1 GSF-National Research Center for Environment and Health, Institute for Stem
Cell Research, Neuherberg, Germany
2 Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
3 MD Anderson Cancer Center, Houston, TX, USA
4 GSF-National Research Center for Environment and Health, Institute of
Developmental Genetics, Neuherberg, Germany
5 GSF-National Research Center for Environment and Health, Institute of Human
Genetics, Neuherberg, Germany
* Author for correspondence (e-mail: magdalena.goetz{at}gsf.de)
Accepted 7 October 2004
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SUMMARY |
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Key words: Forebrain, Cortex, Neurogenesis, Proliferation, Regionalization, DNA-binding domains, Paired domain, Homeodomain, Mouse mutant
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Introduction |
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In contrast, the recently discovered potent neurogenic role of Pax6 appears
more restricted to the telencephalon, and seemingly contributes to the
maintenance of neurogenesis into adulthood exclusively in this part of the
brain (Alvarez-Buylla et al.,
2001; Gage, 2002
;
Hack et al., 2004
;
Heins et al., 2002
;
Nakatomi et al., 2002
). The
absence of functional Pax6 not only severely impairs neurogenesis in the
dorsal telencephalon, its overexpression is also sufficient to drive embryonic
precursors, postnatal cortical astrocytes and adult neural stem cells towards
neurogenesis (Hack et al.,
2004
; Heins et al.,
2002
). In addition, Pax6 is involved in the reduction of
proliferation in the telencephalon, since its loss-of-function results in an
increased number of precursors
(Estivill-Torrus et al., 2002
;
Götz et al., 1998
;
Heins et al., 2002
), while its
gain-of-function reduces the number of progeny generated by a single
progenitor cell (Heins et al.,
2002
). To gain a better understanding of potentially separate
roles exerted by distinct DNA-binding domains of the Pax6 transcription
factor, we examined here the function of its three DNA binding domains, the
PD, PD(5a) and HD in the developing forebrain.
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Materials and methods |
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Immunohistochemistry and in situ hybridization
Embryonic brains were fixed in 4% paraformaldehyde in PBS (PFA) and
sections were cut with a vibratome or with a cryostat after cryoprotection.
Sections were processed for immunohistochemistry
(Götz et al., 1998;
Hartfuss et al., 2001
), using
the primary antibodies against the phosphorylated form of Histone H3 (PH3,
rabbit, Biomol, 1:200), NeuN (mouse IgG1, 1:50, Chemicon), Mash1 (mouse IgG1,
1:2, kindly provided by F. Guillemot), Gsh2 (rabbit, 1:1000, kindly provided
by K. Campbell), Ngn2 (mouse IgG2a, 1:10, kindly provided by D. Anderson),
Olig2 (rabbit, 1:1000, kindly provided by D. Rowitch), Ki67 (rat Tec-3, 1:50,
Dako), ß-galactosidase (rabbit, 1:300, Cappel; mouse IgG2a, 1:500,
Promega), O4 (mouse IgM, 1:1000, kindly provided by J. Price), GFAP (mouse
IgG1, 1:200, Sigma), RC2 (mouse IgM, 1:500, kindly provided by P. Leprince),
Pax6 (rabbit, 1:300, Babco), reticulon1 (mAb 9-4, 1:10, kindly provided by T.
Hirata) and nestin (mouse IgG1, 1:4, Dev. Hybridoma Bank). The respective
secondary antibodies were used from Jackson Immunoresearch, Inc. and Southern
Biotechnology Associates, Inc. Specimens were mounted in Aqua Poly/Mount
(Polysciences, Northampton, UK) and analyzed at a Confocal Microscope (Leica
TCS 4NT; Leica Microsystems; Heidelberg). Digoxigenin-labeled RNA probe for
SFRP2 was made and used as described
(Chapouton et al., 2001
).
Retrovirus preparation and retroviral infection
The entire coding sequence of Pax6 (1873bp fragment) and Pax6(5a) (1915 bp
fragment) was cloned in sense orientation into the BglII unique
restriction site of the retroviral vector 1704 between the upstream LTR and
the EMC IRES sequence (gift of J. E. Majors)
(Ghattas et al., 1991). BOSC23
helper-free packaging cells (Pear et al.,
1993
) were used for viral production by means of transient
transfection with the respective viral plasmid and resulted in a typical titre
of 1x105/ml. Primary cells from cerebral cortex were isolated
and cultured as described previously
(Heins et al., 2002
), and
infected 2 hours after plating at a concentration giving no more than 50
clones per coverslip and analyzed after 1 week in vitro.
Data analysis
The quantification of neurons was performed in sections of the cerebral
cortex at E14 from wild-type (WT) and homozygous mutants at corresponding
rostral, intermediate and caudal levels stained for NeuN. The thickness of the
NeuN-positive cortical plate was measured in confocal pictures taken in a
defined (lateral) area of the cortex by first drawing a line at right angles
to the ventricular surface (VS) from the VS to the pial surface. The length of
this line was determined by the ImageJ program and served as measure of the
total cortical thickness. A second line was drawn from the apical and basal
side of the NeuN-positive band, and its length served as a measure for the
thickness of the band of neurons, the cortical plate. The width of the
cortical plate was calculated as the proportion of the overall thickness of
the cerebral cortex (Table
1).
|
For the clonal analysis in vitro, all clones per coverslip were assessed for their cell type or size (Fig. 5) and the mean was calculated per coverslip (excluding coverslips with more than 50 clones; Fig. 5C,D). Group comparisons were made with the unpaired t-test and P-values smaller than 0.05 were considered significant (*), P-values smaller than 0.001 were considered highly significant (***).
|
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Results |
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Neurogenesis
Neurogenesis was examined in these mutants by immunohistochemistry for
neuronal markers, such as NeuN (Fig.
2) or ßIII-tubulin (data not shown). Neurons accumulate in
the cortical plate (CP) forming a distinct band underneath the pial surface
(Fig. 2A) at midneurogenesis,
embryonic day (E) 14, in the WT cortex. The NeuN-positive band is distinctly
smaller in the cortex of homozygous
Pax6Aey18/ mouse mutants
(Fig. 2A,B), reflecting a
significant decrease in the number of neurons generated
(Table 1). Since the
neurogenesis phenotype of Pax6Aey18/ mice
was reminiscent of the functional null allele Pax6Sey
(Fig. 2E), we quantified the
thickness of the cortical plate relative to the total cortical thickness (see
Materials and methods) in the lateral cortex at rostral, intermediate and
caudal levels (Table 1).
Notably the significant reduction of the thickness of the NeuN-positive band
compared to WT in the Pax6Aey18/ cortex
(P<0.001) and the comparable reduction observed in
Pax6Sey/ cortex
(Table 1), suggests that
Pax6Aey18 is a functional null allele for cortical
neurogenesis. In contrast, we found no reduction in neurogenesis in
Pax6(5a)/ mice
(Table 1;
Fig. 2C) or
Pax64Neu/ mice
(Table 1;
Fig. 2A,D). Thus, the PD, but
not the HD of Pax6 is required for the regulation of neurogenesis in the
cerebral cortex.
|
Patterning
Next we examined the role of the PD and HD in mediating dorso-ventral
patterning in the forebrain. Previous analysis of the functional null
Pax6Sey/ described an almost complete lack
of neurogenin (Ngn) 2 expression in the dorsal telencephalon, while expression
of the transcription factors Mash1, Gsh2 and Olig2, that is normally
restricted to the ventral telencephalon, expands into the dorsal telencephalon
in the absence of Pax6 function (Fig.
3D,D', and data not shown) (see
Stoykova et al., 2000;
Torresson et al., 2000; Yun et al.,
2001
). Precisely this phenotype was observed in the cortex of
Pax6Aey18/ mice
(Fig. 3A,A'; see Fig. S1
in supplementary material) while no changes in dorso-ventral expression
patterns of these transcription factors were observed in the cortex of the
Pax6(5a)/ (Fig.
3B,B') and the HD mutant
Pax64Neu/
(Fig. 3C,C'; Fig. S1).
Since Ngn2 is a direct target of the canonical form of the PD
(Scardigli et al., 2003
),
these findings further support the crucial role of the canonical PD of Pax6 in
its regulation. Consistent with the gradient of Pax6 in the cortex, Ngn2
expression was still visible in the caudal-most and medial regions of the
cortex in the Pax6Sey/ and
Pax6Aey18/, where Pax6 expression is lowest
(see Fig. S2A,B,E in supplementary material).
|
|
The cell types generated by infected precursors were identified by
immunocytochemistry using anti-NeuN for neurons, O4 for oligodendrocytes,
anti-GFAP for astrocytes and RC2 or anti-nestin for undifferentiated
precursors. Clones were classified as pure neuronal clones when all cells of a
cluster were NeuN-positive (examples in
Fig. 5A), as mixed when only
some cells of a cluster were NeuN-positive, and as non-neuronal when no cell
of a cluster was NeuN-positive (Heins et
al., 2001; Heins et al.,
2002
). Note that Pax6 containing the canonical PD potently
increased neurogenesis and the number of neuronal clones as described
previously (Heins et al.,
2002
), while Pax6(5a) overexpression had no effect on neuronal or
glial cell fate (Fig. 5C).
While the canonical form of Pax6 almost completely suppressed the generation
of oligodendrocytes (0% of clones, n=81) or astrocytes (3% of clones,
n=249), precursors infected with the Pax6(5a) form still generated
control levels of oligodendrocyte clones (7% of clones, n=103) or
astrocytes (11% of clones, n=137). One transcription factor involved
in cell fate and the regulation of patterning is Mash1
(Casarosa et al., 1999
) that
normally is reduced after Pax6 overexpression
(Heins et al., 2002
). Only
cells infected with the virus containing Pax6 with the canonical PD
downregulated Mash1 (2% of clones, control 14%) while no change in
Mash1-immunoreactive cells could be observed in the Pax6(5a)-infected cells
(18% of clones). The absence of any effect of Pax6(5a) on cell fate was
further confirmed in rescue experiments performed in cortical cells from
Pax6Sey/ mice
(Fig. 5C), where the low degree
of neurogenesis could only be rescued by introduction of the canonical form of
Pax6, while the Pax6(5a) form showed no effect at all
(Fig. 5C). Taken together,
these data demonstrate that even after overexpression in a Pax6 functional
null background, Pax6(5a) is not able to influence cell fate of cortical
progenitors. This suggests that the targets important for the mediation of the
potent neurogenic role of Pax6 are regulated by the consensus site that is
exclusively bound by the canonical PD of Pax6, i.e. the P6CON site.
In contrast to the lack of effect on cell fate, Pax6(5a) retroviral
transduction exerted a potent effect on cell proliferation. The clone size
reflects the number of the progeny generated by a single infected precursor
and is significantly reduced by overexpression of both forms of Pax6, the
canonical PD or the PD with the 5a insert
(Fig. 5D). Pax6(5a)
overexpression leads to a reduction in clone size independent of their cell
type, i.e. in both neuronal and glial clones. While neuronal clones were
typically small (see examples in Fig.
5A) (Heins et al.,
2001; Heins et al.,
2002
), oligodendrocyte-containing clones are much larger and had a
mean clone size of 16 cells in cultures infected with the control virus but
only 11 cells in Pax6(5a)-infected cultures. Thus, Pax6(5a) reduces cell
proliferation independent of the cell fate, while the canonical form of Pax6
couples both effects.
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Discussion |
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The canonical form of the paired domain is necessary and sufficient for the regulation of neurogenesis and cell proliferation
The novel Pax6 mutation Pax6Aey18 (J.G., unpublished)
with a PD lacking most of its DNA-binding domain but intact HD and TA resulted
in the same telencephalic phenotype as in the Pax6 functional null allele
Pax6Sey/
(Estivill-Torrus et al., 2002;
Götz et al., 1998
;
Stoykova et al., 1996
;
Stoykova et al., 2000
). Even
at the quantitative level, no differences in neurogenesis and cell
proliferation were detectable between
Pax6Aey18/ and
Pax6Sey/ cortices, suggesting that the PD
alone is necessary and sufficient for all of these aspects of cortical
development. In addition, both genotypes expressed similar severity in the
disturbance of patterning in the telencephalon. Our results therefore
demonstrate that targets of the PD are necessary and sufficient to regulate
cell proliferation and cell fate, comprising neurogenesis and patterning, in
the developing forebrain.
Targets of the 5aCON site are involved in the regulation of cell proliferation in the developing telencephalon
Given the predominant role of the PD in exerting the Pax6 functions in
forebrain development, we further determined the specific role of the 5a
splice insert into the PD that shifts DNA binding from the N-terminal to the
C-terminal domain of the PD (Epstein et
al., 1994), by analysis of the Pax6(5a)/
mice (Singh et al., 2002
). We
did not detect any changes in neurogenesis, cell proliferation and
regionalization in the Pax6(5a)/. This finding has to
be interpreted with caution, since the mRNA of the canonical form of
Pax6 was upregulated (1,4-fold) in amounts sufficient to compensate
for the lack of Pax6(5a) in Pax6(5a)/ mice. Messenger
RNA of Pax6(5a) comprises about 10-20% of the total Pax6
mRNA (E10-E12, see Fig. S3 in supplementary material), a ratio previously
observed to be rather effective for transcriptional regulation
(Chauhan et al., 2004
). From
our in vivo analysis we can therefore only conclude that there are no specific
roles of the Pax6(5a) isoform that could not also be exerted by the canonical
PD in the developing forebrain. Indeed, Pax6(5a) affects solely a subset of
the functions of the canonical form of Pax6. Overexpression of Pax6(5a) in
individual cortical precursor cells showed a specific and cell-autonomous
effect on the number of progeny of a single precursor, without affecting cell
fate even in the absence of functional Pax6 in the
Pax6Sey/ background. This phenotype, the
reduction of clone size, can be due to three mechanisms an increase in
cell death, an increase in postmitotic cells or an increase in asymmetric
rather than symmetric cell divisions. We observed no difference in cell death
as analyzed by DAPI staining, but discovered a significant reduction of
proliferating cells in the Pax6-transduced clones already 2 days after
infection (71% proliferating cells amongst all control virus infected cells
compared to 53% after Pax6 overexpression). Thus, Pax6 and Pax6(5a)
overexpression increases the number of cells leaving the cell cycle either due
to an increase in asymmetric cell division or a lengthening of cell cycle
(Calegari and Huttner, 2003
).
Both effects could also explain the increase in the number of precursors seen
in the Pax6 loss-of-function mutations
(Götz et al., 1998
;
Estivill-Torrus et al., 2002
).
Further experiments are needed to clarify the exact role of Pax6 on cell cycle
length or the mode of cell division in proliferating precursors. Since the
Pax6(5a) form is sufficient to induce these changes in proliferation and binds
exclusively to the 5aCON site, these data demonstrate that targets of the
5aCON site are specifically involved in the regulation of cell proliferation,
while targets of the P6CON site seemingly regulate neurogenesis. The latter is
consistent with the role of Ngn2, a target containing the P6CON site, in
neurogenesis (Bertrand et al.,
2002
).
PD, PD5a and HD act in a context-specific manner during forebrain and eye development
The effect on cell proliferation depends on the CNS region while
Pax6 negatively regulates proliferation in the telencephalon, it promotes
proliferation in the vertebrate (Marquardt
et al., 2001) and invertebrate
(Dominguez et al., 2004
) eye.
This is also the case for the Pax6(5a) isoform. The deletion of exon 5a
results in reduced number of iris and lens fiber cells in the eye
(Singh et al., 2002
),
consistent with its proliferation promoting effect. Similarly, in the
Drosophila eye eyegone and the murine Pax6(5a)
positively affect cell proliferation
(Dominguez et al., 2004
).
Thus, the positive effect of the 5aCON targets on cell proliferation in the
eye is widespread across vertebrates and invertebrates, while the
Pax6(5a)-mediated reduction of cell proliferation observed in precursors of
the developing mouse telencephalon seems to be more specific and may have
evolved more recently. The HD of Pax6 plays an important role in the eye
(Favor et al., 2001
), but not
in the developing forebrain. Favor and colleagues
(Favor et al., 2001
) showed
that the Pax64Neu mutation leads to severe defects in eye
formation with homozygous mice developing no eye, except a remnant of the
retinal neuroepithelium (pseudo-optic cup), suggesting that targets of the HD
are important for both the early role of Pax6 in the surface ectoderm for lens
formation, and later processes during retina specification
(Ashery-Padan and Gruss, 2001
;
Ashery-Padan et al., 2000
;
van Heyningen and Williamson,
2002
). Our analysis has further shown reduced proliferation and
Ngn2 immunoreactivity in the remnant of the retinal neuroepithelium in the
Pax64Neu/ (data not shown), similar to the
phenotype in the PD mutant Pax6Aey18/. Both
the PD and HD are thus important for the regulation of proliferation and cell
fate in the retina, while the HD plays no role in these aspects in the
telencephalon. In conclusion our results thus imply the selective use of PD,
PD5a and HD targets as one mechanism that may contribute to the
region-specific differences in Pax6 function within the CNS as well as in
different organs.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/24/6131/DC1
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