1 Department of Pediatrics and Genetics and the Norris Cotton Cancer Center,
Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, 1 Medical Center
Drive, Lebanon, NH 03756, USA
2 Nina Ireland Laboratory of Developmental Neurobiology, Department of
Psychiatry, LPPI, University of California, 401 Parnassus, Box 0984, San
Francisco, CA 94143-0984, USA
Author for correspondence (e-mail:
mark.a.israel{at}dartmouth.edu)
Accepted 2 September 2004
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SUMMARY |
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Key words: Id4, Id2, Cortex, Hippocampus, G1-S, Cell cycle, Neurogenesis, CNS development
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Introduction |
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During early telencephalic development, there is an exponential expansion
of neuroepithelial progenitors in the VZ as the result of rapid, symmetric
cell divisions. The nuclei of these neuroepithelial cells go through
characteristic movements within the VZ, known as the interkinetic nuclear
movements. M-phase nuclei are found at the ventricular surface, while S-phase
nuclei are found away from the ventricle in the distal region of the VZ
(Takahashi et al., 1993;
Takahashi et al., 1995a
).
These early VZ cells (E11.5-E12.5) divide continuously their cell
cycle time is estimated to be about 8 hours
(Takahashi et al., 1993
;
Takahashi et al., 1992
)
leading to rapid lateral expansion of the neuroepithelium. From the VZ
emerge the cells that give rise to the secondary proliferative population
(SPP) in the subventricular zone (SVZ)
(Takahashi et al., 1995b
) and
differentiating neurons of the telencephalon.
Many genes known to affect proliferation and differentiation of the
developing mammalian nervous system are transcription factors. In particular,
members of the basic helix-loop-helix (bHLH) family have received much
attention because of the potency with which they are able to instruct cellular
differentiation (Guillemot,
1999). Consistent with this role in cell fate determination and
differentiation, many bHLH genes are expressed in a tissue-specific manner
(class B bHLH proteins). A sub-class of HLH genes, which lack the basic
DNA-binding domain, is known as Inhibitors of DNA binding or ID genes. The
proteins encoded by these genes act as dominant-negative regulators of bHLH
proteins by forming inactive heterodimeric complexes
(Benezra et al., 1990
;
Norton, 2000
;
Yokota, 2001
).
In mammals, there are four known ID gene family members. Id1, Id2,
Id3 and Id4 are expressed in progenitors of the central nervous
system (CNS) in overlapping but distinctive patterns
(Andres-Barquin et al., 2000;
Jen et al., 1997
). Consistent
with their expression patterns, the activities of ID1 and ID3 seem
functionally redundant in the nervous system. Deletion of either Id1
or Id3 individually does not lead to an observable phenotype, but
when they are both inactivated, telencephalic neurogenesis and angiogenesis
are greatly perturbed (Lyden et al.,
1999
). Mice lacking Id2 are viable, although they have an
abnormal immune system and a lactation defect
(Mori et al., 2000
;
Yokota et al., 1999
). Id1,
Id2 and Id3 have been shown to interact with cell cycle
regulatory molecules (Zebedee and Hara,
2001
). For example, they negatively regulate the expression of
cyclin D1, p16Ink4a and p21CIP1/WAF1/SDI1
(Alani et al., 2001
;
Lasorella et al., 1996
;
Prabhu et al., 1997
). In
addition, both genetic and biochemical evidence indicates that ID2 inhibits RB
(Retinoblastoma) protein function to enhance the G1 to S transition in
proliferating cells (Iavarone et al.,
1994
; Toma et al.,
2000
).
The role of the most recently identified member of the ID family,
Id4, in nervous system development has not been explored. In vitro
studies suggest that ectopic expression of Id4 prevents
oligodendrocytic differentiation (Kondo
and Raff, 2000). However, its role in regulating neural stem cell
proliferation and differentiation is not known. Here, we identify a role of
Id4 during cortical development in vivo and reveal a novel function
for Id4 in regulating cortical neurogenesis.
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Materials and methods |
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The PCR cycling conditions were 95°C for 30 seconds, 60°C for 40 seconds and 72°C for 1 minute for 30 cycles. The amplified PCR products were analyzed on gels to separate the wild-type (166 bp) and targeted (220 bp) allele fragments.
Ventricular surface area measurements
Coronal and saggital sections of Id4+/ and
Id4-/- tissues at matching levels of the CNS were stained
with Cresyl Violet and compared by measuring the VZ surface area using the
ImagePro program. Three separate measurements of the same sections were
averaged and compared. In each age group, three separate littermate brain
pairs, sectioned in coronal and saggital planes at similar angles and matched
to comparable levels along the rostrocaudal and mediolateral axis were
analyzed.
In situ hybridization
In situ hybridization on frozen sections was carried out using
35S-labeled antisense-riboprobes
(Yun et al., 2001). Each probe
was examined on three to five different mutant-control littermate pairs from
at least two independent crosses at each age shown. Photographs were taken
using dark-field optics on an Olympus SZH10 microscope. The cDNAs used in this
study included: Wnt3a (A. McMahon, Harvard University, USA);
Emx1 (J. Rubenstein, UCSF, USA); Dbx1 (T. Jessell, Colombia
University, USA); Ngn2 and NeuroD (F. Guillemot, NIMR, UK);
Tbr2 (A. Bulfone, TIGEM, Italy); and Id2 and Id4
(M. Israel, Dartmouth Medical School, USA).
Immunofluorescence
Labeling of S-phase cells by BrdU incorporation was performed by injecting
BrdU (1 mg/10 g body weight) into pregnant females and waiting for 45-60
minutes before sacrificing. A standard immunofluorescence protocol was used.
Primary antibodies used in this study included: phosphorylated Histone 3 (PH3)
(rabbit IgG; Upstate Biotech), BrdU (rat; Harlen), MAP2 (mouse IgG; Sigma,
catalog number M1406), PCNA (mouse IgG; Novocastra), TBR1 (gift of M. Sheng,
Harvard Medical School, USA). Confocal images of PCNA staining were collected
as 2 µm optical slices.
FACS scan analysis
E12.5 dorsal telencephalon from Id4-/- and control
littermates was dissected in cold PBS. Cells were dissociated in 0.05%
trypsin, rinsed, and resuspended in 100 µl of PBS. Cells were fixed in 70%
ethanol, rinsed, resuspended in 1 ml of solution containing 100 µg/ml
RNaseA/0.2% Triton X-100/50 µg/ml Propidium Iodide, and incubated at
37°C for 30 minutes. Stained cells were scanned in a Becton Dickinson (San
Jose, CA) FACScan flow cytometer, and the cell cycle profile was quantitated
using ModFit software.
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Results |
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Reduced dorsomedial progenitor zones in the Id4-/- mutant
To determine whether the smaller cortex in Id4-/-
animals reflected the loss of a specific region or an overall reduction in all
cortical regions, we examined the expression of molecular markers that
distinguish different progenitor zones. Genes expressed at the dorsal and
ventral extremes of the pallial (cortical) progenitor zone appeared normal.
For example, Wnt3a expression in the cortical hem
(Lee et al., 2000) was
unchanged in mutant animals (Fig.
2A,A'). In addition, no consistent differences were found in
the expression patterns of genes marking the ventral and lateral pallium
progenitors (Dbx1, Emx1, Tbr2 and Wnt7b;
Fig. 2B-D', and not
shown), or the mantle (not shown) (Puelles
et al., 2000
; Yun et al.,
2001
) in Id4-/- animals. To quantitate the
difference in the surface area of the cortical proliferative zone, we measured
the cortical VZ width on matched coronal sections from littermate control and
Id4-/- animals. We found progressive shortening of the VZ
surface area (Fig. 2G) and
consistent reduction in the hippocampal primordium (approximately 50%,
Fig. 2H) in
Id4-/- animals. These findings, in the context of the
histological analysis shown in Fig.
1, indicate that the hypoplasia of the Id4-/-
telencephalon most severely affects dorsomedial structures.
|
To determine whether the molecular abnormalities associated with the loss of Id4 also contributed to cortical hypoplasia by enhancing progenitor cell death, we examined apoptosis in the developing telencephalon of Id4-/- animals. We did not detect differences in the number of apoptotic cells in the Id4-/- and control telencephalon at E11.5, E12.5 or E15.5 (data not shown). Furthermore, the insertion of DNA encoding GFP into the Id4 locus allowed the identification of cells in which Id4 transcription would occur. GFP-expressing cells were clearly present in Id4-/- mutants at E12.5 (Fig. 2F'), indicating that the loss of Id4 function does not result in extensive cell death.
Precocious differentiation of early cortical progenitors
To determine whether the dorsomedial cortical progenitor zones are reduced
as a result of precocious differentiation or compromised proliferation of
neural stem cells, we first examined early neurogenesis in the mutant
telencephalon. At E11.5, cortical neurogenesis is just beginning in the dorsal
and medial pallium, whereas in the more ventral regions of the cortex, the
ventral and lateral pallium, neurons are produced from E10.5. Expression of
pan-neuronal markers at E11.5 (MAP2 and ßIII-tubulin,
Fig. 3A,A', and not
shown) was reproducibly increased throughout the postmitotic zone of the
Id4-/- cortex, suggesting precocious neurogenesis.
Therefore, we examined the expression of other genes that either regulate
neurogenesis in cortical progenitors or are markers of cortical neurons. At
E11.5, neurogenin 2 (Ngn2; Neurog2 Mouse Genome
Informatics), a proneural bHLH gene (Fode
et al., 2000; Gradwohl et al.,
1996
), was expressed in a similar pattern in both wild type and
Id4-/- animals. However, the expression of markers
identifying early differentiating neurons, NeuroD
(Fig. 3C,C') and
Tbr2 (arrow in Fig.
2D,D'), was consistently increased in the mantle zone of the
dorsomedial cortex in Id4-/- animals. Furthermore,
scattered Tbr2-expressing cells were found ectopically in the VZ
(dorsal to its normal expression domain in the VP and LP
(Bulfone et al., 1999
;
Yun et al., 2001
)) of the
dorsal and medial pallium (arrowhead in
Fig. 2D,D'). Each of
these findings is indicative of precocious differentiation of early cortical
progenitors.
|
Prolonged G1-S transition in the Id4-/- cortex
To determine whether defects in progenitor proliferation also contributed
to the Id4-/- mutant phenotype, we examined markers of
cell cycle phases and found that G1-S transition of the Id4-/-
neuroepithelial cells is compromised at E12.5. We compared progenitor cells in
wild-type and Id4-/- cortex by analyzing the expression of
markers that identify all cells actively proliferating (PCNA, proliferating
cell nuclear antigen), or cells in M-phase (PH3: Phosphorylated Histone 3), or
cells in S-phase, by a short pulse of BrdU (Bromodeoxyuridine)-labeling. At
E12.5, the density of proliferating cells in the VZ is indistinguishable in
wild-type and Id4-/- animals (PCNA+ cells in
Fig. 4A,A',D'), but
the VZ is approximately 15% thicker in the Id4-/- mutant
(see Fig. 1C,C',
Fig. 4A,A'). To determine
whether this increased thickness could be attributed to cells at a particular
phase of the cell cycle, we first examined cells in M phase. We found that the
actual number of cells in M-phase was indistinguishable in the wild-type and
Id4-/- animals at this age
(Fig. 4C,C', and not
shown). BrdU labeling at E12.5 identified cells that were in S phase
(Fig. 4B,B'). Although
the thickness of the S-phase zone varied within the cortical VZ (thinner
dorsomedially) in the mutant animal, the most striking difference between the
control and Id4-/- animals we found was in the thickness
of the zone corresponding to the G-phases of the cell cycle. The G-phase zone
is sandwiched between the BrdU-positive S-phase cells
(Fig. 4B,B') and the
PH3-positive M-phase cells (Fig.
4C,C'). The thickening of this zone (distance between the
arrowheads in Fig. 4B,B')
clearly accounts for the overall increased thickness of the VZ in mutant
animals. The presence of cycling cells in this region, as identified by PCNA
expression (Fig.
4A,A',D'), suggested that progenitor cells in the
Id4-/- animal might make a prolonged transition between
G1-S and/or G2-M phases of the cell cycle. We, therefore, evaluated the cell
cycle distribution of cortical cells isolated from E12.5 dorsal telencephalon
by FACS analysis. In multiple experiments (n=7) examining cells from
E12.5 or E13.5 littermate embryos, we invariably observed a reduction in the
percentage of cells in S-phase and a corresponding increase in the percentage
of cells in G0/G1, as indicated in representative experiments from E12.5 shown
in Table 1. In wild-type
animals at E12.5, virtually all VZ cells are dividing and no G0 cells are
found among them (Takahashi et al.,
1995a). Our finding that the thicker mutant VZ is composed of
PCNA+ proliferating cells (Fig.
4A',D') indicates that the increased percentage of
cells in G0/G1 detected by FACS analysis
(Table 1) represents a
prolonged G1 in the mutant cells at this age. Although the increased number of
prematurely differentiating cells observed at this age may also contribute to
the higher percentage of G0/G1 cells in the ID4 mutants, the fact that most
cells in the G-phase zone are PCNA positive
(Fig. 4A',D')
indicates that a contribution of prematurely differentiating cells to the
thickening of the VZ is unlikely. Together, these two independent lines of
investigation indicate that Id4 is required for normal G1-S
transition in early neural stem cells. Thus, the reduced brain size in the
Id4-/- cortex results from both premature differentiation
and compromised cell cycle transition of early neural stem cells.
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Discussion |
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Requirement for Id4 during early stem cell proliferation
Neural epithelium of the developing cortex expands laterally through the
rapid division of early stem cells. Cortical neurogenesis commences at around
E11.5 and the onset of an observable Id4-/- phenotype coincides
with this major milestone in cortical development. At E12.5, the mutant
cortical VZ lateral expansion is greatly compromised, and a progressively
reduced cortical VZ area is found from this time forward. For example, at
E15.5, the cortical VZ surface area is less than 50% of the control littermate
brains (60% mediolaterallyx
70% rostrocaudally). At E12.5, the
Id4 mutant VZ is thicker and this thickening can be attributed to a
prolonged G1 phase of the cell cycle (Fig.
4 and Table 1).
This phenotype is consistent with in vitro studies indicating that ID genes
enhance proliferation by facilitating the G1-S transition. For example, ID2
has been shown to bind to the RB (Retinoblastoma) protein thereby enhancing
the G1-S transition (Iavarone et al.,
1994
), and other ID genes have been shown to regulate the
expression of the G1 cyclin-dependent kinase inhibitors p16 and p21 at the
transcriptional level (Alani et al.,
2001
; Lasorella et al.,
1996
; Prabhu et al.,
1997
). ID genes are in turn regulated during the cell cycle where
their peak level of expression is observed early in G1, after serum
stimulation (Christy et al.,
1991
; Hara et al.,
1994
). However, our analysis of these G1-S regulators in the
Id4-/- telencephalon did not reveal significant changes in
their expression levels (data not shown), suggesting that Id4 does
not regulate their expression during cortical development. We are currently
examining the possibility that ID4 interacts with these molecules at the
protein:protein level to affect their function.
Our findings provide evidence supporting an integrated proliferative
regulatory mechanism that coordinates the timing, the number and the neuronal
specificity of cortical neurons, as proposed by Caviness and his colleagues in
their theoretical model (Caviness et al.,
2003). Their model predicts that increasing the proportion of
daughter cells exiting the cell cycle early will lead to reduced lateral
expansion of the neuroepithelium, as a result of the reduced proportion of
daughter cells available to expand the proliferating population. It also
predicts that the early cortical plate will be transiently thicker (because of
an increased output of postmitotic cells), but significantly thinner at the
end of the neurogenic period because of the loss of stem cells early on. In
addition, an increase in the proportion of daughter cells exiting the VZ is
associated with a lengthening of the cell cycle time, particularly G1, during
normal development. With the exception of a thicker, rather than thinner,
cortical plate in the Id4 mutant at E18.5, the Id4 mutant
phenotype is consistent with this model. Hence, our findings generally support
the existence of an integrated regulatory mechanism, and identify Id4
as a key regulator that controls the time domain, the output domain, and
neuronal specification during cortical development.
Role of ID family members in telencephalic development
We were intrigued by the region-specific requirement for ID4 function, and
the relatively mild mutant phenotype at P0 despite the early loss of stem
cells due to both premature differentiation and compromised cell cycle
transition. As Id1, Id2 and Id3 are also expressed in the
developing telencephalon, these ID genes may functionally compensate for
Id4 in non-affected areas. For example, mutational studies of
Id1 and Id3 have revealed that they are functionally
redundant during telencephalic development. To examine this possibility, we
monitored expression levels of other ID genes in Id4 mutant
telencephalon. Indeed Id1, Id2 and Id3 expression levels
were increased in the Id4 mutant, when compared with a control
littermate (Fig. 2E' and
not shown). In particular, Id2 expression was altered both spatially
(Fig. 2E,E') and
quantitatively (not shown). Hence, we examined possible functional
redundancies between Id2 and Id4 by analyzing Id2/4
double null embryos. We were unable to detect evidence of altered
telencephalic development in Id2-/- animals (data not
shown). Interestingly, the brain size of the Id2/4 double null animal
was only slightly smaller than that of the Id4 single mutant (data
not shown), and no obvious defects were found in other areas of the
telencephalon. This observation indicates that Id2 is not necessary
to compensate for Id4 in other areas, and supports the idea that ID4
function is unique and necessary for the development of the telencephalic
midline tissue. Id2/4 double mutant analysis also suggests the
existence of other compensatory mechanisms, either at the molecular level by
Id1 or Id3, or at the cellular level (see below).
Role of Id4 in differentiating neurons
In contrast to the reduced proliferation observed in the early
neuroepithelium, the number of proliferating cells outside of the VZ in the
Id4-/- brain after E15.5 is dramatically increased
(Fig. 4). These ectopically
positioned cells proliferate, as indicated by both S-phase and M-phase
markers, and differentiate, as indicated by NeuroD expression, in ectopic
positions. These results indicate a potential compensation mechanism at the
cellular level for the reduced proliferation of early VZ progenitors, and may
account for the relatively mild Id4 mutant phenotype observed at
birth.
Ectopically positioned proliferating cells found in the
Id4-/- cortex are reminiscent of changes observed in
animals lacking the Rb gene. In Rb-/- animals,
ectopically positioned proliferating cells are found in the cortex, and this
phenotype has been interpreted as indicating that differentiating mantle cells
have aberrantly re-entered the cell cycle in the absence of functional RB
(Clarke et al., 1992;
Lee et al., 1992
;
Lipinski et al., 2001
;
MacPherson et al., 2003
). We
therefore examined Rb levels in Id4-/- animals,
but we did not observe a significant difference in its expression, either at
the RNA or protein level (not shown). ID2 has been shown to bind RB protein
and to inhibit its function post-translationally
(Iavarone et al., 1994
).
Hence, a possible explanation for the observed phenotype is that the increased
level of Id2 expression in the Id4 mutant
(Fig. 2E') leads to
inactivation of RB. However, direct examination of this hypothesis indicates
that this phenotype is not the result of an epigenetic event (increased
Id2 levels). The Id2/4 double mutant cortex also contained
many ectopically proliferating cells in the mantle region (not shown). These
observations suggest that if the Id4 phenotype were mediated through
the RB pathway, ID4 most likely acts to enhance RB-mediated inhibition of
proliferation, in contrast to ID2, which negatively regulates RB. Among the
many possibilities, direct interactions can be envisioned in which ID4
competes with ID2 for RB binding in differentiating neurons. Or, ID4 may
directly interact with other molecules in the cell cycle machinery, and
depending on the interacting partner and cellular context, either enhance or
inhibit cell cycle transition.
Id4 interaction with bHLH factors
ID genes have been shown to inhibit differentiation in many different cell
types in vitro and in vivo (Norton,
2000). The main mechanism of this potent inhibitory effect is
attributed to its ability to form dominant-negative dimers with bHLH proteins.
In the developing cortical progenitor zone, at least three proneural bHLH
genes, Ngn1, Ngn2 and Mash1, are important for progenitor
proliferation, cell fate specification, and differentiation
(Fode et al., 2000
;
Nieto et al., 2001
;
Parras et al., 2002
;
Ross et al., 2003
). As ID
proteins are thought to act as negative regulators of bHLH proteins by
sequestering available E-proteins, we have postulated that proneural bHLH
proteins are hyper-active in the Id4 mutant cortex. Consistent with
this model, the Id4 mutant phenotype correlates well with both
loss-of-function and gain-of-function studies of these proneural genes
(Fode et al., 2000
;
Parras et al., 2002
;
Sun et al., 2001
). For
example, ectopic expression of Ngn1 clearly promotes neuronal
differentiation (Sun et al.,
2001
), and the simultaneous loss of Ngn2 and
Mash1 function results in reduced neurogenesis (and NeuroD
expression), particularly in the dorsomedial cortex
(Fode et al., 2000
). In
Id4-/- animals, where proneural bHLH proteins are
presumably free from ID4 inhibition, neural stem cells prematurely
differentiate and this is most obvious in the dorsomedial region
(Fig. 2D,D',
Fig. 3A,A',C,C'). It is unclear, at present, why the dorsomedial region of the telencephalon is
particularly sensitive to both positive and negative HLH protein activity, but
these in vivo studies reveal an important requirement for bHLH transcription
factor function in this area, and emphasize the role of Id4 in
regulating its development.
CNS development requires complex molecular interactions that control progenitor proliferation and differentiation, and Id4 can now be recognized to be important for these processes. Our finding that Id4 inhibits neural progenitor differentiation is consistent with the assignment of this function to other ID family members. In addition, we provide in vivo evidence for a novel function of Id4 in preventing aberrant cell cycle entry in differentiating neurons. These observations underscore the importance of cell context for ID gene function, and in particular for Id4, which either enhances or blocks cell cycle transition in the same developing tissue at different times. This study reveals a crucial role for Id4 in cortical development, and identifies the molecular pathways over which its effects are mediated, while suggesting important new areas for future study.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Psychiatry and Neurosciences, Division
Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima
University, Hiroshima, 734-8551, Japan
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