely extend long neurites (C), often forming randomly
misoriented dwarfed processes (E). Cultures were maintained for 40 hours (B,C)
and 72 hours (D,E), respectively. Scale bars: 40 µm in B,C; 20 µm in
D,E.
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Fig. 5. Cell-autonomous effect of Pax6 on elongation of bipolar parallel
fibers. EGL cells in microexplant cultures were transfected with either GFP
cDNA alone (wt, rSey2) or GFP with Pax6 or Pax6-EnR cDNAs
(rSey2+Pax6, wt+Pax6-EnR). (A) Morphology of granule cells shown by
GFP fluorescence (green) together with immunofluorescence with Zic1 (red) and
Pax6 (blue). Scale bar: 20 µm. (B) The length (left) and the number (right)
of neurites quantified in GFP expressing cells. Each column in the right graph
represents the number of neurites as indicated.
(*P<0.001, **P<0.0001, in
comparison with the wild-type GFP control; ***P<0.07 in
comparison with the mutant GFP control).
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Fig. 6. Altered morphology and behavior of EGL cells transduced by dominant
negative Pax6 virus. (A) Viral constructs used for expressing Pax6-EnR and
EnR. Infected cells express a marker gene, alkaline phosphatase (AP), through
an IRES sequence. HD, homeodomain; PD, paired domain. (B,D) Cells infected
with EnR viruses appear in the IGL and display a granule cell morphology with
small, round soma, parallel fibers and claw-like dendrites. (C,E) The EGL
cells infected with Pax6-EnR viruses fail to migrate and cluster around the
injection site. Neither parallel fibers nor mature granule cells in the IGL
are observed. Scale bars: 40 µm in B,C; 20 mm in D,E. (F) The percentages
of infected cells found in each layer.
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Fig. 7. Aberrant formation of cytoskeletal structures in Pax6 mutant
(rSey2/rSey2) EGL cells. (A,B) PKH26-labeled
cells emigrating from a microexplant of E21 EGL observed at 1 DIV. Wild-type
cells (A) have long straight neurites, while mutant cells (B) protrude short
neurites, occasionally bifurcated and/or extruding lamellipodia in the midst
of the shafts (arrow). (C,D) Scanning electron micrographs of microexplant
cultures (3 DIV). Wild-type cells (C) possess thin growth cones at the tip,
most of which simply taper off. Pax6 mutant cells (D) expand large
growth cones with massive lamellipodia. (E,F) Distribution of F-actin (green)
and microtubules (red) in growth cones. In comparison with wild type (E), an
excess amount of F-actin accumulates in the large growth cones of
Pax6 mutant EGL cells, accompanied by invasion of microtubules
(F).
(G) Quantification of growth cone size. Live cells labeled with PKH26 were
observed at 1 DIV. The growth cone area was measured as the number of pixels
in the graphic image. Data are indicated as mean±s.e.m.
(P<0.005). Scale bars: 10 µm.
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Fig. 8. Functional interaction of Pax6 with the Rho/ROCK pathway in
axonogenesis of the granule cell. (A-F) Morphology of neurons from wild-type
(A-C) and Pax6 mutant (D-F) EGLs. Dissociated EGL cells were labeled
with PKH26 and cultured at a low density for 20 hours. Most wild-type cells
(A) have bipolar processes (arrows), whereas excess neurites are present in
Pax6 mutant cells (D) as well as Y-27632-treated cells (B)
(arrowheads). See text for details. Scale bar: 20 µm. (G,H) Quantitative
analyses of the number (G) and length (H) of axons induced by
Y-27632-treatment or by C3 misexpression. Data are indicated as
mean±s.e.m. P<0.001 in comparison with the respective
untreated controls for wild-type cells (white bars) and mutant cells (gray
bars).
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