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First published online 3 January 2007
doi: 10.1242/dev.02764
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1 Genes and Development Group, Centres for Integrative Physiology and
Neuroscience Research, Hugh Robson Building, George Square, University of
Edinburgh, Edinburgh EH8 9XD, UK.
2 Division of Reproductive and Developmental Sciences, Genes and Development
Group, Centres for Integrative Physiology and Reproductive Biology, Hugh
Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD,
UK.
3 MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU,
UK.
* Author for correspondence (e-mail: Martine.Manuel{at}ed.ac.uk)
Accepted 20 November 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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wild-type chimeras indicates that the defect is cell autonomous. We
analyzed cortical arealization in PAX77 mice and found that, whereas the loss
of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels
predicted to shift rostral areas caudally has very little effect. These
findings indicate that Pax6 levels are stabilized by autoregulation, that the
proliferation of cortical progenitors is sensitive to altered Pax6 levels and
that cortical arealization is not.
Key words: Pax6, Cortex, Overexpression, Autoregulation, Proliferation, Neurogenesis, Lamination, Regionalization, Thalamocortical, Chimeras
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Neumann and Cohen,
1997; Tabata and Takei,
2004; Yucel and Small,
2006). The potential of a limited set of transcription factors to
direct the generation of an enormous diversity of neuronal phenotypes might be
enhanced greatly if different levels of transcription factors cause different
developmental outcomes. If levels of expression are important, then two
predictions can be made: (i) developing neurons should contain mechanisms to
stabilize levels of expression of transcription factors in the face of
altering exposure to environmental signals as the embryo grows and changes;
and (ii) neurogenesis should be affected if levels of transcription factor
expression are altered. Here, we tested these predictions for the
transcription factor Pax6.
Mice homozygous for a loss-of-function mutation of Pax6 lack eyes
and nasal structures, and die at birth with serious brain abnormalities,
including forebrain patterning and growth defects
(Bishop et al., 2000;
Bishop et al., 2002;
Estivill-Torrus et al., 2002;
Grindley et al., 1997;
Hill et al., 1991;
Hogan et al., 1986;
Kroll and O'Leary, 2005;
Mastick et al., 1997;
Muzio et al., 2002;
Pratt et al., 2000;
Schmahl et al., 1993;
Stoykova et al., 1996;
Warren and Price, 1997). In
normal mice, corticogenesis occurs from E10.5 to E17.5 by a process of
progenitor proliferation in the ventricular zone (VZ) followed by migration of
neural precursors to the overlying cortical plate. Pax6 is expressed
in the VZ throughout corticogenesis (Caric
et al., 1997; Estivill-Torrus
et al., 2002; Walther and
Gruss, 1991) and is implicated in progenitor proliferation,
migration, differentiation, lamination and arealization
(Bishop et al., 2000;
Estivill-Torrus et al., 2002;
Heins et al., 2002;
Schuurmans et al., 2004;
Tarabykin et al., 2001).
Previous studies identified a reduced progenitor population and defective
differentiation as primary defects resulting from the loss of Pax6
(Heins et al., 2002;
Quinn et al., 2006).
Most studies on the functions of Pax6 in brain development have studied the
consequences of its removal. Whether the level at which it is expressed in the
brain is important is unclear, although its expression in a gradient in the
cortex suggests that it might be. Levels of expression in the eye are crucial,
with both reduced and increased gene dosage causing defects in development
(Schedl et al., 1996). Here,
we tested whether increased levels of Pax6 affect cortical progenitor
proliferation, cortical lamination and cortical arealization using the PAX77
mouse line produced by Schedl et al.
(Schedl et al., 1996). In
addition to their two endogenous Pax6 alleles, mice hemizygous for
the PAX77 transgene carry five to seven copies of the human
PAX6 locus, including its upstream and downstream regulatory regions
(the mouse and human Pax6 proteins are identical). The PAX77
transgene is functional, as demonstrated by its ability to rescue the eye and
brain defects in mice carrying loss-of-function mutations of Pax6
(Schedl et al., 1996).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), designated PAX77+, carry five to seven
copies of a 420 kb human PAX6 YAC (Y593) with all copies integrated
at the same locus. We refer to the array of integrated YAC Y593 copies as the
PAX77 transgene. PAX77 homozygous mice, designated
PAX77+/+, carry 10-14 copies of human PAX6 and
were genotyped by fluorescent in situ hybridization using the Fat5 probe
(Schedl et al., 1996). The
PAX77 line was maintained on a CD1 background. The morning of the vaginal plug
was deemed E0.5. The first 24 hours after birth was deemed P0. Animal care
followed institutional guidelines and UK Home Office regulations.
Western blots
For each protein sample, two E12.5 telencephalons of the same genotype were
combined and lysed in TENT buffer (20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 150 mM
NaCl; 1% (v/v) Triton-X100). Subsequent processing followed Pinson et al.
(Pinson et al., 2006), using
anti-Pax6 serum 13 (1:200; from S. Saule, Institut Curie, Paris, France)
(Carriere et al., 1993) and
anti b-actin (1:5000; Sigma). Densities of bands on X-ray films were
quantified with a GS-710 densitometer and Quantity One software (BioRad).
Autoregulation of Pax6
We generated reporter transgenic mice containing one copy of a modified
version of the PAX6-containing YAC used to generate the PAX77 line.
The modified YAC (Y1123) contains the upstream and downstream regulatory
regions sufficient to recapitulate full PAX6 expression, but the
PAX6 gene is rendered non-functional by the insertion of a tau-green
fluorescent protein (tau-GFP) reporter construct
(Tyas et al., 2006).
Expression of tau-GFP from Y1123 in these mice, named DTy54, reports
faithfully the sites and levels of endogenous Pax6 expression
(Tyas et al., 2006). Here, we
studied the expression from Y1123 in Pax6+/+,
Pax6Sey/Sey (designated Pax6-/-) or
PAX77+ embryos. Embryos were fixed (4% paraformaldehyde)
and embedded in 4% low-melting-point agarose. Vibratome sections (200 µm)
were counterstained with TOPRO3 (Molecular Probes, NL) and imaged (Leica
confocal microscope). To prepare isolated cells, heads from E14.5
Pax6+/+, Pax6-/- or PAX77 embryos also
carrying Y1123, or from wild-type embryos were dissociated (Papain
Dissociation System, Worthington Biochemical). Cells were analyzed on a
Beckman-Coulter XL flow cytometer (10,000-20,000 cells per sample).
Quantitative reverse transcription PCR
Telencephalic cDNA samples were collected from E14.5 wild-type or
PAX77+ embryos. Quantitative reverse transcription PCR
(qRT-PCR) used the following primer pairs: mouse Pax6
(5'-AACACCAACTCCATCAGTTC-3' and
5'-ATCTGGATAATGGGTCCTCT-3'; 153 bp product) and 18S
ribosomal RNA (5'-GTGGAGCGATTTGTCTGGTT-3' and
5'-CAAGCTTATGACCCGCACTT-3'; 321 bp product). qRT-PCR was performed
using Qiagen Quantitect SYBR Green PCR kit (Qiagen, USA) and a DNA Engine
Opticon Continuous Fluorescence Detector (GRI, UK). The abundance of each
transcript in the original RNA sample was extrapolated from PCR reaction
kinetics using Opticon software.
Cortical progenitor proliferation
E12.5 or E15.5 pregnant females were sacrificed 1 hour after injection with
200 µl of 10 mg/ml bromodeoxyuridine (BrdU, Sigma; in 0.9% NaCl,
intraperitoneal). Wax sections (10 µm) were immunostained with anti-BrdU or
anti-phosphorylated histone H3. Cells were counted in 100 µm-wide sampling
boxes in the VZ and subventricular zone (SVZ) of the rostral, central and
caudal cortex of three wild-type and three PAX77+ embryos.
Each count was repeated on three to five non-adjacent sections from each
embryo for BrdU analysis and on 10-19 non-adjacent sections for phosphorylated
histone H3 analysis.
Cortical layer thickness
A total of three wild-type and three PAX77+ P7 pups
were anesthetized and perfused with 4% paraformaldehyde. Coronal wax sections
(10 µm) were stained with cresyl violet. For each brain, the thickness of
the cortical layers was measured using the Image Tool software (University of
Texas Health Science Centre at San Antonio, San Antonio, TX, USA) on 16-28
non-adjacent sections at equivalent rostral, central and caudal levels.
Chimeric embryos
Chimeras were produced by embryo aggregation
(West and Flockhart, 1994).
Embryos differed at the Gpi1 locus (encoding glucose phosphate
isomerase) and one of each pair carried a reiterated ß-globin TgN
(Hbb-b1) transgene (abbreviated to Tg), identifiable by DNA in
situ hybridization (Keighren and West,
1993; Lo, 1986;
Lo et al., 1987). Outbred CD1A
females (homozygous Gpi1a/a CD1 strain mice) were induced
to ovulate and were mated to hemizygous PAX77+ males on a
CD1A background to produce PAX77+ and wild-type embryos,
all of which were Gpi1a/a;Tg-/-.
(C57BL/6xCBA/Ca)F1 females (Gpi1b/b;
Tg-/-) were induced to ovulate and were mated to `BTC'-strain
males (Gpi1b/b;Tg+/+ on a mixed
[C57BL/6xCBA/Ca] background) to produce embryos, all of which were
wild-type, Gpi1b/b; Tg+/-. Chimeras were
transferred to pseudopregnant Gpi1c/c (`CF1' hybrid
strain) females. E16.5 foetuses were dissected into cold PBS, and their limbs
and tails were removed and analyzed by GPI1 electrophoresis
(West and Flockhart, 1994).
Heads were fixed and processed for analysis by in situ hybridization and other
body tissues were digested to obtain DNA for PCR genotyping to distinguish
PAX77wild-type and wild-typewild-type chimeras. A total of three
to five non-consecutive coronal sections from each chimeric brain were
examined for each hemisphere and each region along the anterior-posterior
axis. Percentages of Tg- cells were determined by counting numbers
of hybridization signals and nuclei in 100 µm-wide boxes. The contribution
of Gpi1a/a cells to chimeras used here was 42-86%.
To determine proportions of Tg- and Tg+ cells in
M-phase in PAX77wild-type chimeras, ß-globin DNA in situ
hybridization was followed by immunostaining with anti-phosphorylated histone
H3. Nickel was added to the diaminobenzidine visualization solution to obtain
a grey precipitate. Cells were counted in 100 µm-wide sampling boxes in the
VZ of the rostral cortex of each E16.5 chimeric or wild-type embryo. Each
count was repeated on four to five non-adjacent sections. The contribution of
Gpi1a/a cells to the chimeras used here was 42-57%.
Immunohistochemistry
Processing for Emx2 detection was as described in Mallamaci et al.
(Mallamaci et al., 1996), for
Ephrin B2 detection was as in Pratt et al.
(Pratt et al., 2004) and for
all other immunochemistry reactions was as in Martynoga et al.
(Martynoga et al., 2005).
Immunostaining reactions were performed as in Martynoga et al.
(Martynoga et al., 2005),
except that antigen retrieval was not necessary for Emx2 detection. Primary
antibodies were BrdU (1:200; Becton Dickinson), phosphorylated histone H3
(1:500; Sigma), Pax6 (1:500; DSHB), Emx2 (1:500, a gift from G. Corte,
National Institute for Cancer Research, Genova, Italy) and Ephrin B2 (1:500;
R&D Systems AF496,). To analyze the cortical Pax6 gradient, a biotinylated
secondary antibody was used with Alexa Fluor 488-conjugated streptavidin
(Molecular Probes, Inc.). Serial sagittal sections through four cortical
hemispheres from wild-type and PAX77+/+ E12.5 embryos were
analyzed (Leica confocal microscope). The fluorescence intensity was
quantified on each section in 50 µmx50 µm boxes placed at rostral
and caudal cortical poles, and the ratio between the two was calculated. The
highest anterior/posterior ratio for each cortical hemisphere was used to give
a value for the maximum steepness of its Pax6 gradient. An average was
calculated from all four hemispheres.
In situ hybridization
For Id2 in situ hybridization, embryos were dissected (cold PBS),
fixed overnight (4% paraformaldehyde), wax-embedded and sectioned (10 µm).
In situ hybridization was performed
(Porteus et al., 1992) using a
digoxigenin-labeled anti-sense Id2 RNA probe (from D. O'Leary, the
Salk institute, La Jolla, USA).
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). Images
were captured (Leica DMLB microscope), and linear and area measurements
obtained (Image Tool software). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Negative autoregulation of the Pax6 locus
Our finding that Pax6 protein levels do not increase in proportion to gene
copy number suggests that a negative autoregulation of the expression of the
gene exists. To test this, we examined the expression of tau-GFP from one copy
of Y1123 (Material and methods) (Tyas et
al., 2006) in embryos that were Pax6+/+,
Pax6-/- or PAX77+ with confocal
microscope settings held constant throughout
(Fig. 2A-C). The signal was
much weaker in the cortex of PAX77 mice
(Fig. 2B), and was more intense
in Pax6-/- cortex (Fig.
2C), than in wild-type cortex. Previous studies of
Pax6-/- brains have shown that, although their morphology
is distorted, mutant counterparts of major brain structures are recognizable
and Pax6 mRNA expression is restricted to regions corresponding to
Pax6-expressing regions in wild-type mice
(Stoykova et al., 1996;
Warren and Price, 1997). We
found that, in Pax6-/- brains, the expression of tau-GFP
from Y1123 is also restricted to these regions;
Fig. 2C shows expression
limited to dorsal telencephalic cells in mutants, as in wild type
(Fig. 2A). Reduced GFP
expression in PAX77 embryos and increased expression in
Pax6-/- embryos was not confined to the forebrain, but was
seen in all regions that express Pax6 (see Fig. S1 in the supplementary
material).
Results from flow cytometry on dissociated cells from E14.5 brains are shown in Fig. 2D-H. Data in each histogram in Fig. 2D-G is from a single embryo. Analysis of non-transgenic embryos provided frequency distributions of background fluorescence intensity (Fig. 2D): distributions were similar in different individuals and gate B was set to include an average of 2% of cells (range 1-5%) in a set of 12 samples from non-transgenic embryos. Cells falling within gate B in Y1123 transgenic embryos (Fig. 2E-G) were considered to express tau-GFP. Each frequency distribution shown in Fig. 2D-G was similar to that from all the other embryos of the same genotype (n=4-6 embryos per genotype). For each sample, the average fluorescence intensity of the population of cells within gate B was obtained and data were combined for each genotype group (Fig. 2H); combining values for the median fluorescence intensity of each sample gave the same outcome (data not shown). Embryos of all three genotypes contained populations of GFP-expressing cells, but the average fluorescence intensity of this population was higher on a Pax6-/- background (Fig. 2E,F,H) and lower on a PAX77 background (Fig. 2E,G,H). These results indicate that GFP-expressing cells increase their expression levels in the absence of Pax6 proteins, whereas an 1.5- to 3-fold non-ectopic overexpression of Pax6 suppresses GFP expression. We conclude that the level of activity of the Y1123 reporter is inversely related to the level of endogenous Pax6 production.
We carried out qRT-PCR to test whether raised levels of PAX6 protein lowered the expression of Pax6 mRNA. We found significantly reduced levels of mRNA for mouse Pax6 in the telencephalon of PAX77+ E14.5 embryos, to one-third of wild-type levels (Student's t-test, P<0.03; n=3 embryos of each genotype), indicating that Pax6 negatively regulates its own mRNA production and/or stability.
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;
Heins et al., 2002). First, we
tested whether overexpression of Pax6 has an effect on cortical-cell
proliferation. The proliferative zone of the neocortex contains two
populations of progenitors: apical progenitors (APs) undergo mitosis at the
ventricular surface and express high levels of Pax6; and basal progenitors
(BPs) are derived from APs and divide in the SVZ and basal VZ. Pax6 is
downregulated during the transition from AP to BP
(Englund et al., 2005). BrdU
was used to label cortical progenitors in S-phase of the cell cycle
(Fig. 3D,E,G,H), and
phosphorylated histone H3 was used as a marker of mitotic progenitors
(Fig. 3J,K). We determined the
proportion of cells in S-phase (pScells) in the VZ along the cortex
of PAX77+ and wild-type embryos at E12.5, early in
corticogenesis, and were unable to detect any significant difference
(Fig. 3A-F). We then determined
the pScells and the density of cells undergoing mitosis
(dMcells) along the cortex of wild-type and
PAX77+ embryos at E15.5, a late stage of corticogenesis.
We found a significant reduction of the pScells
(Fig. 3G-I) and the
dMcells (Fig. 3J-L)
among APs in the rostral and central cortex of PAX77+
embryos compared to wild type (Student's t-test, P<0.05).
No significant difference was detected in the caudal cortex. Thus, Pax6
overexpression alters the proliferation of rostral and central APs at late
stages of corticogenesis, whereas early progenitors seem unaffected. We did
not find any significant alteration of pScells and
dMcells among BPs in the rostral, central and caudal cortex of
PAX77+ embryos compared to wild type (data not shown). We tested whether overexpression of Pax6 causes an increase in neurogenesis by analyzing the expression of the neuronal marker ß-tubulin III in the cortex of wild-type and PAX77 embryos using immunohistochemistry and flow cytometry. We found no difference in the proportions of ß-tubulin III-expressing cells in PAX77 embryos compared to wild type (see Fig. S2 in the supplementary material).
Effects of overexpression of Pax6 on late cortical progenitors are cell autonomous
In PAX77 mice, Pax6 levels are increased at all of its sites of expression,
and so a late-onset cortical defect might arise as a secondary consequence of
defects elsewhere (e.g. PAX77 mice are microphthalmic and may have other as
yet undetected extra-cortical abnormalities). We tested whether the defects
detected in mutants reflect a requirement for a correct level of Pax6 within
cortical progenitors themselves rather than in their environment. To
discriminate between these two possibilities, we analyzed foetal chimeras in
which the mutant cells have the potential to interact with wild-type cells so
that any defects that they retain are therefore more likely to be cell
autonomous.
Embryos derived from a wild typexPAX77+ cross
were aggregated to wild-type embryos, carrying the Tg transgene as a marker,
to produce control chimeras
[wild-type;Tg-wild-type;Tg+] or mutant chimeras
[PAX77+;Tg-wild-type;Tg+].
Cells derived from the wild-type embryo could be identified, after DNA in situ
hybridization against Tg, by the presence of a brown spot in the
nucleus (Fig. 4A,B). The global
percentage of cells derived from each of the two embryos used to generate each
chimera was estimated by quantitative analysis of GPI1 isozyme composition of
the limbs (West and Flockhart,
1994), with the percentage of the GPI1A isozyme representing the
contribution of cells derived from the
wild-typexPAX77+ cross.
We analyzed three control and four mutant chimeras at E16.5 and determined the percentage of Tg- cells in the proliferative zones (VZ and SVZ), the intermediate zone (IZ) and the cortical plate (CP) at rostral, central and caudal positions of each chimeric cortex. For each chimera, the observed contribution of Tg- cells to each cortical region (obsTg-) was compared to the expected contribution of Tg- cells (expTg-) given by the percentage of GPI1A for that chimera. The mean ratio obsTg-:expTg- in the different cortical regions of the control chimeras was always slightly greater than 1 (between 1.08 and 1.23, Fig. 4C-E), reflecting the fact that the brown spot identifying Tg+ cells was not always present in the plane of section analyzed, and the number of Tg- cells was therefore slightly overestimated. This applied equally to control and mutant chimeras. Our results showed that the Tg- cells in the mutant chimeras were significantly under-represented in the proliferative zones of the rostral (Student's t-test, P<0.01), central (P<0.05) and caudal (P<0.05) cortex compared with the control chimeras (Fig. 4C). The Tg- cells in the mutant chimeras were also significantly under-represented in the IZ of the rostral cortex (Student's t-test, P<0.05) compared with control chimeras (Fig. 4D). We did not detect any difference in the contribution of Tg- cells to the cortical plate between mutant and control chimeras (Fig. 4E), consistent with there having been no earlier defect in production of postmitotic cells. Overall, these results indicate that, late in corticogenesis, cortical progenitors must express normal levels of Pax6 to contribute normally to the proliferative zones of the cortex.
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Pax6 overexpression decreases the thickness of superficial cortical layers
The formation of superficial cortical layers occurs during late stages of
corticogenesis (Caric et al.,
1997). We tested whether the defect in late cortical progenitor
proliferation observed in PAX77 embryos results in postnatal lamination
defects. We compared the thickness of deep (V and VI) and superficial (II-IV
combined) cortical layers and of the marginal zone (future layer I) in
PAX77+ and wild-type brains at P7
(Fig. 5). The thickness of
superficial layers II-IV was significantly decreased in the rostral and
central PAX77 cortex compared to the wild-type cortex (Student's
t-test, P<0.01, n=3 brains of each genotype;
Fig. 5C,D). The thickness of
deep cortical layers and that of layer I are not significantly altered in the
PAX77 cortex. This is consistent with the late cortical progenitor
proliferation defects observed in PAX77 embryos.
Cortical arealization is largely unaffected by Pax6 overexpression
Previous work has suggested that Pax6 plays a crucial role in
cortical arealization by conferring rostro-lateral identities to cortical
progenitors (Bishop et al.,
2000; Bishop et al.,
2002; Muzio et al.,
2002). The arealization of the neocortex is altered in
Pax6-/- mice: caudo-medial areas expand while
rostro-lateral areas shrink. Moreover, expression of the gene encoding the
transcription factor Emx2, which confers caudo-medial identities, is
upregulated, suggesting that Pax6 downregulates Emx2
(Muzio et al., 2002).
Accordingly, we predicted that the overexpression of Pax6 would have an
opposite effect on cortical regionalization (i.e. the downregulation of
Emx2 and a caudo-medial shift of rostro-lateral areas at the expense
of caudo-medial regions) (Fig.
6). To address the issue of how the observed overexpression of
Pax6 in the brains of PAX77 mice [1.5- to 2-fold and 3-fold for Pax6 and
Pax6(5a), respectively; Fig. 1]
would be expected to impact cortical regionalization, we measured the
steepness of the cortical Pax6 gradient by quantitative comparison of
fluorescent Pax6 immunoreactivity between rostral and caudal cortical poles of
each of a series of sagittal sections (Materials and methods). The
fluorescence intensity was used as an indication of relative Pax6 expression
levels. We found that the average ratio between rostral and caudal Pax6 levels
is 3.37 (±0.26 s.e.m., n=4) in the wild-type cortex and 4.16
(±0.4 s.e.m., n=4) in the PAX77+ cortex
(not significantly different, Student's t-test). As shown in
Fig. 6A, this indicates that
the overall steepness of the Pax6 gradient is increased in the cortex of PAX77
embryos and the increase of Pax6 levels observed in the brains of PAX77
transgenic embryos would be expected to displace anterior cortical regions
that normally express the highest levels of Pax6 to the posterior pole of the
cortex.
|
; Bishop et al.,
2002; Mallamaci et al.,
1998; Muzio et al.,
2002; Rubenstein et al.,
1999). Emx2 is normally expressed in a caudo-medialhigh
to rostro-laterallow gradient in the embryonic cortex and this
gradient is conserved in PAX77+ (and
PAX77+/+, data not shown) cortex at E12.5 with no obvious
difference in the intensity of label between the different genotypes
(Fig. 7A,B). In wild-type
embryos at E16.5, Ephrin B2 is strongly expressed in a caudal domain of the
neocortex (up to the arrowhead in Fig.
7C). This caudal domain of strong expression of Ephrin B2 is
present in the cortex of PAX77+ (and
PAX77+/+, data not shown) embryos and does not appear
contracted (Fig. 7D). In
wild-type embryos at E18.5, Id2 is strongly expressed in a rostral
domain in layers 2 and 3 of the neocortex
(Fig. 7E, arrow marks the limit
of this domain) and in a caudal domain in layer 5
(Fig. 7E, white arrowhead marks
the boundary of this domain). These patterns were not altered in
PAX77+ (data not shown) nor in
PAX77+/+ embryos (Fig.
7F). Cortical areas are also characterized by the specific connections they establish with the thalamus. The somatosensory and visual cortical areas normally establish axonal connexions with the thalamic ventro-posterior geniculate (VP) and dorsal lateral geniculate (dLG), respectively. We examined the organization of thalamocortical projections by injecting fluorescent carbocyanine dyes (DiA and DiI) into visual and somatosensory cortical areas at E17.5 (see Fig. S3A in the supplementary material). Neither PAX77+ (see Fig. S3C in the supplementary material) nor PAX77+/+ (data not shown) embryos showed any consistent differences in projections from that of wild type (see Fig. S3B in the supplementary material) and in neither wild-type nor mutant mice did DiA at the caudal pole back label neurons in the VP.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
|
). Our
identification of negative feedback in the regulation of Pax6 expression in
the developing brain highlights the probable importance of maintaining
appropriate levels of expression in the face of rapid intra- and
extra-cellular changes as embryos grow in size and complexity.
Our evidence for negative autoregulation of an intact locus in vivo comes
from the finding that GFP production from the Y1123 transgene, which reports
faithfully the sites and levels of activation of Pax6
(Kleinjan et al., 2006;
Tyas et al., 2006), is
upregulated on a Pax6-/- background, where no endogenous
Pax6 or Pax6(5a) protein is present (so freeing reporter expression from
regulation), and is reduced in PAX77 embryos in which Pax6 levels are
increased. Furthermore, we show that mouse Pax6 mRNA levels are
reduced in mice that overexpress PAX6. This finding complements nicely
previous studies reporting an increased signal from in situ hybridizations for
Pax6 mRNA in the telencephalon of Pax6-/- mutants
(Muzio et al., 2002) and
increased expression of Pax6 in Pax6(5a)-/-
mutants (Haubst et al.,
2004).
Despite negative feedback, Pax6 protein levels are elevated in PAX77 embryos; thus, negative autoregulation is not able to overcome the effects of introducing five to seven additional copies of the PAX6 locus in PAX77 mice. We found evidence that it constrains the elevation of Pax6 levels in these mice. Western blots showed that absolute levels of Pax6 proteins are increased in the brain of PAX77+ embryos by approximately 1.5- to 3-fold. Because PAX77+ mice contain five to seven extra copies of the gene (seven to nine copies in total, as opposed to two), a slightly larger (3.5- to 4.5-fold) increase in protein levels might have been predicted if each extra copy were functioning at a wild-type level. That they are not is demonstrated by the reduction in expression from the Y1123 transgene on a PAX77 background, which is roughly halved (Fig. 2).
These findings indicate that a negative-feedback mechanism is in place to
stabilize Pax6 protein levels. Previous in vitro studies have shown that Pax6
proteins can bind to isolated Pax6-promoter sequences, but that this
results in activation of transcription
(Aota et al., 2003;
Okladnova et al., 1998;
Plaza et al., 1993;
Plaza et al., 1995). Further
evidence for such activation comes from in vivo studies showing that, in some
tissues (e.g. lens and olfactory placodes, but not in cortex), Pax6 protein is
required for transcription of the Pax6 gene
(Aota et al., 2003;
Grindley et al., 1995;
Kim and Lauderdale, 2006;
Kleinjan et al., 2004). The
mechanisms regulating the levels of expression of Pax6 are likely to be
complex. Our findings implicate negative autoregulation, be it direct or
indirect, as a potent stabilizing component in vivo.
Pax6 overexpression affects late cortical progenitor proliferation cell autonomously
We showed that controlled overexpression of Pax6 in vivo specifically
affects the proliferation of late cortical progenitors. This effect is
strongest in rostral and central parts of the cortex, where levels of Pax6 are
highest. At E15.5, there are significant reductions in the proportions of
progenitor cells in S-phase and in their densities in M-phase both rostrally
and centrally; reductions are slightly greater rostrally. In E16.5 chimeras,
there are significant reductions in the numbers of mutant cells in rostral and
central proliferative layers; again, the reduction is slightly larger
rostrally. In addition, the proportion of mutant cortical progenitors in
M-phase in the rostral cortex of the chimeras is lower than normal. Consistent
with these findings, superficial cortical layers II-IV, which arise mainly
from E15.5 onwards (Gillies and Price,
1993), are significantly thinner in the rostral and central cortex
of postnatal Pax6-overexpressing mice. Our in vivo findings complement a
previous study reporting that overexpression of Pax6 in vitro by viral
transduction of dissociated cortical cells at E14.5 results in an early exit
from the cell cycle (Heins et al.,
2002).
In E15.5 caudal cortex, where Pax6 levels are lowest, there are no
differences in the proportions of progenitor cells in S-phase and their
densities in M-phase between PAX77 and wild-type mice. In the caudal cortex of
E16.5 chimeras, however, the numbers of mutant cells in the proliferative
layers are significantly reduced, although the extent of the reduction is
smaller than in central and rostral regions. A possible explanation for these
regional differences is that the rostral cortex is more advanced than the
caudal cortex at each embryonic age (Bayer
and Altman, 1991) and so the emergence of defects in the caudal
proliferative layers is delayed.
|
Overexpression of Pax6 has little effect on cortical regionalization
Previous studies have suggested that Pax6 plays an important role in
regulating cortical regionalization by promoting rostro-lateral identities and
repressing the expression of Emx2, which confers caudo-medial identities
(Bishop et al., 2000;
Bishop et al., 2002;
Muzio et al., 2002;
Muzio and Mallamaci, 2003). We
predicted that an 1.5- to 3-fold overexpression of Pax6 in the cortex would
result in a downregulation of Emx2, such that caudal levels would decrease to
the very low levels normally found rostrally, and in a caudal shift of rostral
areas (Fig. 6). Unexpectedly,
we found unaltered expression of Emx2 in the cortex of PAX77 embryos. The
analysis of the expression of cortical markers, the topography of
thalamocortical connections and the position of the barrel field did not
reveal any difference between wild-type and PAX77 mice, suggesting that
cortical regionalization is largely unaffected by Pax6 overexpression. The
only defect we observed is a reduction in the size of PMBSF in PAX77 mice.
Previous studies have shown that the loss of Pax6 results in the
upregulation of Emx2 expression
(Muzio et al., 2002). One
possibility to explain both this previous finding and our new result is that
Pax6 is needed to permit the repression of Emx2, but that its level
of expression does not determine the level of Emx2 expression.
Existing evidence indicates that the loss of Emx2 and overexpression of Emx2
in vivo shift cortical areas either caudally or rostrally, respectively
(Bishop et al., 2000;
Hamasaki et al., 2004;
Muzio et al., 2002). It seems
likely that Emx2 plays a dominant role in determining cortical arealization,
whereas Pax6 might have little or no direct role. Increased expression of Pax6
might not affect arealization because it has little, if any, effect on Emx2
levels. Loss of Pax6 might indirectly result in a rostral shift of cortical
areas (Bishop et al., 2000;
Muzio et al., 2002) as
consequence of the upregulation of Emx2. However, there are other ways in
which the loss of Pax6 might indirectly affect cortical arealization. The
reduction of rostro-lateral cortical areas in Pax6-/-
embryos could be a consequence of the absence of olfactory placodes, which
emerge at E9.5 and are a region of mesenchymal-epithelial induction
orchestrating regional differentiation and pathway formation in the forebrain
(Anchan et al., 1997;
Balmer and LaMantia, 2005;
LaMantia, 1999;
LaMantia et al., 2000;
LaMantia et al., 1993).
Olfactory placodes fail to form in Pax6-/- embryos
(Hogan et al., 1986) and the
consequent loss of signalling molecules might influence the development and
regionalization of the cortex. The olfactory placodes form normally in PAX77
mice (see Fig. S4 in the supplementary material), which might explain the
absence of cortical regionalization defects.
On the other hand, we can not altogether exclude the possibility that increasing cortical Pax6 levels does have some effect on cortical arealization. The reduction in the size of the primary somatosensory area in PAX77 mice could reflect the opposing effects of an upregulated Pax6 gradient and an unaffected Emx2 gradient compressing the somatosensory cortex between them, or might result from the increased steepness of the Pax6 gradient. It is also possible, however, that the reduction in size of the somatosensory cortex in PAX77 mice is a consequence of defects in the development of cortical layers II-IV, as discussed above.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/545/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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