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The Journal of Neuroscience, August 1, 2002, 22(15):6526-6536
Brain Factor-1 Controls the Proliferation and
Differentiation of Neocortical Progenitor Cells through Independent
Mechanisms
Carina
Hanashima1, *,
Lijian
Shen1, 2, *,
Suzanne C.
Li1, and
Eseng
Lai1
1 Cell Biology Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021, and
2 Program of Physiology, Biophysics and Molecular Medicine,
Graduate School of Medical Sciences, Weill Medical College, Cornell
University, New York, New York 10021
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ABSTRACT |
The winged helix gene Brain factor-1 (BF1) has a
pleiotropic role in the development of the cerebral hemispheres of the
brain. Mice lacking BF1 have defects in the morphogenesis of the
structures of the dorsal telencephalon (e.g., neocortex) and the
ventral telencephalon (e.g., the basal ganglia). This study focuses on the functions of BF1 in the dorsal telencephalon. We showed previously that telencephalic progenitor cells lacking BF1 differentiate into
neurons prematurely. Here, we demonstrate that the loss of BF1 also
results in an early lengthening of the cell cycle in neocortical
progenitors. To investigate the mechanisms by which BF1 regulates
progenitor cell proliferation and differentiation in the developing
brain, we have replaced the endogenous BF1 protein with a DNA binding
defective form of BF1 in mice, BF1NHAA. The
BF1NHAA protein restores the growth of the dorsal
telencephalon, by improving the proliferation of progenitor cells.
However, the BF1NHAA protein does not correct the
early neuronal differentiation associated with the loss of BF1. In
contrast, replacement of endogenous BF1 with wild-type BF1
corrects the defects in both the proliferation and differentiation of
neocortical progenitors. These results demonstrate that BF1 controls
progenitor cell proliferation and differentiation in the neocortex
through distinct DNA binding-independent and binding-dependent mechanisms.
Key words:
BF1; foxg1; winged helix; neocortex; brain development; telencephalon; neurogenesis
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INTRODUCTION |
The neocortex is the brain structure
associated with higher cognitive functions. Evolution of the mammalian
brain is characterized by a disproportionate expansion of the neocortex
relative to other structures (Clark et al., 2001
). Neocortical neurons
are generated from a sheet of neuroepithelial cells of the dorsal
telencephalon, during the period of neurogenesis. The length of the
cell cycle in progenitor cells and the duration of the neurogenetic
interval primarily determine the total number of neurons that will
comprise the neocortex (Takahashi et al., 1993, 1996a; Caviness et al., 1995). Between embryonic day 11 (E11) and E18 in the mouse, the G1
phase of the cell cycle becomes progressively longer, resulting in a
slower rate of cell division (Takahashi et al., 1993, 1996b). Concomitantly, a steadily increasing fraction of cells withdraw from
the cell cycle and ventricular zone to differentiate, leading to the
depletion of the progenitor cell population and the cessation of
neurogenesis. To understand how the growth of neocortex is regulated,
we are examining the mechanisms that control the neurogenetic program
in the telencephalic neuroepithelium.
Expression of the Winged Helix gene Brain factor-1
(BF1/foxg1) within the neural tube is limited
to the progenitor population that comprises the telencephalon (Tao and
Lai, 1992). BF1 is an early marker of cells specified as telencephalic
neuroepithelium. It continues to be expressed in neurons and glia
derived from the telencephalon in the adult brain (Dou et al., 1999).
BF1 plays a critical role in the development of the cerebral
hemispheres. Embryos lacking BF1 die at birth with hypoplasia of the
cerebral hemispheres (Xuan et al., 1995). In the dorsal telencephalon, the major anomaly is an accelerated rate of neuronal differentiation within the population of progenitors of the neocortical
neuroepithelium. This results in the premature depletion of the
progenitor cell pool and ultimately, in the generation of fewer
neurons. BF1(
/) mutants also have reduced progenitor
cell proliferation in the anterior third of the dorsal telencephalon.
Members of the Winged Helix (WH) family are characterized by a highly
conserved DNA binding domain, functioning both as transcriptional activators and repressors. BF1 has been shown to act primarily as a
transcriptional repressor (Li et al., 1995; Bourguignon et al., 1998).
This activity requires an intact WH domain and the ability to bind to
DNA. Recently, however, we have obtained evidence that BF1 may also
function through additional mechanisms that are independent of DNA
binding (Dou et al., 2000). In vitro studies showed that a
DNA binding defective mutant BF1 protein
(BF1NHAA) could antagonize the
antiproliferative activity of TGF
, as well as transcriptional
activation by TGF. These results led us to postulate that BF1 may
control the development of the neocortex through both DNA binding
dependent and DNA binding independent mechanisms. To test this
possibility in vivo and to dissect the molecular pathways
controlled by BF1 in the developing brain, we examined the consequences
of replacing the endogenous BF1 protein in mice with a DNA binding
defective form.
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MATERIALS AND METHODS |
Targeting of the tetracycline transactivator gene to the
BF1 locus. The targeting construct was made by replacing the
lacZ sequence in the BF1 targeting vector
previously described (Xuan et al., 1995) with the sequence encoding the
tetracycline transactivator (tTA) from pUHD 15-1 (Gossen and Bujard,
1992). The SalI site in the tTA sequence was eliminated by
mutagenesis. The tTA sequence was inserted between a
SalI-ApaI fragment containing the BF1
promoter and an EcoRI-KpnI fragment containing
an SV40 intron and poly(A) sequence (Hebert and McConnell, 2000). The
SalI-BamHI fragment was then inserted into the
SalI-BamHI sites in the pHBL3 plasmid. Linearized targeting vector was electroporated into W9.5 embryonic stem
(ES) cells. Targeted ES clones were identified by PCR and Southern blot as previously described. Three correctly targeted clones
were injected into blastocysts to generate chimeric mice that were bred
with C57/BL6 mice.
Generation and screening of tetO transgenic lines.
IRES3lacZ was generated by inserting a lacZ
cassette (Xuan et al., 1995) into the Nco-Sma sites of the
IRES-poly plasmid (generously provided by T. Jessell). An
Xba-Xho fragment of IRES3lacZ was ligated to the
3' end of Xba site of pUHD 10.3 plasmid to generate a
plasmid containing the multiple tet-Operator sites and promoter, the
IRES lacZ cassette, with an intervening Xba site.
The BF-1NHAA mutant sequence (Dou et
al., 2000) was cloned into this Xba site. Linearized plasmid
was injected into oocytes. Transgenic offspring were identified by
Southern analysis. Twelve transgenic lines were examined for the
responsiveness of the tetO promoter to activation by tTA. Males from
each line were mated with females heterozygous for the tTA gene at the
BF1 locus (BF1 (+/tTA)).
Embryos were obtained from timed pregnancies, with noon of the plug
date defined as E0.5. Embryos were fixed in 4% paraformaldehyde and
embedded in OCT for frozen sections. -gal staining in 10 µm frozen
sections was performed as previously described (Xuan et al., 1995).
Embryos were examined at E13.5 by staining for -galactosidase
activity. Nine lines with -gal activity were studied further.
Immunohistochemistry and in situ hybridization.
Immunohistochemical detection of BrdU and MAP2 were performed as
described previously, (Xuan et al., 1995) except for the following
changes. For BrdU, frozen sections (10 µm) were treated with 1.25 µg/ml proteinase K at 37°C for 17 min for E13.5 embryos. Shorter
treatment was performed for younger embryo tissues. Sections were then
postfixed with 4% paraformaldehyde for 10 min. For the secondary
antibody, biotinylated rabbit anti-rat antibodies were diluted 1:1000
in PBS with 1.5% normal rabbit serum. For MAP2, goat anti-mouse
antibodies conjugated with peroxidase were diluted 1:100 in PBS
containing 0.5% Triton X-100 and 1% normal goat serum.
Immunohistochemical localization of phosphorylated histone H3 was
performed with mouse antibody from Upstate Biotechnology (Lake Placid,
NY), followed by detection with the ABC system (Vector
Laboratories, Burlingame, CA).
In situ hybridization using digoxigenin-labeled probes was
performed as previously described (Yang et al., 1999) with the following modifications. Endogenous peroxidase activity was quenched with 1.5% H2O2 in methanol
for 15 min. Digoxigenin-labeled cRNA probes (100 ng/ml) were hybridized
at 60°C. Sections were incubated with anti-DIG-POD antibody (Ab)
(1:500) overnight in a humidified chamber at 4°C.
Biotinyl-tyramide was applied at 37°C for 15 min. Bone
morphogenetic protein (BMP) probe
templates were kindly provided by B. Hogan (Vanderbilt
University) (BMP2,4,6,7). The BF1
Sfi-Sma probe (nucleotide 1391-1850) was used to detect
transcripts in the NHAA rescue and wild-type (WT) rescue lines.
Timed BrdU labeling. The quantitation of BrdU-labeled nuclei
after a pulse of BrdU is a useful tool to monitor the proliferative activity of a population of cells. However, it is a relatively insensitive method to detect changes in the rate of cell proliferation. In the neocortical neuroepithelium, a significant increase in the
length of the cell cycle from 12 to 15 hr would result only in a 20%
reduction in the fraction of labeled nuclei and be difficult to detect.
We took advantage of the fact that nuclei of neural progenitor cells
move within the ventricular zone in a regulated manner during the cell
cycle. The nuclei in cells undergoing mitosis are positioned next to
the apical (ventricular) surface. During G1, the nuclei migrate to the
basal surface, where they initiate DNA replication in the S phase.
Subsequently, the nuclei migrate back toward the apical surface during
G2. Thus, the position of the nucleus in the ventricular zone reflects
its position in the cell cycle. By monitoring the number of
BrdU-labeled nuclei at the apical surface, we can more readily detect
changes in cell cycle length between two populations of cells.
Previous studies have established that the length of the cell cycle in
neocortical progenitors increases progressively between E11 and E17,
from 8 to ~18 hr. Nearly all of this increase is attributable to the
prolongation of the G1 phase of the cell cycle (Takahashi et al.,
1993). The S phase is relatively constant at 4 hr in length, whereas G2 + M is ~2 hr long. BrdU given intraperitoneally at 25 µg/gm to a
pregnant mouse will result in a pulse of labeling for ~4 hr in
developing embryos (Takahashi et al., 1992) because BrdU is rapidly
cleared. A single injection will label a cohort of nuclei that are in
the S phase of the cell cycle during the labeling period. These nuclei
will span an 8 hr window (4 hr labeling period plus the 4 hr length of
the S phase) within the cell cycle. By examining embryos at various
time points after BrdU labeling, we will be able to follow this cohort
of labeled nuclei as they progress through the cell cycle. We selected
the 15 hr time point because it is approximately the length of the cell
cycle at E14.5 (Takahashi et al., 1993).
Quantitation of BrdU-labeled nuclei. BrdU-labeled nuclei in
frozen sections were identified by immunohistochemistry using a
monoclonal rat anti-BrdU antibody (Harlan Bioproducts for Science, Indianapolis, IN). Both the total number of nuclei and the
number of BrdU-labeled nuclei along the apical surface of the
ventricular zone were counted. Nuclei were counted under a Nikon light
microscope in 10 µm sections, 40-60 µm apart, with 6-10 sections
counted from each embryo (spacing and number of sections increased with embryo age). For the 15 hr labeled sections in Figure 5, data were
collected from four to six embryos at each age [two from each of the
genotypes ((BF1(+/), BF1(/), NHAA
rescue)]. We counted >950 nuclei for each genotype at E11.5, and
>1100 nuclei at E12.5, E13.5, and E14.5. Nuclei were counted from the
posterolateral region, as shown in Figure 5. For the 2 hr pulse
labeling in Figure 6, data were collected from six embryos [three from
BF1(+/), three from BF1(/)]. A minimum of
2800 nuclei was counted from each region of the telencephalon (anterior
and posterior as shown in Fig. 6) for each genotype. Data from each set
is reported as the mean of the calculated percentage of BrdU-labeled
nuclei, ±SEM.
Quantitation of BF1 proteins in the embryo. The
telencephalic vesicles from each E12.5 embryo were obtained by
dissection of the head. Surface ectoderm was removed. The tissue was
homogenized by sonication in 100 µl of 50 mM
Tris buffer, pH 8, 50 mM NaCl, 1% Triton-X 100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors (P8340;
Sigma, St. Louis, MO). Ten micrograms of protein lysate were loaded on
an SDS gel. Immunoblotting was performed with affinity-purified rabbit
anti-BF1 polyclonal antibody at 1:1000 dilution (Yao et al., 2001).
This antibody was raised to the C-terminal peptide of BF1, and
therefore should recognize WT BF1 and
BF1NHAA equally well.
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RESULTS |
Targeting of the tetracycline transactivator gene to the
BF1 locus
To examine the molecular pathways regulated by BF1 in the
developing embryo, we developed a transgenic mouse model to direct the
expression of a mutant form of BF1 in the developing telencephalon. This model uses the tetracycline-regulated system developed by Gossen
and Bujard (1992) and Kistner et al. (1996). We first generated a mouse
line expressing the tetracycline transactivator in BF1-expressing cells
by targeting the tTA gene to the BF1 locus, through homologous recombination in ES cells (Fig.
1A). Germline
transmission of this ES cell line generates mice in which one copy of
the BF1 gene is replaced with the tTA gene
[BF1(+/tTA) line]. In
situ hybridization studies demonstrate that the tTA gene is
expressed in a pattern that closely matches that of the BF1
gene. The expression pattern of tTA is identical to that of BF1 in the telencephalic neuroepithelium, the olfactory
epithelium, in the pharyngeal pouches and ectodermal placodes (Fig.
1B-E) (data not shown). Expression of tTA is not
detectable in the optic vesicle. Because BF1 expression
levels in the optic vesicle are lower than at other sites and the tTA
probe is less sensitive than the BF1 probe, it is possible
that tTA is expressed in the optic vesicle of the
BF1(+/tTA) mice at levels
below the sensitivity of our assay.
BF1(+/tTA) mice have a normal
life span and are fertile. They have no observed anomalies in brain
development as assessed by light microscopy of embryos from E12.5 to
postnatal day 0.
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Figure 1.
Targeting of the tetracycline transactivator (tTA)
to the BF1 locus. A, Map of the targeting
vector used to replace the BF1 gene in ES cells by
homologous recombination. In the BF1 (+/ tTA)
heterozygote at E13.5, the expression pattern of the tTA gene matches
that of BF1 in the telencephalon (B, C) and
olfactory epithelium (D, E), as detected by in
situ hybridization. Mating of the
BF1(+/tTA) mice with a transgenic animal with
a target gene under the control of multiple tetO sites results in
offspring that express the target gene in BF1-expressing cells. An
IRES-lacZ cassette placed downstream of the target gene
directs translation of the -galactosidase gene, which serves a
reporter of target gene expression (F). Staining
for -gal activity in a BF1(+/tTA):tetO-
BF1NHAA IRES lacZ embryo at
E13.5 demonstrates the expression of the target gene in the
telencephalic neuroepithelium (H). No
-gal activity is detected in embryos lacking the tTA
(G) or in BF1(+/tTA):
tetO- BF1NHAA IRES lacZ
embryos harvested from pregnant dams that were fed doxycycline (20 µg/ml) in the drinking water (I). Scale
bar, 200 µm.
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Directed expression of target genes in the
telencephalic neuroepithelium
A DNA binding defective form of BF1
(BF1NHAA) was previously generated by
site-directed mutagenesis of the Winged Helix domain (Dou et al.,
2000). The three-dimensional structure of the winged helix identifies
two amino acids in Helix 3 that make critical base contacts with the
DNA. The corresponding two residues in BF1, N(165) and H(169), were
mutated to alanines to generate the BF1NHAA protein. We have shown that this
mutant protein (BF1NHAA) is unable to bind
to high-affinity BF1 binding sites and is inactive in a transcriptional
repression assay. Mutation of the corresponding two amino acids in Qin,
the chick homolog of BF1, disrupt its ability to transform chick embryo
fibroblasts, a function that is mediated through a transcriptional
repression mechanism (Ma et al., 2000).
To express BF1NHAA in the developing
cerebral hemispheres under the control of the tTA protein, we generated
several transgenic lines with this target gene fused to a promoter
containing multiple tet-Operator sites. An IRES lacZ
cassette was placed downstream of the target gene to facilitate the
detection of cells expressing the target gene under the control of the
tet-transactivator (Fig. 1F) (Mountford and Smith,
1995). Twelve transgenic lines were generated, of which nine exhibited
lacZ expression in the telencephalon when mated with the
BF1(+/tTA) line to
generate BF1(+/tTA): tetO
BF1NHAA embryos. LacZ expression in these
embryos indicates that the target gene,
BF1NHAA, is transcribed under
the control of the tTA protein (Fig. 1H). No lacZ
expression is detected in the absence of the tTA protein (Fig.
1G). The level of lacZ expression varies between transgenic lines, most likely reflecting the different response of the tetO promoter at different integration sites. However, the pattern of
expression is constant among the different transgenic lines, matching
that of the tTA gene. In some of the high expresser lines, we observe
ectopic expression in scattered cells of the diencephalon and midbrain.
Based on the level of lacZ expression, we have grouped the transgenic
lines into high (three lines), medium (three lines), and low (three
lines) level expressers. In this report we will focus on representative
lines of the medium (#25, 77) and high expresser class (#83, 99). The
level of BF1NHAA protein expression in
these lines is described below. Expression of the lacZ reporter in
embryos is abolished when pregnant mice are given doxycycline in the
drinking water beginning on the day the coital plug is identified (Fig.
1I), providing additional evidence that the target
gene is under the control of the tTA protein. Doxycycline
concentrations of 2 µg/ml are sufficient to repress lacZ expression
to undetectable levels.
Replacement of endogenous BF1 with tTA-directed expression of
BF1NHAA
To examine the activity of the BF1
NHAA protein in a BF1 null background, we
generated mice in which this mutant protein replaces the endogenous BF1
protein. We first crossed the tetO
BF1NHAA line with the
BF1(+/lacZ) line (Xuan et
al., 1995), to generate animals with the tetO BF1NHAA transgene and with one
copy of BF1 replaced with the lacZ gene. These
mice are phenotypically normal, and express lacZ under the control of
the BF1 promoter. These males were then mated with female
BF1(+/tTA) mice (Fig.
2E). One quarter of the
offspring will have no copies of the BF1 gene
[BF1(-tTA/-lacZ)],
and one half of these will also be hemizygous for the tetO BF1NHAA gene. These embryos
will be referred to as BF1 mutant
(BF1(-tTA/-lacZ))
and NHAA rescue (BF1 mutant + BF1NHAA), respectively.
BF1(-tTA/-lacZ)
mutants are indistinguishable from the previously described BF1(-lacZ/-lacZ)
mutants (Xuan et al., 1995). Newborn mice die within minutes of birth,
have a flattened forehead, and irregularly shaped small eyes. NHAA
rescue animals also die within minutes of birth and have eye anomalies.
However, the forehead of these mice is notably more rounded in shape
(data not shown).
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Figure 2.
Replacement of endogenous BF1 with
BF1NHAA. Mating of
BF1(+/lacZ):tetO
BF1NHAA IRES lacZ males with
BF1 (+/tTA) females will yield offspring, one
quarter of which will lack BF1. One half of these (or one eighth of the
total) will also have the tetO BF1NHAA
IRES lacZ construct. We refer to these as "NHAA rescue"
embryos (E). Expression of the
BF1NHAA transcript in these embryos was
monitored by -gal staining and by in situ
hybridization (B,#77; C, #99;
D, #83). This was compared with the level of the
BF1 transcript in BF1 (+/) heterozygotes
(A). The level of BF1NHAA
protein was determined by immunoblotting extracts from dissected
telencephalon (#99 line) at E12.5, and compared with that in WT
embryos, heterozygotes, and BF1 (/) mutants
(F). Scale bar, 200 µm.
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To compare the approximate expression level of the tTA-directed
BF1NHAA transcript with that of
endogenous BF1, we performed in situ hybridization with a probe for BF1 that is within the
protein coding sequence (BF1 Sfi-Sma fragment).
Hybridization signals were compared between BF1
heterozygotes and NHAA rescue embryos at E12.5. Sections shown were
processed simultaneously under identical conditions. For transgenic
lines in the moderate expresser group, expression levels of the
BF1NHAA transcript in a mutant
background are slightly lower than endogenous levels of
BF1 transcripts in BF1 (+/) heterozygotes (Fig.
2B). High expresser lines have levels three to four
times higher (Fig. 2C,D). Among the different tetO
BF1NHAA lines, the relative
levels of BF1NHAA expression by
in situ hybridization correlates well with the relative
level of lacZ expression detected by -gal staining. However, -gal
activity in high expresser tetO
BF1NHAA lines is severalfold
lower than in BF1(+/lacZ)
heterozygotes, indicating that translation of the lacZ gene from the IRES is much less efficient than translation initiated from
the 5' end of the transcript.
To determine the level of BF1NHAA protein
expression, we immunoblotted extracts from the forebrain of E12.5
embryos with an anti-BF1 antibody. We find that expression of the
BF1NHAA protein in a high expresser line
(#99) is higher than levels of endogenous BF1 in WT embryos. Expression
levels in the medium expresser group (#25, 77) are threefold to
fourfold lower and are slightly lower than the level of BF1 in
BF1(+/) heterozygous embryos (data not shown). Thus, there
is a good correlation between BF1 mRNA and protein levels expressed
under the control of the tTA protein. NHAA rescue embryos from both the
high and medium expresser lines have very similar phenotypes.
BF1NHAA protein improves the growth of the
dorsal telencephalon
We found that the cerebral hemispheres of the NHAA rescue embryos
were intermediate in size between that of normal mice and BF1 mutants. The basal ganglia, which are derived from the
basal telencephalon, were absent in the NHAA rescue embryos, a result similar to that observed in the BF1 (/) mutant. However,
a marked improvement in the growth of the dorsal telencephalon was
observed in the NHAA rescue. In the BF1 mutant, the dorsal
telencephalon is much smaller than normal (Fig.
3D,H). By contrast, the
dorsal telencephalon of the NHAA rescue, is nearly equal in size to the dorsal telencephalon of normal embryos (Fig. 3, compare A,
B). The neocortex of the BF1 mutant also has an
irregular contour (Fig. 3H), reflecting variations in
the rate of progenitor cell proliferation within the anterior
telencephalic neuroepithelium. This anomaly is corrected in the NHAA
rescue. The neocortex of NHAA rescue embryos has a smooth contour
resembling that of normal embryos. These results show that the
BF1NHAA protein can substitute for the WT
protein to enhance the growth of the dorsal telencephalon. However, the
development of the dorsal telencephalon is not completely normalized by
the BF1NHAA protein. Anomalies remain,
notably in the morphology of the dorsomedial telencephalon (Fig.
3F, arrows).
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Figure 3.
Improved growth of the dorsal telencephalon in
NHAA and WT BF1 rescue embryos at E13.5. Coronal
(A-D) and horizontal
(E-H) sections show that the size of the dorsal
telencephalon in the NHAA rescue is similar to that of the dorsal
telencephalon in BF1 (+/) heterozygotes and in the BF1 WT
rescue. The NHAA rescue has a thicker cortical plate than that found in
BF1(+/) heterozygotes or in the BF1 WT rescue.
Asterisks indicate the position of the cortical plate,
which expresses -galactosidase at lower levels than in the
ventricular zone and contains the differentiated neurons of the dorsal
telencephalon (F, H). dt, Dorsal
telencephalon; bt, basal telencephalon. Scale bar, 200 µm.
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Aberrant cell cycle progression throughout the dorsal telencephalon
of the BF1 mutant
We have previously described anomalies in progenitor cell
proliferation in the anterior third of the dorsal telencephalon of mice
lacking BF1. We detected a significant reduction in the fraction of BrdU-labeled nuclei in this region of the mutant
telencephalon (Xuan et al., 1995). To determine whether cell
proliferation was altered in the remainder of the dorsal telencephalon
of BF1 (/) mutants, we devised a method to monitor the
lengthening of the cell cycle during the period of neurogenesis. We
examined the fraction of BrdU-labeled nuclei at the apical surface of
the ventricular zone 15 hr after a single injection of BrdU. This
method is based on interkinetic nuclear migration of the progenitor
cells of the neuroepithelium (Takahashi et al., 1996b). Nuclei move
from the apical surface to the basal surface and back during every cell cycle (Fig. 4A). To
determine the position of mitotic nuclei in the telencephalic
neuroepithelium of BF1(/) mutants, we localized the
phosphorylated form of histone H3 by immunohistochemistry. We show that
mitotic nuclei are restricted to the apical surface of the ventricular
zone in the BF1(/) mutant as well as in their normal
littermates at E13.5 (Fig. 4B,C). After a short BrdU
labeling period of 2 hr, we found that BrdU-positive cells are present throughout the apical and basal regions of the ventricular zone, in
both BF1 mutants and their normal littermates (Xuan et al., 1995) (see Fig. 6). This suggests that BrdU-labeled nuclei are able to
move from the apical to the basal region in the BF1 mutant. Taken together, these results indicate that interkinetic nuclear migration is not disrupted by the loss of BF1.
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Figure 4.
Interkinetic nuclear migration of neocortical
progenitors is not altered in the BF1 (/) mutant.
Mitosis takes place at the apical surface, adjacent to the ventricle.
Immunohistochemical localization of phosphorylated histone H3
demonstrates mitotic nuclei at the apical surface of the ventricular
zone in the telencephalon of both normal BF1(+/)
heterozygotes and BF1(/) mutants at E13.5.
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We estimated that a single injection of BrdU in a pregnant mouse labels
a cohort of nuclei spanning an 8 hr interval in the cell cycle, based
on the length of the S phase and the clearance rate of BrdU (Takahashi
et al., 1992; Caviness et al., 1995). When the length of the cell cycle
is 8 hr (at E11.5), this cohort spans the entire cell cycle (Fig.
5I), and BrdU-labeled
nuclei are distributed throughout the ventricular zone (Fig.
5A). As the cell cycle length increases, the labeled cohort
represents a progressively smaller fraction of the total interval. When
the average cell cycle length increases to ~15 hr, few labeled nuclei will be in the G2 and M phase of the cell cycle when this cohort is
examined 15 hr after labeling (Fig. 5I). This will
result in a marked reduction of BrdU-labeled nuclei at the apical
surface of the ventricular zone (Fig. 5D). Thus, the
fraction of BrdU-labeled nuclei at the apical surface in this assay
correlates with the fraction of progenitor cells with a cell cycle
length of ~15 hr or longer.
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Figure 5.
Aberrant pattern of BrdU labeling in the
ventricular zone of the BF1 (/) mutant and restoration
of normal patterns in the NHAA rescue. Diagram of the cell cycle in
neocortical progenitor cells at E11.5 and E14.5
(I). Arrows depict the
approximate position in the cycle of cells that will be labeled with
BrdU after a single intraperitoneal injection of BrdU to the pregnant
mouse. The position of this cohort of BrdU-labeled nuclei is also
depicted 15 hr later, when the embryos are examined. When the cell
cycle length is the same as the labeling period or slightly longer,
labeled nuclei will be present in all phases of the cell cycle. Under
these conditions, BrdU-labeled nuclei are distributed throughout the
ventricular zone. This is observed from E11.5 to E13.5 in WT embryos
(A-C) and at E11.5 in BF1 (/)
mutants (E). When the cell cycle length increases
to ~15 hr or greater, labeled nuclei will be absent from the G2 and M
phase of the cell cycle (I). This will
result in a marked reduction in the number of BrdU-labeled nuclei at
the apical surface of the ventricular zone, as seen at E14.5 in WT
embryos (D) and at E12.5 to E14.5 in
BF1 (/) mutants (F-H). In NHAA
rescue embryos, 15 hr labeling at E13.5 (J) shows
BrdU-labeled nuclei distributed throughout the ventricular zone, as
found in normal embryos. BrdU-labeled nuclei are not excluded from the
apical surface until E14.5 (K).
Arrowheads point to the apical surface of the
ventricular zone. Shown in this figure are high magnification views of
the posterolateral region of the telencephalic neuroepithelium
approximately at the positions indicated by the
arrowheads in the horizontal sections of Figure 3. The
posterior telencephalon is on the right side of each section.
L, Quantitation of the percentage of BrdU-labeled nuclei
at the apical surface of the ventricular zone in the posterolateral
telencephalon, 15 hr following a single injection of BrdU from E11.5 to
E14.5. Nuclei lining the ventricular surface were counted. The
percentages of BrdU-labeled nuclei are plotted as the mean ± SEM.
Scale bar, 50 µm.
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|
We examined the BrdU labeling pattern in BF1 (+/) embryos
from E11.5 through E14.5, after an injection of BrdU given 15 hr earlier. We focused on the posterolateral telencephalon, as indicated by the arrowheads in Figure 3. From E11.5 through E13.5, we observe BrdU-labeled nuclei throughout the ventricular zone (Fig.
5A-C). However, at E14.5, BrdU-labeled nuclei are absent
from the apical surface of the neocortical neuroepithelium (Fig.
5D). The cell cycle length at E14.5 has been previously
estimated to be 15.1 hr (Takahashi et al., 1993). We next examined the
pattern of BrdU labeling in the BF1(/) mutant embryos.
At 11.5, BrdU-labeled nuclei are present throughout the ventricular
zone (Fig. 5E). However, at E12.5 and later, BrdU-labeled
nuclei are sharply reduced in number at the apical surface of the
neocortical neuroepithelium in the lateral and posterior telencephalon
(Fig. 5F-H). This reduction was quantified by
counting BrdU-labeled and unlabeled nuclei at the apical surface of the
ventricular zone (Fig. 5L). The fraction of BrdU labeled
nuclei at the apical surface begins to decline at E13.5 and is reduced
to 18% at E14.5 in the BF1 (+/) heterozygotes. In
contrast, this fraction is reduced to ~10% by E12.5 in the BF1(/) mutant. We interpret these results to suggest
that the cell cycle length increases at an earlier stage in development than normal in the absence of BF1. At E12.5, the fraction of cells with
an elongated cell cycle in the neocortical neuroepithelium of the
BF1(/) mutant is comparable to that found in normal
E14.5 embryos.
BF1NHAA improves the proliferation of
telencephalic progenitor cells
To determine the basis for the increased size of the neocortex in
mice expressing BF1NHAA, we examined the
proliferative behavior of telencephalic progenitor cells by BrdU
labeling. We find that BF1NHAA improves
progenitor cell proliferation in the rostral region of the dorsal
telencephalon by two criteria. We compared the pattern of BrdU-labeled
nuclei after 15 hr, with that observed in BF1 mutants and
their normal littermates (Fig. 5). In the NHAA rescue, we find that the
fraction of BrdU-labeled nuclei at the apical surface of the
ventricular zone of the telencephalon is higher than in the mutant and
comparable to that of normal embryos at E12.5 and E13.5 (Fig.
5J,L). The fraction of BrdU-labeled nuclei at apical surface
decreases at E14.5 to the level found in normal embryos (Fig.
5K,L).
We also determined the BrdU labeling index following a 2 hr labeling
period in both the anterior third (anterior) and the posterolateral
(posterior) region of the dorsal telencephalon in horizontal sections
(Fig. 6J). The
BF1 mutant has marked reduction in the fraction of
BrdU-labeled nuclei in the anterior dorsal telencephalic
neuroepithelium compared to the posterior dorsal telencephalon (Xuan et
al., 1995) (Fig. 6D-F), in contrast to a more
uniform fraction of BrdU-labeled nuclei throughout the neocortical
neuroepithelium of the normal BF1(+/) heterozygote (Fig.
6A-C). NHAA rescue embryos have a significant
improvement in the BrdU labeling index in the anterior telencephalon,
which is similar to that observed in normal embryos (Fig.
6G-J). Thus, by two assays, we find that the
BF1NHAA protein restores the proliferation
of progenitor cells throughout the dorsal telencephalon to near normal
levels.
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Figure 6.
The BF1NHAA protein improves
the proliferation of telencephalic progenitors in the anterior
telencephalon. Horizontal sections of E13.5 embryos from
BF1(+/) heterozygotes (A-C),
BF1(/) mutants (D-F), and NHAA
rescue (G-I). The positions of higher
magnification views of the anterior and posterior regions of the
telencephalic neuroepithelium telencephalon are indicated with
rectangles. J, The fraction of
BrdU-positive nuclei after a 2 hr pulse of BrdU was determined in
the anterior and posterior regions. The reduction in BrdU labeling in
the anterior telencephalon of the BF1(/) mutant is
corrected to near normal levels in the NHAA rescue. Scale bar:
A, D, G, 400 µm;
B, C, E, F,
H, I, 50 µm.
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BF1NHAA does not correct the premature neuronal
differentiation associated with the loss of BF1
In the BF1 mutant, neuronal differentiation is
initiated at the usual time, E11.5. However, by E12.5 we detect a large
increase in the number of MAP2-positive cells that demarcate a thicker mantle zone (preplate and cortical plate) in the BF1 mutant
compared with normal littermates. This is especially prominent in the
lateral and posterior regions of the dorsal telencephalon (Xuan et al., 1995). These findings are consistent with a role for BF1 in reducing the rate of neuronal differentiation in the population of cortical progenitor cells. Although accelerated neuronal differentiation must be
associated with an increased rate of cell cycle exit, we do not observe
a significant reduction in the fraction of BrdU-labeled nuclei in the
posterolateral telencephalon of the BF1 mutant. We postulate
that this is the result of the rapid migration out of the ventricular
zone to the cortical plate of progenitor cells that withdraw from the
cell cycle. The observation that the neuroepithelium is thinner in this
region is consistent with this hypothesis.
We compared the thickness of the mantle zone to the thickness of
the ventricular zone (VZ) as an indicator of the extent of neuronal
differentiation (mantle:VZ ratio). In the dorsal telencephalon of
normal embryos, neuronal differentiation begins earlier in the
posterior region, resulting in a higher mantle:VZ ratio posteriorly. At
E12.5, the mantle:VZ ratio ranges from 0.1 anteriorly to 0.4 posteriorly. In the BF1 mutant at E12.5, accelerated
neuronal differentiation results in a marked increase in the thickness of the mantle layer. The mantle:VZ ratio in the BF1 mutant
ranges from 0.5 anteriorly to 4 posteriorly (Xuan et al., 1995).
Expression of the BF1NHAA protein does not
change this ratio significantly. The mantle:VZ ratio in the NHAA rescue
embryo at E12.5 ranges from 0.5 anteriorly to 3 posteriorly. In the
lateral telencephalon, shown in a coronal section at the level of the
eye, the mantle:VZ ratio is 0.15 in the BF1 (+/)
heterozygote (Fig. 7A,D) and
1.4 in the NHAA rescue (Fig. 7B,E). The abnormally thick
mantle zone in NHAA rescue embryos can also be seen in Figures 2 and 3.
Thus, the accelerated neuronal differentiation associated with the loss
of BF1 is not corrected by the expression of
BF1NHAA.
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Figure 7.
Expression of the neuronal marker, MAP2, in the
dorsal telencephalon at E12.5. MAP2 expression in coronal sections in
BF1 (+/) heterozygotes (A, D), NHAA rescue
embryos (B, E), and BF1 WT rescue embryos
(C, F) identifies differentiated cells of the
preplate and cortical plate (mantle zone). The thickness of the mantle
zone in the NHAA rescue is much greater than in the BF1
heterozygote or in the BF1 WT rescue. The mantle
zone:ventricular zone ratio is 0.15 in D, 1.4 in
E, and 0.3 in F. The mantle zone is
thinnest anteriorly and thicker posteriorly within the telencephalon.
Therefore we show coronal sections from comparable planes at the level
of the eye, in the heterozygote, the NHAA rescue, and the
BF1 WT rescue. Scale bar: A-C, 400 µm;
D-F, 50 µm.
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The accelerated neuronal differentiation in the neocortical
neuroepithelium of the NHAA rescue suggests that the regulation of
neuronal differentiation requires the DNA binding activity of BF1.
However, it remained possible that this deficit is the result of an
imperfect replacement of BF1 activity with the tTA system rather than
to the loss of DNA binding activity in the BF1NHAA protein. To address this issue, we
replaced the endogenous BF1 gene with that of WT BF1 under
the control of the tTA protein. We find that "WT BF1 rescue"
embryos exhibit normal growth of the dorsal telencephalon (Fig.
3C,G). In two lines that express WT BF1 at levels lower than
that of moderate BF1NHAA expresser lines,
the growth of the dorsal telencephalon is comparable with that in
normal embryos. Both the rate of progenitor proliferation as assessed
by BrdU incorporation studies, and the timing of neuronal differentiation are corrected (data not shown). The thickness of the
mantle zone in the WT rescue is comparable to that in WT embryos and
significantly thinner than in the NHAA rescue (Fig. 7C,F). These results support the conclusion that BF1
controls the timing of neuronal differentiation in the neocortex
through a DNA binding-dependent mechanism.
Ectopic expression of BMP genes in the
dorsal telencephalon of the BF1 (/) mutant and the
NHAA rescue
We have shown that BMP4 is ectopically
expressed in the telencephalic neuroepithelium of the BF1
(/) mutant (Dou et al., 1999). Here we report that
BMP6, BMP7, and
BMP2 (Fig. 8)
(data not shown) are also ectopically expressed in the telencephalon of
embryos lacking BF1. Instead of being restricted to the
dorsomedial telencephalon and the medial wall of the telencephalic
vesicles, in a pattern complementary to that of BF1 (Furuta
et al., 1997), BMPs 2, 4, 6, and
7 are expressed broadly in the telencephalic neuroepithelium. BMP expression is expanded dorsolaterally and ventrolaterally from its normal domain in the medial wall. Because the
ventral telencephalon is almost entirely missing in the BF1 (/) mutant, the lateral dorsal telencephalon assumes a ventral position in the embryo. Coronal sections through the anterior telencephalon show that ectopic BMP expression
is detectable throughout the mutant neuroepithelium (Dou et al., 1999)
(data not shown). In more posterior sections of the telencephalon,
ectopic BMP expression is not present uniformly
throughout the neuroepithelium, but is highest in the dorsolateral and
ventrolateral telencephalon (Fig. 8B,E). This ectopic
expression overlaps the expression domain of the lacZ reporter (Dou et
al., 1999) (data not shown).
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Figure 8.
Ectopic expression of BMPs is not
corrected in the NHAA rescue. BMP6
(A-C) and BMP7
(D-F) expression is restricted to the
dorsomedial telencephalic neuroepithelium in the BF1(+/)
heterozygote at E11.5 (arrows in A,
D). In the BF1 (/) mutant and the NHAA
rescue at E11.5, ectopic BMP6 and
BMP7 is detected in the lateral telencephalon.
The level of BMP6 and
BMP7 is highest dorsolaterally and
ventrolaterally (arrowheads). Scale bar, 200 µm.
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|
To determine the potential role of increased BMP activity in regulating
the proliferation and differentiation of telencephalic progenitor cells
in the BF1 (/) mutant, we examined the expression of
BMPs in NHAA rescue embryos. We find that
BMPs 2, 4, 6, and 7 are
ectopically expressed in the telencephalic neuroepithelium in a pattern
similar to that found in the BF1 (/) mutant (Fig. 8C,F) (data not shown). These findings show that DNA
binding activity is required for BF1 to repress the expression of
BMP genes. They also suggest that increased BMP
activity in the telencephalic neuroepithelium does not result in the
reduced progenitor cell proliferation rate observed in the
BF1 (/) mutant.
| |
DISCUSSION |
Replacement of endogenous BF1 with a DNA binding
defective form
We have adapted the tetracycline-regulated system developed by
Gossen and Bujard (1992) and Kistner et al. (1996) to control the expression of genes within the telencephalic neuroepithelium. A
mouse line, BF1(+/tTA), was
generated that expresses the tTA protein under the control of the BF1
promoter by targeting the tTA sequence to the BF1 locus by homologous
recombination. We also generated transgenic lines that contain
constructs expressing either WT BF1 or the DNA binding defective form,
BF1NHAA, under the control of
the tet-Operator. These new lines and the previously described
BF1(+/lacZ) line, were used
to generate embryos that lack endogenous BF1 and express either WT BF1
or BF1NHAA under the control of the tTA
protein in the telencephalic neuroepithelium. Examination of these
embryos reveals that some critical functions of BF1 in the developing
brain are mediated through DNA binding-independent mechanisms, whereas
others do require the ability to bind to DNA.
The utility of the BF1/tTA system is supported by the following
results: (1) The BF1NHAA
transcript is expressed in a pattern that closely matches that of the
endogenous BF1 protein. (2) We show that protein levels of
BF1NHAA in embryos that demonstrate a
phenotypic rescue, range from just below the level of BF1 in
BF1(+/) heterozygotes to slightly higher than the level of
BF1 in wild-type embryos. Thus, the
BF1NHAA protein functions at protein
levels in the physiological range. (3) The phenotypic changes observed
in the NHAA rescue are dependent on the expression of adequate levels
of the BF1NHAA protein. Three low
expresser lines have a phenotype that is indistinguishable from that of
the BF1 (/) mutant. Furthermore, when pregnant dams of
high expresser lines are given doxycycline to shut down the activity of
the tTA protein, the BF1NHAA
transcript is not expressed (Fig. 1I), and NHAA
rescue embryos are phenotypically indistinguishable from BF1
(/) mutants (data not shown). (4) Replacement of endogenous BF1
with WT BF1 using the tTA system rescues the growth and development of
the dorsal telencephalon. This occurs despite the relatively low
expression levels in the two transgenic lines that we have generated.
In contrast, rescue of ventral telencephalon development is poor. Further studies will be required to determine whether higher levels of
WT BF1 will restore the development of the ventral telencephalon with
the tTA system.
BF1 maintains the proliferation rate of telencephalic progenitor
cells during the neurogenetic period
We provide evidence that BF1 regulates the length of the cell
cycle throughout the neocortical neuroepithelium after E11.5. These
findings extend our previous work that showed that BF1 is required for
normal progenitor cell proliferation in the anterior third of the
dorsal telencephalon. The requirement for BF1 in progenitor cell
proliferation is not observed at earlier stages in development. Cell
proliferation in the neocortical neuroepithelium is unaffected by the
loss of BF1 from E8.5 when BF1 is first detectable in the neural plate,
to E11.5, a period of rapid cell proliferation (Wilson, 1982). Loss of
BF1 results in anomalies in the cell cycle only after E11.5, when the
cell cycle length begins to increase and neuronal differentiation is
initiated. Instead of a gradual increase in the cell cycle length
between E11.5 and E17.5, neocortical progenitors of mice lacking BF1
display a rapid increase in cell cycle length. This result suggests
that one function of BF1 may be to regulate the response of
telencephalic neuroepithelial cells to mitogenic or antiproliferative
signals. We have reported evidence that supports the latter
possibility. Telencephalic neuroepithelial cells lacking BF1
proliferate in culture at a rate similar to that of WT cells in
response to the growth factor, FGF-2. In contrast, primary cultures of
cortical progenitor cells lacking BF1 are more sensitive to the
antiproliferative activity of TGF and activin (Dou et al.,
2000).
BF1 regulates neocortical progenitor cell proliferation through a
DNA binding-independent mechanism
By examining the consequences of replacing endogenous BF1 with the
DNA binding defective BF1NHAA protein, we
show that the maintenance of progenitor cell proliferation in the
developing telencephalon by BF1 is mediated through a DNA binding-independent mechanism. BF1NHAA
enhances the growth of the telencephalic vesicles, compared with embryos lacking BF1. The size of the neocortex in the NHAA rescue embryos is restored to that of normal embryos. We show that the BF1NHAA protein is able to substitute for
the WT BF1 protein to maintain the proliferation of neocortical
progenitors, to normalize BrdU incorporation in the anterior dorsal
telencephalon and to reverse the accelerated lengthening of the cell
cycle in the dorsal telencephalon, associated with the loss of
BF1. Misexpression studies in Xenopus have led to
the proposal that repression of the cell cycle inhibitor p27 is an
important mechanism by which BF1 affects cell proliferation (Hardcastle
and Papalopulu, 2000). We find that mice lacking both BF1
and p27 have reduced telencephalic progenitor cell
proliferation as found in the BF1 mutant (C. Hanashima and
E. Lai, unpublished observations). This result together with the
findings in the NHAA rescue, suggest that repression of p27
expression may not be an essential mediator of the control of cell
proliferation by BF1. Our new findings support previous in
vitro studies that demonstrated that BF1 antagonizes the
antiproliferative activity of TGF through a DNA binding-independent
mechanism (Dou et al., 2000). We speculate that antagonism of the
antiproliferative activity of TGF family members may be an important
mechanism by which BF1 controls neocortical neurogenesis.
Control of neuronal differentiation in the neocortex: the role of
transcriptional repression by BF1
In contrast with the regulation of progenitor cell proliferation,
the inhibition of neuronal differentiation by BF1 is dependent on its
DNA binding activity. NHAA rescue embryos have a thicker mantle zone in
the dorsal telencephalon, compared with normal embryos of the same age.
The BF1NHAA protein does not inhibit the
withdrawal of neocortical progenitors from the cell cycle and delay
their differentiation into neurons. Our results suggest that regulation
of gene expression is the primary mechanism by which BF1 controls
neuronal differentiation. Because transcriptional repression has been
shown to be the predominant DNA binding-dependent activity of BF1, we
investigated potential regulatory targets that could mediate its
ability to inhibit neuronal differentiation.
We present evidence that multiple BMP genes are
targets of repression by BF1. BMPs 2, 4, 6, and 7 are ectopically expressed in the
telencephalic neuroepithelium of mouse embryos lacking BF1.
The BF1NHAA protein does not repress
ectopic expression of BMPs associated with
the absence of BF1. This is consistent with a requirement for DNA binding activity for BF1 to repress BMP
expression. BMP4 has been reported to inhibit the
proliferation of neocortical progenitor cells as well as to promote
their differentiation into neurons and glia (Li et al., 1998; Mabie et
al., 1999). Our results suggest that repression of
BMP expression by BF1 is not required for normal
proliferation rates of telencephalic progenitor cells. NHAA rescue
embryos exhibit normal growth of the dorsolateral telencephalon despite
having ectopic BMP expression. The inability of
the BF1NHAA protein to correct the ectopic
expression of BMPs may contribute to the
anomalies of the dorsomedial telencephalon in the NHAA rescue embryos.
Studies with additional regional markers are in progress to delineate
the mechanisms by which BF1 regulates dorsoventral patterning of the telencephalon.
BMPs exert their effects by binding to and activating a
complex of cell surface receptors. The type I receptors,
BMPR-1A and BMPR-1B, are expressed in the
neuroepithelium (Dewulf et al., 1995) and are believed to be the
primary transducers of BMP signals in the developing brain.
Expression of a constitutively activated BMP receptor
(caBmpr-1b) in the neuroepithelium results in premature neuronal differentiation in mid-gestation embryos, supporting a role
for this receptor in mediating a terminal response to BMPs, resulting in apoptosis or differentiation of neural progenitors (Panchision et al., 2001). Antagonism of BMP activity in
neocortical progenitor cells with a dominant-negative BMP
type I receptor or with noggin, results in the inhibition of neuronal
differentiation, demonstrating that endogenous BMP signals
trigger the differentiation of neocortical neurons (Li et al., 1998; Li
and LoTurco, 2000). Taken together with our studies, we infer that
ectopic BMP expression resulting from the
absence of BF1, promotes the differentiation of telencephalic
progenitors into neurons and that repression of
BMP gene expression by BF1 mediates its ability
to inhibit neuronal differentiation.
The key finding in this study is that the control of progenitor
cell proliferation and differentiation in the neocortex by BF1 are
mediated by distinct mechanisms. BF1 promotes the proliferation of
telencephalic neuroepithelial cells through a DNA binding-independent mechanism, whereas BF1 delays neuronal differentiation in the neocortex
through a DNA binding-dependent mechanism. This study also provides the
first genetic evidence that a WH gene has essential functions that do
not require the DNA binding activity of the WH domain. Finally, the
targeting of the tetracycline transactivator to the BF1 locus creates a
mouse line that will be a useful tool for the study of forebrain development.
| |
FOOTNOTES |
Received Oct. 17, 2001; revised March 11, 2002; accepted March 18, 2002.
*
C.H. and L.S. contributed equally to this work.
This work was supported by National Institutes of Health (NIH) Grants
HD29584 and EY11124 (E.L.), a grant from the Muscular Dystrophy
Association (S.L.), and a Cancer Center Support Grant from the
NIH. We thank Leah Brunk, Marissa DeVito, and Dina Lomonte for
excellent technical support and Willie Mark and the members of the
Transgenic Mouse Core Facility for help with the mice. We are grateful
to H. Bujard, S. McConnell, B. Hogan, and T. Jessell for providing
plasmids and Lorenz Studer for critically reviewing this manuscript.
Correspondence should be addressed to Eseng Lai, Cell Biology Program,
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY
10021. E-mail: lai{at}ski.mskcc.org.
| |
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TOP
ABSTRACT
INTRODUCTION
