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
Accepted 14 May 2001
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SUMMARY |
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Key words: Pax6, Cerebellum, Cell polarization, Granule cell, Parallel fiber, Cytoskeleton
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INTRODUCTION |
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A variety of molecules are known to act during axon formation and migration
(Goodman and Shatz, 1993;
Goodhill, 1998
). These include
long-range and short-range guidance molecules, signaling molecules that
interpret and decode these cues, and cytoskeletal components that regulate
cell morphology. Several signaling molecules, including ROCK (Bito et al.,
2000
), Unc51.1 (Tomoda et al.,
1999
) and DDR1 (Bhatt et al.,
2000
), have been implicated in
the early steps of axonogenesis of granule cells in vitro. ROCK is a
serine/threonine kinase activated downstream of Rho GTPase. Intensive studies
have elucidated the importance of regulation by Rho family small GTPases in
cytoskeletal reorganization during neuronal morphogenesis and migration (Luo
et al., 1996
; Threadgill et
al., 1997
; Zipkin et al.,
1997
; Steven et al.,
1998
; Albertinazzi et al.,
1999
; Nakayama et al.,
2000
). Inhibition of ROCK
activity in cultured granule cells triggers excessive sprouting of axons,
suggesting that the Rho/ROCK pathway negatively controls axon outgrowth
probably through organizing actin dynamics, especially during the initial step
of polarity induction (Bradke and Dotti,
1999
; Bito et al.,
2000
). However, Unc51.1 and
DDR1 have been shown to act as positive regulators of axon outgrowth as
revealed by dominant-negative inhibition of these molecules (Tomoda et al.,
1999
; Bhatt et al.,
2000
). It remains elusive,
however, how the activities of these molecules are orchestrated during
cellular morphogenesis of the granule cell.
Differentiation of granule cells is thought to depend on both genetic
programs intrinsic to the granule cells and epigenetic influences of adjacent
siblings as well as of other cell types in the developing cerebellum (Trenkner
et al., 1984; Dahmane and
Ruiz-i-Altaba, 1999
;
Wechsler-Reya and Scott,
1999
). Studies using culture
systems have demonstrated that granule cells autonomously develop polarity to
form parallel fibers in the absence of spatial cues (Nagata and Nakatsuji,
1991
; Powell et al.,
1997
). One candidate molecule
implicated in such an intrinsic mechanism is a paired domain-containing
homeobox gene, Pax6. In the Pax6 mutant mouse, small eye
(Sey/Sey), the EGL is malformed and the horizontal array of newly
forming parallel fibers is disorganized (Engelkamp et al.,
1999
). Besides this anomaly in
the EGL, multiple neurological defects are also seen in the mutant cerebellum,
including attenuated foliation, misplacement of a subset of EGL cells and
deformity in the vermis-forming territory (Engelkamp et al.,
1999
). It remains unknown
which of these defects are direct or indirect consequences of lack of
Pax6 function.
Pax6 is known as a morphogenetic gene with a myriad of activities
in patterning (Stoykova et al.,
2000; Toresson et al.,
2000
; Yun et al.,
2001
) and cell-type
specification (Burrill et al.,
1997
; Ericson et al.,
1997
; Osumi et al.,
1997
) during early development
of the CNS. It has also been shown that Pax6 is involved in various
aspects of axon pathfinding and migration of CNS neurons. These include radial
migration of cortical neurons (Schmahl et al.,
1993
; Caric et al.,
1997
), formation of
thalamo-cortical projections (Kawano et al.,
1999
; Pratt et al.,
2000
), and formation of
longitudinal and medial-ventral tracts in the forebrain (Mastick et al.,
1997
; Vitalis et al.,
2000
). However, disruption of
these processes in the Pax6 mutant CNS appears to be an indirect
consequence of the loss of a prerequisite role of Pax6 in conferring
regional identity to the neurons, which either express Pax6 or
interact with Pax6- expressing cells. In the forebrain, for example,
Pax6 expression defines the midbrain-forebrain boundary and provides
local guidance information to the post-optic commissure axons (Mastick et al.,
1997
).
In this study, we present evidence for a novel role for Pax6 as a critical regulator in cell polarization in the cerebellar granule cell. Pax6 mutation caused severe deficits in initial polarization of axons and the cytoskeletal reorganization in growth cones. Furthermore, cell body migration was aberrant in Pax6 mutant granule cells. These phenotypes appeared to be due to the absence of intrinsic activity of Pax6 in the granule cell. It is thus suggested that Pax6-mediated transcriptional control is essential to achieve proper granule cell polarization during parallel fiber formation both in vivo and in vitro.
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MATERIALS AND METHODS |
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Microexplant and reaggregate cultures
Microexplant cultures of wild-type and
rSey2/rSey2EGL at E21 were prepared as
described by Nagata and Nakatsuji (Nagata and Nakatsuji,
1991). Reaggregate cultures
were basically performed as described (Nagata and Nakatsuji,
1994
). Briefly, the EGLs from
E21 wild-type and rSey2/rSey2 embryos were
dissociated, and fractions containing small neurons were collected and labeled
with PKH26 (Sigma). Unlabeled host cells were prepared from P2 EGL and mixed
with labeled cells at a ratio of 20:1. The cell mixture was centrifuged in an
Eppendorf tube and incubated for 1 hour to make reaggregates that were then
cut into 300-400 µm pieces and placed on culture dishes coated with
poly-D-lysine/laminin. Cultures were maintained at 37°C in 5%
CO2 and analyzed after 1-3 days.
Immunohistochemistry and fluorescent microscopy
Cultures and fresh-frozen cryosections (10 µm) were fixed with 4%
paraformaldehyde (PFA) for 15 minutes at room temperature. Primary and
secondary antibodies used for staining were as follows: mouse monoclonal
antibody (mAb) against TAG-1 (4D7; 1:4, DSHB); mAb against Zic1 (1:50, kindly
provided by J. Aruga); mAb against MAP2 (1:200, Sigma); mAb against Tau1
(1:200, Boehringer Manheim); mAb against -tubulin (1:200, Sigma);
rabbit polyclonal antibody (pAb) against Pax6 (Inoue et al.,
2000
); pAb against 440-kD
AnkyrinB (1:200, kindly provided by M. Kunimoto); and goat
anti-rabbit or anti-mouse secondary antibodies conjugated with FITC, AMCA
(Chemicon) or Alexa568 (Molecular Probes). F-actin was stained with Oregon
Green 488-phalloidin (1:100, Molecular Probes). After washing, slides were
mounted in a glycerol-based medium SlowFade (Molecular Probes) and analyzed
using a Nikon E800 microscope with x20 and x40 PlanFluor
objectives.
For quantitative analysis of the number and length of axons, the trace from the contour limit of the cell soma to the tip of the major process of the neuron was defined as its axon length. Processes longer than 3 µm emerging from the soma, or those longer than 10 µm bifurcating from the shaft were counted as axons. To quantify the relative size of the growth cones, live cells labeled with PKH26 were kept at 37°C and observed using water-immersed 40x Fluor objective (0.80W). The size of growth cones was measured using the graphic image of IpLab (Scananalytic).
Dil labeling of EGL cells
E21 cerebella fixed with 4% paraformaldehyde (PFA) were embedded in 3% agar
and cut coronally at a thickness of 200 µm using a vibratome. Small
crystals of the lipophilic dye DiI were placed on the EGL and left for 10 days
at 4°C to allow diffusion of the dye. After washing away the crystals,
sections were mounted and analyzed using a fluorescent microscope.
Transmission and scanning electron microscopies (TEM and SEM)
Cultured cells were fixed with 4% PFA and 5% glutaraldehyde in phosphate
buffer (PB; 0.1M, pH 7.4) at room temperature overnight. They were washed with
PB and post-fixed with 1% osmium tetroxide in PB for 1 hour. For TEM, the
samples were washed well with distilled water (DW) and stained en bloc with 1%
uranyl acetate for 1 hour. They were then dehydrated through a graded ethanol
series, cleared with hydroxypropyl metacrylate and embedded in Epon 812. They
were cut with a ultramicrotome and observed under an TEM (JEM-1200EX, JEOL; or
EM-002B, Topcon, Japan) at an accelerating voltage of 80 kV. For SEM, the
samples washed with DW were subsequently immersed in 2% tannic acid for 1
hour, and then 1% osmium tetroxide in DW for 1 hour (Katsumoto et al.,
1981). They were dehydrated
and immersed in t-butyl alcohol. The samples were dried at critical
point (JFD-300, JEOL, Japan), coated with platinum/palladium and observed
under an SEM (H-800, Hitachi, Japan) at an accelerating voltage of 15 kV.
In situ hybridization
In situ hybridization on cryosections was performed by a method described
previously (Lin and Cepko,
1998). The rat Pax6 cDNA used
as a probe was described previously (Matsuo et al.,
1993
).
Transfection of plasmid cDNA
Plasmid cDNAs used for transfection were as follows: mouse Pax6 and
Pax6-EnR cDNAs subcloned in a pCAX expression vector (full-length cDNA of
mouse Pax6 was kindly provided by P. Gruss); ROCK-Delta3 (Ishizaki et al.,
1997); C3-GFP (Watanabe et
al., 1999
); and EGFP-N1
(Clontech).
For expression in a microexplant culture, a modified calcium phosphate
procedure developed by A. Ghosh was used (Threadgill et al.,
1997). Transfections were
performed 6-12 hours after plating when emigrating cells were observed.
Plasmids used were either 2 µg GFP-N1 alone or a mixture of 0.8 µg
GFP-N1 and 2.4 µg plasmid of interest per well on multi-well plates
(Nunclon Multidish four wells, Nunc). Analysis was carried out 24-36 hours
after transfection. Under these conditions, usually 10-40 neurons expressed
GFP. The efficacy of co-transfection was >80%.
Transfections in low-density cultures of dissociated cells were performed
as previously described (Bito et al.,
2000). Small cerebellar neurons
were labeled with PKH26 and plated at 2x105 cells per well in
polylysine/laminincoated wells. Cells were fixed 20 hours after transfection
for fluorescent microscopy. Some cells were stained with F-actin or with
anti-Tau instead of PKH26, all of which gave basically the same results.
Construction and infection of recombinant retrovirus
The Pax6-EnR chimera was created by fusing the N-terminal domain of Pax6,
which contains two DNA binding sites (amino acids 3-306), with the Engrailed
repressor domain. Using PCR-based mutagenesis, NcoI and
EcoRI sites were introduced in the Pax6 cDNA at the first methionine
and at the amino acids 307-308, respectively. Primers used were as follows;
5'-GACTCGAGCCATGGAGAACAGTCACAGCGG-3',
5'-CTGATATCGACAGGTGTGGTGGGCTG-3'. The NcoI-EcoRV
fragment of the PCR product was ligated into a pBluescript IISK+. The insert
was cut with NcoI and EcoRI and then ligated into a
NcoI-EcoRI-digested EnR-pSLAX21 (kindly provided by T.
Furukawa). The entire Pax6-EnR fusion and EnR were cloned into a retroviral
vector pLIA as described by Furukawa et al. (Furukawa et al.,
1997). The viruses were
produced in a Phoenix cell line (generously provided by G. Nolan), and were
concentrated at 1x106-1x108 pfu/ml according
to the procedure described by Cepko (Cepko,
1998
).
To inject virus, neonatal ICR mice were anesthetized on ice. A Hamilton microliter syringe (Hamilton, Whitter, CA) was inserted through the skin and the skull to a position above the EGL and 1 µl of virus solution was injected over 2 minutes. The pups were revived at 36°C and returned to the litter. At 10 days post-transfection, infected cerebella were dissected, sectioned at 50 µm using vibratome and stained for AP.
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RESULTS |
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Pax6 is expressed in the granule cells during
development
To explore Pax6 activity during granule cell differentiation, we
examined Pax6 expression in developing rat cerebella. Pax6
transcripts were detected in the EGL throughout pre- and postnatal development
(Fig. 2A-D). At E20, intense
expression of Pax6 mRNA was observed in the EGL cells and in
dispersed cells in the cerebellar cortical primordia
(Fig. 2A). After P5,
Pax6 expression was seen in postmigratory granule cells in the
emerging IGL at reduced levels compared with premigratory EGL cells
(Fig. 2B-D; Stoykova and Gruss,
1994). Observation at higher
magnification revealed that a subset of cells in the molecular layer (ML) also
expressed Pax6 mRNA (arrows in
Fig. 2E). Expression of Pax6
protein showed basically the same profile
(Fig. 2F); immunoreactivity was
prominent in the EGL cells and declined in the IGL cells. Cells in the ML that
expressed Pax6 protein had fusiform nuclei, suggesting that they were granule
cells migrating from the EGL to the IGL. Thus, Pax6 mRNA and protein
are expressed in granule cells throughout the course of their differentiation
and migration.
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Pax6 mutant cells differentiate as granule cells but fail to
form bipolar axons and migrate in random orientation
The expression pattern of Pax6 in developing cerebella prompted us
to examine its potential role during neurite outgrowth and migration of
granule cells. As the homozygous mutants die at birth during the early phase
of granule cell differentiation, we performed microexplant cultures of E21 EGL
in which cell-autonomous migration and cellular morphogenesis of the granule
cells can be reconstituted (Nagata and Nakatsuji,
1991). By 1 day in culture (1
DIV), wild-type cells extruded long neurites radially and emigrated out from
the EGL explant. The radial neurites formed parallel bundles after 2 DIV. TEM
and time-lapse tracing showed that most neurons maintained close contact with
the bundles of preexisting long neurites and migrated along straight
trajectories (Fig. 3A,C). In
contrast, only a few radial fibers were seen in the Pax6 mutant EGL
culture. TEM revealed that many round cells failed to form long leading
processes in the mutant cultures (Fig.
3B). Active migration was observed, although the cells dispersed
randomly (Fig. 3D). The radial
migration of cultured granule cells has been shown to be dependent on laminin
substrate (Nagata and Nakatsuji,
1990
). We therefore checked
the expression of putative binding partners of laminin in granule cells, L1
and ß1-integrin. Both are expressed equally in wild-type and mutant
cells, precluding the possibility that the disorganized appearance of
Pax6 mutant cells was due to mere non-responsiveness to laminin
substrate (data not shown).
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The morphology and behavior of the mutant cells raised the possibility that
differentiation of the granule cells might be disturbed or delayed in the
absence of Pax6 expression. To test this, we examined the expression
of several neuronal and granule cell markers by immunofluorescence. We first
confirmed that 70-80% of emigrating cells from the wild-type and mutant
explants were granule cells and/or granule cell progenitors, as revealed by
Zic1 expression (data not shown; Aruga et al.,
1994). These cells strongly
expressed Pax6 protein in wild-type cultures, while no Pax6 antigen was
detectable in mutant cultures (Fig.
3E,F). A subset of cells that emigrated from the wild-type explant
expressed TAG-1, an early marker for parallel fiber axons, on their long
bipolar neurites (Fig. 3G). In
contrast, although equal numbers of Pax6 mutant cells showed strong
expression of TAG-1 around their cell bodies, extension of TAG-1-positive long
neurites was rarely seen (Fig.
3H). The expression of the microtubule-associated protein, MAP2,
was mostly confined to the leading processes at 2 DIV in both wild-type and
mutant explants (Ono et al.,
1994
). While wild-type cells
regularly aligned long leading processes distally to the explant, mutant cells
randomly misoriented short processes regardless of the orientation of other
cells (Fig. 3I,J). Pax6 mutant cells occasionally had multiple processes that expressed
MAP2, suggesting an abnormal control of the leading process formation. After 3
DIV, many cells in both the wild-type and the mutant cultures had ceased
expressing TAG-1, suggesting they had exited the early phase of granule cell
differentiation (data not shown). These cells indeed strongly expressed 440
kDA AnkyrinB, a specific marker for the parallel fiber axons
(Kunimoto, 1995
), together
with Tau 1, a general marker for axons
(Fig. 3K,L and data not shown).
In the wild-type culture, the massive radial neurites appeared to
differentiate into AnkyrinB-positive parallel fibers. Pax6
mutant cells also synthesized AnkyrinB but formed large meshes of
irregular neurites rather than organized parallel bundles. Little difference
was observed in the relative numbers of GFAP-positive glia, most of which were
found in close proximity to the explant, between the wild-type and the mutant
cultures (around 10%; data not shown). After 3 DIV, we observed apoptotic cell
death more frequently in mutant cultures, although it affected neither the
apparent density nor the total number of surviving cells (data not shown).
Taken together, these results suggest that Pax6 mutant cells can
recapitulate the typical cascade of gene expression during granule cell
differentiation, but fail in correctly polarizing their parallel fiber axons
and subsequently migrate in a disorganized manner.
Pax6 regulates morphogenesis and migration of granule cells
in a cell-autonomous manner.
The specific expression of Pax6 in developing granule cells
suggests that abnormal polarization of
rSey2/rSey2 granule cells might be a direct
consequence of loss of intrinsic Pax6 activity within the
differentiating granule cells. Alternatively, it may indicate that
Pax6 is involved in cell-cell interaction of granule cells with the
same and/or other cell types in the cerebellum. To distinguish between these
possibilities, we created a mixed culture of
rSey2/rSey2 mutant EGL cells with wild-type
cells. Cellular morphology was visualized by labeling cells with a fluorescent
membrane-soluble dye PKH26 (Fig.
4A). The control wild-type cells labeled with PKH26 extended long
neurites and migrated radially along the fibers
(Fig. 4B). We occasionally
observed cells with characteristic T-shaped axons resulting from vertical
reorientation (Fig. 4D). On the
other hand, Pax6 mutant cells rarely elongated long neurites and
scattered randomly in spite of the regular radial fibers formed by surrounding
wild-type cells (Fig. 4C,E).
These results suggest that the abnormal morphology and migration of
Pax6 mutant cells are cell-autonomous, as they could not be rescued
by co-culture with wild-type cells.
|
To assess whether Pax6 activity is sufficient to correct the anomalies of the mutant cells, we transfected Pax6 cDNA into mutant cells. The number and the length of neurites of Zic1-positive cells were quantified by co-transfecting green fluorescent protein (GFP) cDNA in explant cultures (Fig. 5). When GFP cDNA was transfected alone, the proportion of Zic1-positive cells with longer neurites was significantly higher in wild-type cells than in mutant cells. Notably, we found a considerable number of neurons with more than two neurites in mutant cultures. In contrast, expression of Pax6 in mutant cells restored wild-type phenotypes; most of the cells co-transfected with Pax6 and GFP cDNAs became bipolar and extended neurites to lengths comparable with wild-type cells.
|
These results strongly suggest that Pax6 functions intrinsically
in the granule cell upstream of the formation of long bipolar neurites and
orderly migration. To verify that Pax6 function is truly involved in
the genetic cascade of granule cell differentiation in normal developing
cerebellum, we misexpressed a dominant-negative form of Pax6 in
granule cell precursors in situ as well as in vitro. The DNA-binding domain of
Pax6 was fused to the repressor domain of Drosophila
Engrailed (EnR), producing a fusion protein that should block
transcriptional activation by intrinsic Pax6 (Furukawa et al.,
1997; Matsunaga et al.,
2000
). We first confirmed that
misexpression of the Pax6-EnR cDNA in the microexplant culture of
wild-type cells resulted in multipolar morphology similar to
rSey2/rSey2 cells
(Fig. 5). Thus, perturbation in
the maintenance of Pax6 function to activate transcription of the
downstream target gene(s) was sufficient to cause the morphological deficits
observed in Pax6 mutant EGL cells even within the wild-type genetic
background.
By utilizing a replication-incompetent retrovirus vector (pLIA), we next
misexpressed the dominant-negative gene in neonatal EGL cells and assayed
morphology and location of the infected cells after 10 days by staining for
the marker gene AP (Fig. 6). During postnatal development, granule cell precursors represent almost the
sole mitotic cells in the EGL infectable by retrovirus (Zhang and Goldman,
1996). Granule cell
progenitors infected with control vector viruses or those carrying Engrailed
repressor domain alone differentiated normally and correctly migrated to the
IGL (Fig. 6B,F). They elongated
bipolar parallel fibers in the molecular layer, formed glomeruli at the tip of
dendrites and adopted a T-shape morphology, as visualized by AP staining
(Fig. 6D). By contrast, most of
the cells infected with viruses transducing the Pax6-EnR transgene
remained in the EGL (Fig.
6C,F). Short, bushy processes were observed around their cell
bodies in the EGL and few or no parallel fibers were formed in the ML
(Fig. 6E). While descendant
cells carrying the control viruses spread out over a broad area, those
misexpressing Pax6-EnR tended to remain clustered around the injection site,
possibly owing to the failure in tangential migration of the EGL cells in the
absence of the leading process formation. It was also notable that a
significantly smaller number of infected cells were observed with Pax6-EnR
viruses, suggesting that Pax6 might also contribute towards
maintaining survival of granule cells during differentiation.
|
Taken together, these results indicate that Pax6 functions as an intrinsic factor required for elongation of bipolar axons and subsequent orderly migration of granule cells in the developing EGL.
Aberrant sprouting of axons accompanies enlargement of growth cones
in the Pax6 mutant granule cells
The next obvious question was which step of cellular morphogenesis and
migration was disturbed in the Pax6 mutant granule cells. As shown in
Fig. 3, Pax6 mutant
EGL cells expressed a series of differentiation markers, precluding the
possibility that the onset of molecular switches in granule cell
differentiation was simply retarded in the absence of Pax6 function.
Taking into account that Pax6 is a transcription factor, and that its action
is cell-autonomous in the granule cell, we hypothesized that Pax6 regulates
transcription of cytoplasmic and/or transmembrane protein(s) implicated in
cellular morphogenesis of granule cells during axon formation and
migration.
We noticed that Pax6 mutant cells had multipolar processes with
occasional bifurcated branches or lamellipodia
(Fig. 7A,B). Growth cones at
the tips of axons were also found to be larger in the mutant cells compared to
the wild-type (Fig. 7C-G).
Double-immunofluorescence staining revealed entry of actin and microtubule
networks into those growth cones (Fig.
7E,F). These phenotypes were reminiscent of those seen following
treatment with an inhibitor of p160 ROCK, a Rho GTPase-associated kinase (Bito
et al., 2000).
|
The above observation raised an intriguing possibility that Pax6 might regulate the expression of factor(s) implicated in the Rho/ROCK pathway. To assess this possibility, we first examined initial outgrowth of axons in dissociated EGL cells (Fig. 8). By 20 hours after plating, most wild-type cells extruded bipolar neurites, presumably forming future parallel fiber axons (Fig. 8A,G). In contrast, a significant proportion of Pax6 mutant cells possessed an increased number of processes (Fig. 8D,G). Differences in neurite lengths between wild-type and mutant cells were not prominent at 20 hours of culture. Wild-type cells treated with Y27632, a specific inhibitor of ROCK, exhibited multipolar morphology similar to Pax6 mutant cells (Fig. 8B). The only noticeable difference in their phenotypes was a slight increase in axon length in Y27632-treated cells (Fig. 8H). Overexpression of a RhoA-inhibiting enzyme, C3, resulted in further enhancement of similar phenotypic changes (Fig. 8C). We also tested modulators of other small GTPases, including dominant-negative and constitutively-active forms of Cdc42 and Rac1, but they exhibited phenotypes apparently distinct from Pax6 mutant cells (data not shown). As these observations suggested that Pax6 might be involved in the Rho/ROCK pathway, we blocked the Rho/ROCK pathway in Pax6 mutant cells by treatment with Y27632 or by misexpression of C3, and quantified the number and the length of neurites in comparison with untreated mutant cells. We hypothesized that if the phenotype of Pax6 mutant cells was caused by loss of expression of factor(s) implicated in the Rho/ROCK pathway, there should be little additional change in the phenotypes of Pax6-deficiency by treatment with Y27632 or C3. However, the effects of Y27632 and C3 were additive in the Pax6 mutant cells; the number and the length of neurites were significantly augmented by treatment with Y27632 or C3 (Fig. 8D-H). Furthermore, misexpression of the constitutively-active form of ROCK (Delta 3) had a comparable inhibitory effect on neurite formation in both wild-type and mutant cells (data not shown). These results suggested that the phenotypes caused by the absence of Pax6 function was unlikely to be mediated by a modification of endogenous ROCK activity.
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DISCUSSION |
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Cell-autonomous function of Pax6 during parallel fiber
formation and tangential migration of granule cells
Pax6 mutant animals show a wide variety of neurological
abnormalities in migration and axonal pathfinding of CNS neurons. However,
most of the phenotypes characterized so far result from indirect effects of
Pax6 mutation. In sharp contrast, granule cells require the intrinsic
activity of Pax6 to properly form bipolar axons and for subsequent
cell body migration, implicating Pax6-involvement in the transcriptional
control required for polarization of axons. Disrupted formation of long
bipolar neurites in mutant cells was rescued by forced expression of
Pax6, but not by co-culturing with wild-type cells, indicating
cell-autonomy of Pax6 function during granule cell morphogenesis.
Combinatorial expression of different markers for granule cells and parallel
fiber axons ensured that Pax6 mutant EGL cells were competent to
differentiate as granule cells and to form parallel fiber axons. It is thus
plausible that Pax6 is involved in polar elongation of axons after
the initial stage of their formation.
Another striking feature of Pax6 mutant EGL cells is their unsteady zigzag migration path in culture. While wild-type EGL cells elongated long leading processes along preexisting radial axons, Pax6 mutant cells extended short leading processes in random orientations that often detached from the axons, as visualized by immunofluorescence against MAP2 and by TEM studies. Our co-culture experiments demonstrated that this disorganized migration of Pax6 mutant cells was not due to the absence of a radial neurite scaffold, but rather to a cell-autonomous defect of migrating cells. Thus, besides its role in axon elongation, Pax6 might also be involved in regulation of tangential migration of granule cells guided by leading processes that contact with preformed axon fibers. In contrast to the mutant cells in culture, the EGL cells that misexpress a dominant-negative Pax6 failed to move away from the site of their birth in developing cerebella. The appearance of the thickened EGL of mutant animals also suggests that tangential migration of premigratory EGL cells are disturbed in the absence of Pax6 function. This is not surprising if one considers that tangential migration of postmitotic EGL cells is guided by leading processes via contact-mediated association with pre-existing parallel fibers. Hence, densely packed EGL cells might offer massive resistance to the passage of Pax6-deficient cells that randomly move on the free surface of a culture dish.
Which step of parallel fiber formation formation of the bipolar
leading and trailing processes, tangential migration, or axon elongation
does Pax6 specifically regulate? As the leading and trailing
processes of bipolar EGL cells are later transformed into parallel fiber axons
(Ono et al., 1994), it is
difficult to draw a sharp distinction between these continuous steps in
granule cell morphogenesis. From our data, it is likely that Pax6
function is involved in axon elongation as well as during the initial
outgrowth of bipolar neurites, as expression of Pax6 after 1 DIV was
sufficient to rescue attenuated elongation of bipolar neurites in mutant cells
(Fig. 5). Thus, the
cell-autonomous function of Pax6 may be involved in sustaining the
formation of bipolar processes that guide tangential migration and later
elongate to form parallel fiber axons.
Cellular mechanisms downstream of Pax6 function
We also searched for the cellular mechanisms regulating granule cell
morphogenesis impaired in Pax6 mutant cells. Formation of multiple
axonal processes, abnormal lamellipodia and increase in the growth cone size
in mutant cells strongly suggest an important role for Pax6 in the
control of cytoskeletal dynamics during neurite formation. Apparent
similarities in the phenotypes of Pax6 mutant cells with those evoked
by inhibition of the Rho/ROCK activities prompted us to examine a functional
interaction of Pax6 and the Rho/ROCK pathway. Quantitative analysis
of the action of Y-27632 on Pax6 mutant cells excluded direct
involvement of Pax6 in the ROCK pathway. In other words, it is
unlikely that Pax6 regulates expression of signaling molecule(s) in
direct association with ROCK. Some differences in the phenotypes of mutant
cells and ROCK-inhibited cells were observed: a mild increase in axon length,
presumably owing to early initiation of axon outgrowth, was seen in
Y27632-treated cells but not in Pax6-mutant cells
(Fig. 8). In fact, unlike
Y27632 treatment, initiation of neurite formation was not advanced in mutant
cells (data not shown). Nevertheless, several common morphological features
between Pax6- and ROCK-deficient cells suggested that Pax6
might regulate cytoskeletal dynamics via a mechanism closely related to the
ROCK pathway. One possibility to be tested in future studies is that
Pax6 may be involved in a step maintaining neuronal polarity
downstream of ROCK-mediated initial polarization of granule cells. The
enhanced phenotypes induced by the Rho-inhibiting enzyme C3 are consistent
with the idea that multiple co-existing Rho-dependent mechanisms regulate
actin and/or other cytosketetal dynamics (Kaibuchi et al.,
1999; Bito et al.,
2000
; Bradke and Dotti,
2000
). C3 expression yielded
alterations in the number and the length of axons of wild-type and mutant
cells to a similar plateau, implying that loss of Pax6 function might
cause inactivation of a ROCK-independent Rho pathway. Alternatively,
Pax6 might be involved in a cascade independent of Rho/ROCK activity,
as we cannot rule out that phenotypes evaluated by the parameters we used in
this study were saturated in C3-expressing cells.
It is noteworthy that in culture, Pax6 mutant neocortical radial
glia and cerebellar granule cells exhibit similar morphological abnormalities
(Götz et al.,
1998): they form multipolar
short processes instead of long bipolar processes of wild-type cells. This
raises an intriguing possibility that Pax6 might have a general role
in regulating cytoskeletal dynamics during polarization of neural cells in the
CNS. Pax6 may activate transcription of the genes required for
organization of cytoskeletal proteins in granule cells, which should act
independently of the ROCK pathway. Identification of downstream target genes
for Pax6 is in progress to reveal the molecular cascades that control
the dynamic change of granule cell polarity during development.
Contribution of Pax6 in other aspects of cerebellar development
Pax6 expression is first detected in the EGL stem cells in the
upper rhombic lip at as early as E14.5 in normal rat embryos (K. K. and M. K.,
unpublished). The EGL stem cells move rostrally from the upper rhombic lip
along the surface of the cerebellar primordium to form the EGL by E19 in
rodents. This raises the possibility that Pax6 is involved in the
early phase of cerebellar development, including migration of the stem cells
to populate the EGL. Previous studies have indeed shown that a subset of EGL
cells is mislocated in the inferior colliculus in the Pax6 mutant
mouse Sey/Sey (Engelkamp et al.,
1999). This ectopic extension
of the EGL may be the result of loss of Unc5h3 expression in the
Sey/Sey mouse. However, in the rat
rSey2/rSey2 mutants, we found no difference in
the level of expression of netrin and its receptors, including
Dcc, Unc5hs and neogenin, in the EGL (data not shown).
Consistently, the rostral migration of EGL stem cells appeared normal in rats,
suggesting that neither loss of Unc5h3 expression nor mislocation of
EGL cells necessarily occur in the context of Pax6 deficiency. That
the EGL was completely covered by granule cells in Pax6 mutant
animals supports the notion that the initial migration of EGL stem cells does
not require Pax6 function. Histological analysis could distinguish
rostral migration of the EGL stem cells from tangential migration of
premigratory EGL cells, in that the former is not guided by leading processes
but resembles passive dispersal of round cells (Ryder and Cepko,
1994
; Altman and Bayer,
1997
; Komuro et al.,
2001
). It is intriguing that
leading process-guided migration of lower rhombic lip cells to form
precerebellar nuclei is also disrupted in Pax6 mutant animals
(Engelkamp et al., 1999
; Yee
et al., 1999
). Thus, the
ability of Pax6 to regulate migration may be a mechanism common to
neurons that use leading processes in the cerebellar system.
What is the role of early expression of Pax6 in the upper rhombic lip cells? Besides abnormal behavior of EGL cells, multiple morphological defects are observed in Pax6 mutant cerebellum, including mediolateral regionalization of the cerebellar primordium. Early expression of Pax6 might regulate these aspects independently of its action on postmitotic EGL cells.
Concluding remarks
Pax6 plays critical roles in numerous aspects of early patterning
and cell-type specification in the CNS. Our present report pinpoints a novel
function of Pax6 in the cell-autonomous control of cytoskeletal
networks during the polarization of the CNS neuron. As could be speculated
from its pleiotropic actions during development, downstream targets of
Pax6 might well be context dependent within individual
Pax6-expressing cells. It would thus be necessary to identify the
specific target genes in the granule cell to clarify the molecular mechanisms
regulating formation of their bipolar axons. Further studies will be needed to
understand how widely
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ACKNOWLEDGMENTS |
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