1 Developmental Genetics Program and the Department of Cell Biology, The
Skirball Institute of Biomolecular Medicine, New York University Medical
Center, 540 First Avenue, New York, NY 10016, USA
2 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
* Author for correspondence (e-mail: fishell{at}saturn.med.nyu.edu)
Accepted 21 July 2005
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SUMMARY |
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Key words: Notch1, Notch3, Striatum, Patch, Matrix, Neural progenitor
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Introduction |
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The mature striatum is mosaically arranged into two distinct compartments,
the patch (or striosome) compartment and the surrounding matrix. Patch and
matrix neurons each display unique biochemical profiles and have different
functional properties arising from separate corticostriatal and nigrostriatal
afferent and efferent pathways (Gerfen,
1984; Gerfen,
1992
; Ragsdale and Graybiel,
1990
). Like the cerebral cortex
(Angevine and Sidman, 1961
;
Luskin and Shatz, 1985
;
Rakic, 1974
), neurons in the
striatal patches and matrix are generated in a precise developmental sequence,
with the majority of patch neurons being produced prior to those in the matrix
(van der Kooy and Fishell,
1987
). The earliest born patch neurons reside in the
ventrolateral-most region of the striatum, called the subcallosal streak (SCS)
(Song and Harlan, 1994
).
Little is known about the signals that regulate populations of neural
progenitor cells in the basal forebrain and their subsequent differentiation
into early- and late-born cell types in the striatum
(Halliday and Cepko,
1992
).
Notch signaling has been proposed to be a key regulator of the orderly
progression of cell types during forebrain development
(Schuurmans and Guillemot,
2002). Both Notch receptors and their Delta-Serrate-Lag2 (DSL)
ligands are expressed within the proliferative ventricular and subventricular
zones (VZ and SVZ, respectively) during neurogenesis
(Lindsell et al., 1996
).
Gain-of-function studies have revealed that constitutive Notch signaling leads
to cells remaining as progenitors
(Henrique et al., 1997
;
Mizutani and Saito, 2005
;
Ohtsuka et al., 2001
), whereas
decreased Notch activity is correlated with a reduction in neural progenitors
(Hitoshi et al., 2002
;
Yoon et al., 2004
;
Yoon and Gaiano, 2005
) and
increased neuronal differentiation (de la
Pompa et al., 1997
; Ishibashi
et al., 1995
). In addition, Notch signaling is thought to regulate
glial versus neuronal identity (Furukawa
et al., 2000
; Gaiano et al.,
2000
; Morrison et al.,
2000
; Wang et al.,
1998
). Radial glia are stem cells in the nervous system
(Anthony et al., 2004
;
Malatesta et al., 2000
;
Noctor et al., 2001
;
Noctor et al., 2004
), and
brain lipid-binding protein (BLBP), a marker of radial glia, has recently been
shown to be a direct target of the Notch signaling pathway
(Anthony et al.,
2005
). Although the majority of previous experiments have
focused on the role of Notch activity in early developmental events, such as
neurogenesis and cell fate determination, several studies have suggested that
the Notch pathway may also play important roles in postmitotic neurons. In
particular, in vitro experiments have implicated Notch signaling in regulating
the growth of neurites (Berezovska et al.,
1999
; Franklin et al.,
1999
; Redmond et al.,
2000
; Sestan et al.,
1999
).
Because Notch1 null mutants die at embryonic day 9.5 (E9.5)
(Conlon et al., 1995;
Swiatek et al., 1994
), a time
prior to formation of the nervous system, it has been impossible to examine
the role of Notch signaling in neurogenesis and in subsequent stages of
neuronal maturation in vivo. Neural progenitor cells sequentially give rise to
different types of neurons, from which it can be predicted that the loss of
Notch signaling would result in the production of early cell fates at the
expense of later-born cell types in the striatum, because the progenitor
population would become prematurely depleted in the absence of Notch activity.
However, at least one Notch receptor, Notch3, has been reported to
antagonize Notch1 activity on the basis of gain-of-function
experiments (Apelqvist et al.,
1999
; Beatus et al.,
1999
; Beatus et al.,
2001
). Notch3 null mutants are viable
(Krebs et al., 2003
) and
display some defects in vasculogenesis
(Domenga et al., 2004
), but
the function of Notch3 in striatal progenitor cells is at present
unclear. Moreover, the requirement for Notch signaling once cells exit the VZ
is unknown. Both Notch1 and RBP-J
(an intracellular
mediator of signaling through all Notch receptors) null mutants show signs of
precocious neuronal differentiation, although RBP-J
mutants
display more severe defects than Notch1 null mutants, suggesting that
another Notch family member may also play a role in forebrain neurogenesis
(de la Pompa et al.,
1997
).
Like Notch1, Notch3 is expressed by progenitor cells within the
forebrain (Lindsell et al.,
1996). To test the role of Notch1 and Notch3
receptors in regulating neurogenesis in the striatum, we have investigated the
phenotypes occurring in single and compound Notch1 conditional and
Notch3 null mutant animals. We used the Cre-LoxP system
(Sauer and Henderson, 1988
)
and two different Cre-driver lines to produce two distinct conditional
deletions of the Notch1 receptor. In one case, Notch1 is
removed throughout the telencephalon from the beginning of neurogenesis
onwards. In the second case, Notch1 is deleted only after cells have
exited the VZ in the ventral telencephalon. We have assessed striatal
development in Notch1 conditional; Notch3 null double mutant
mice in the context of both of these Cre-driver lines.
We show here that removing Notch1 in the forebrain prior to neurogenesis preferentially affects early-born neurons in the striatum, whereas later born cell types are generated normally. In addition, we demonstrate that Notch3 functionally compensates for the loss of Notch1 in the nervous system and mediates the conservation of late-born neurons in Notch1 conditional mutants. Notably, removal of Notch1 and Notch3 in cells after they have left the ventricular zone has no effect on striatal development. These experiments reveal that Notch signaling is not required in postmitotic neurons for their migration or the subsequent patterning of the striatum.
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Materials and methods |
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BrdU birthdating
Pregnant mice were injected with intraperitoneally with 2 mg of
bromodeoxyuridine (BrdU) (Sigma, St Louis, MO) in a solution of PBS with 7 mM
NaOH. BrdU was administered at E10.5, E11.5, E12.5, E13.5, E14.5 and E15.5,
and the embryos were subsequently allowed to develop until E18.5, at which
point the dams were terminally anesthetized and the embryos were removed and
perfused with 2% paraformaldehyde and postfixed for 2 hours at 4°C. At
least three mutants and three wild-type littermates were analyzed for each
time-point of BrdU administration.
Tissue preparation and in situ hybridization
Embryos were dissected in chilled PBS and fixed in either 2% or 4%
paraformaldehyde for four hours at 4°C, cryoprotected in 30% sucrose,
embedded in Tissue-Tek® OCT, and sectioned at a thickness of 14-16 µm
on a Leica CM3050 S cryostat. RNA in situ hybridization was performed as
previously described (Schaeren-Wiemers and
Gerfin-Moser, 1993; Wilkinson
and Nieto, 1993
). RNA probes were labeled with digoxigenin and
visualized with BM-Purple®, according the manufacturer's instruction
(Roche Biosciences). The following cDNA probes were used: Notch1, Notch2,
Notch3, Hes1, Hes5, Mash1, Neurod and Ebf1. Images were obtained
using a Diagnostics 4.2 camera and Spot Advanced software, and processed using
Adobe Photoshop.
Antibodies and immunohistochemistry
Rabbit anti-DARPP-32 (Chemicon International, Temecula, CA) was used at
1:500, rabbit anti-tyrosine hydroxylase (Chemicon International) was used at
1:500, rabbit anti-glutamate receptor 1 (Chemicon International) was used at
1:50, mouse anti-BrdU (BD Biosciences, San Jose, CA) was used at 1:100, and
rabbit anti-GFP (Molecular Probes, Eugene, OR) was used at 1:1000.
Ephrin-A4/Fc (R&D Systems, Minneapolis, MN) was used at 2 µg/ml.
Secondary antibodies conjugated with Cy3 or Alexa-488 were obtained from
Jackson ImmunoResearch Laboratories (West Grove, PA) and Molecular Probes, and
raised in goats. -human IgG-Alexa-488 (Molecular Probes) was used at
1:200. Fluorescent images were acquired using a cooled-CCD camera (Princeton
Scientific Instruments, NJ) and Metamorph software (Universal Imaging,
Downington, PA), and were processed using Adobe Photoshop.
Western blot
E12.5 mutant and wild-type brain lysates were prepared in 100 µl RIPA
buffer (10 mM Tris/HCl (pH 7.5), 140 mM NaCl, 1 mM orthovanadate, 1% Nonidet
P-40, 2 mM PMSF, 5 mM EDTA, 20 µg/ml aprotinin, 20 µg/ml leupeptin),
spun down and the supernatants were boiled in Laemli sample buffer. Proteins
were resolved by 8% SDS-PAGE and transferred onto a PVDF membrane for western
blot analysis. Rabbit anti-Notch1 (Upstate, Lake Placid, NY) followed by
peroxidase-conjugated anti-Rabbit IgG (Jackson ImmunoResearch Laboratories)
was used to detect the cleaved form of endogenous Notch1, and mouse anti-alpha
tubulin (Sigma-Aldrich) followed by peroxidase-conjugated anti-Mouse IgG
(Jackson ImmunoResearch Laboratories) was used to detect endogenous
tubulin.
Striatal analysis
Coronal sections from E18.5 telencephalon were double immunostained with
antibodies to Darpp32 and BrdU, and fluorescent images were obtained as
described above. Using Metamorph software (Universal Imaging, Downington, PA),
regions of Darpp32 immunoreactivity (which define the subcallosal streak and
striatal patches) were outlined and the number of BrdU-positive cells in each
compartment were counted. The matrix compartment of the striatum was defined
as the region remaining around the clusters of Darpp32-positive cells. Six
striatal sections were analyzed per animal and at least three mutant and three
wild-type littermates were analyzed for each time-point of BrdU administration
(E10.5-E15.5). Microsoft Excel was used to compute the data and perform the
statistical analyses. Student's t-test (one-tailed) was used to
compare the measurements of the mutant and wild-type animals at each
time-point, and statistical significance was determined with P-values
of less than 0.05.
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Results |
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Perturbation of early neurogenesis in Notch1 conditional mutants
The telencephalon of Foxg1Cre; N1 cKOs
exhibits striking morphological defects at E12.5, including a reduction in the
overall size of the developing forebrain. The subcortical regions of the
telencephalon, characterized by the lateral, medial and caudal ganglionic
eminences (LGE, MGE and CGE, respectively), are particularly affected in the
Foxg1Cre; N1 cKOs at E12.5. In
Foxg1Cre; N1 cKOs, the MGE, LGE
(Fig. 2A) and CGE (data not
shown) are severely diminished in size. These ventral eminences comprise
neural progenitors as well as their differentiating progeny. Both progenitor
cells (visualized by Hes1 and Hes5 expression,
Fig. 2A) and newly
differentiating neurons [identified by the expression of the neuron-specific
marker TuJ1, as shown by Yoon et al. (Yoon
et al., 2004)] are reduced in the Foxg1Cre;
N1 cKOs when compared with wild-type littermates at E12.5.
|
Mash1 mRNA is expressed by differentiating cells in the ventral
telencephalon and is upregulated in Foxg1Cre; N1
cKOs at E12.5 (Fig. 2A, right
panels). The increase in Mash1 expression is most apparent in cells
residing within the SVZ. In addition, the mutant SVZ appears to be thicker
than the wild-type SVZ, suggesting that cells precociously transit to the SVZ
in the absence of Notch1. A similar observation was made in
Mash1 mutant mice in which VZ cells in the LGE expressed SVZ markers
prematurely, a defect that the authors attributed to reduced Notch signaling
(Casarosa et al., 1999). Like
the forebrain, the developing retina of Foxg1Cre;
N1 cKOs displays increased proneural gene expression, and
Neurod is substantially elevated compared with wild-type littermates
(Fig. 2A, insets, right
panels). The upregulation of proneural genes in the eye and ventral forebrain,
suggests that cells precociously initiate neuronal differentiation at E12.5 in
the absence of Notch1. Although Foxg1Cre;
N1 cKOs display obvious morphological defects at E12.5, we were
surprised to note that the mutant brains appear relatively normal in their
overall morphology at E14.5 (Fig.
2B). Furthermore, levels of Hes1, Hes5 and Mash1
in the forebrain of Foxg1Cre; N1 cKOs appear
similar to those found in the forebrain of wild-type littermates at E14.5
(Fig. 2B), suggesting that
neurogenesis occurs normally at this time and that telencephalic development
recovers in Foxg1Cre; N1 cKOs. By contrast,
removing Notch1 in the embryonic mouse retina results in
abnormalities that progressively worsen during development and fail to improve
(Fig. 2A,B, insets, and H.A.M.
and G.F., unpublished).
|
There are several obvious defects in early-born striatal neurons in the absence of Notch1. First, the SCS appears thicker in Foxg1Cre; N1 cKOs than in controls, relative to the overall size of the striatum (Figs 3, 4, Fig. 5B). Second, the remaining patch clusters are fewer in number in these mutants, although the patches that do form tend to be larger in size than in wild-type embryos (Figs 3, 4, Fig. 5B). Finally, dopaminergic (Th-positive) fibers from the SN form aberrant projections in the forebrain of Foxg1Cre; N1 cKOs (Fig. 3B). Unlike wild-type littermates, which display an enrichment of Th-positive fibers in the SCS, as well as in numerous other patch compartments in the striatum, Foxg1Cre; N1 cKOs show expanded Th innervation of the SCS and fewer Th fibers forming characteristic clusters than in the wild-type striatum (Fig. 3B, upper panels). Because the dopaminergic fibers that innervate the striatum originate in the SN, an area of the midbrain that still retains Notch1 activity, the aberrant Th immunostaining in the mutants reflects the functional consequences of defects in the patch targets. In addition, dopaminergic innervation of the developing striatum is delayed in Foxg1Cre; N1 cKOs by 2 days when compared with wild-type embryos (data not shown). Much to our surprise, Foxg1Cre; N1 cKOs also display numerous ectopic Th-positive fibers in the cerebral cortex compared with wild-type embryos, which display few, if any, Th projections to the cortex (Fig. 3B, lower panels). Because the size of the SN is indistinguishable between mutants and wild-type littermates (data not shown), one possible explanation is that afferent fibers from the SN fail to find normal or adequate patch targets in the mutant striatum and subsequently form aberrant projections to the frontal cortex.
|
One explanation for the defects observed in the patch and SCS neurons in
the absence of Notch1 is that Notch signaling acts in progenitors to
preserve a sufficient number of progenitor cells to ensure the generation of
all striatal cell types. Without adequate Notch signaling, progenitor cells
may produce postmitotic neurons without replenishing themselves. However, the
defects observed in the patch compartment may also reflect changes in cell
proliferation, cell death, or neuronal differentiation and we have examined
each of these possibilities. We do not observe any obvious differences in the
proliferation of neural progenitors residing in the VZ (as assessed by BrdU
incorporation or markers for cycling cells such as Ki67 or phosphohistone-H3)
between wild-type and mutant mice (data not shown), indicating that the
increase in the SCS is not likely to be due to enhanced proliferation of SCS
neurons or their progenitors. Next, we examined whether cell death is
increased in Foxg1Cre; N1 cKOs using antibodies
that recognize cleaved caspase 3 and TUNEL labeling, both hallmarks of
apoptosis. The number of cleaved caspase 3 immunoreactive cells is increased
in Foxg1Cre; N1 cKOs when compared with wild-type
littermates from E12.5 through E16.5, as is the number of TUNEL-positive
nuclei (Mason et al., 2005).
However, even though cell death is increased in the absence of
Notch1, we could not find a selective increase in cell death at the
time when most patch neurons are generated (E12.5-E13.5) compared with later
time-points, when the bulk of matrix neurons are produced (E13.5 and later)
(Mason et al., 2005
). These
data suggest that there is a generalized increase in cell death in the absence
of Notch1 that equally affects patch and matrix neurons. Indeed,
Notch has been reported to regulate cell survival via mechanisms distinct from
its effects on neurogenesis (Oishi et al.,
2004
). Therefore, we favor a model in which Notch1 acts
in progenitors to control their differentiation as early-born cell types in
the striatum, and in which the loss of Notch1 activity results in a
majority of cells differentiating as SCS neurons at the expense of remaining
patch neurons.
|
The role of Notch3 in the development of the striatum in the absence of Notch1
It seemed likely that the normal development of matrix neurons in the
striatum of Foxg1Cre; N1 cKOs is mediated through
the activity another member of the Notch family of receptors. We examined both
Notch2 and Notch3 expression at E10.5 using in situ
hybridization and found detectable levels of Notch3 mRNA in the VZ
(Fig. 1D). By contrast,
Notch2 is present primarily within the epithelium of the choroid
plexus and not within the VZ at E10.5 (Fig.
1D). Thus, Notch3 seemed to be a more likely candidate
than Notch2 based on their expression patterns. However, previous
gain-of-function studies on Notch3 activity reported that
Notch3 is a weak activator of canonical Notch target genes and can
even inhibit Notch1 signaling
(Apelqvist et al., 1999;
Beatus et al., 1999
;
Beatus et al., 2001
).
Notch3 alone is not essential for striatal development because
Notch3 null mutants display normal patch and matrix compartments
(data not shown). However, to resolve whether or not Notch3
(N3) can functionally compensate for the loss of Notch1, we
examined the development of the striatum in conditional N1; N3 null
double mutants (Foxg1Cre; N1; N3 DKOs). These
double knockouts were generated by crossing N3 null mutants, which
are viable and fertile (Krebs et al.,
2003
), onto the Foxg1Cre; N1 cKO
background.
|
Notch1 and Notch3 act within the VZ to regulate the distinct cell types that form the compartments of the striatum
The defects observed in the Foxg1Cre; N1 cKOs
and the Foxg1Cre; N1; N3 DKOs could result either
from the lack of Notch signals in progenitor cells in the VZ, or during a
later developmental stage, such as migration and differentiation, as
Foxg1Cre results in the permanent removal of
Notch1 throughout the telencephalon. Notch signaling has been
reported to function in postmitotic neurons, such as in controlling neurite
morphology (Berezovska et al.,
1999; Franklin et al.,
1999
; Redmond et al.,
2000
; Sestan et al.,
1999
). In addition, Presenilin 1, a membrane protein responsible
for the cleavage activation of the Notch receptor
(De Strooper et al., 1999
;
Struhl and Greenwald, 1999
)
has been shown to play important roles in neuronal migration
(Louvi et al., 2004
). These
findings raise the possibility that the striatal disorganization observed in
our Foxg1Cre single and double mutants results from the
loss of Notch signaling during the migration and differentiation of neurons in
the striatum.
To test this idea, we selectively removed Notch function after cells exit
the VZ using Dlx5/6-Cre-IRES-EGFP transgenic mice
(Dlx5/6Cre), in which Cre recombinase is absent from the
striatal VZ and is expressed only when cells transit into the subventricular
zone (SVZ) and underlying mantle (Stenman
et al., 2003) (Fig.
7A). When the Dlx5/6Cre line is crossed to the
Z/EG recombination reporter line (Novak et
al., 2000
), EGFP permanently marks cells that have undergone
Cre-mediated recombination during their development. This fate-mapping
experiment reveals that the entire striatum, including both patch and matrix
compartments, has undergone recombination by postnatal day 1 (P1) in Z/EG;
Dlx5/6Cre mice (Fig.
7B). Therefore, the Dlx5/6Cre transgenic mice
will facilitate the removal of floxed Notch1 in all neurons in the
striatum but only after they exit the VZ. Because we have shown that
Notch3 functionally compensates for the loss of Notch1
(Fig. 6), we generated
Dlx5/6Cre conditional mutants on the Notch3 null
background (Dlx5/6Cre; N1; N3 DKOs). Unlike
Foxg1Cre; N1 mutants,
Dlx5/6Cre; N1; N3 DKOs survive into adulthood.
When we examined patch and matrix development in
Dlx5/6Cre; N1; N3 DKOs at P1
(Fig. 7C-D) or in adults (data
not shown), the mutant striatum appeared to be indistinguishable from the
wild-type striatum. The patch marker Darpp32
(Fig. 7C) and the matrix marker
Ebf1 (Fig. 7D) both
show normal patterns of expression in the Dlx5/6Cre double
knockouts at P1. Thus, activity through Notch receptors 1 and 3 is not
critical once cells are in the SVZ and mantle. Taken together, these results
suggest that Notch signaling is essential when cells are in the VZ to regulate
the distinct neuronal cell types found in the striatum. Once cells progress to
the SVZ and mantle, Notch signaling is not required for the subsequent stages
of maturation and morphogenesis that will ultimately form the mature
striatum.
|
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Discussion |
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We show that Notch3 gene function underlies the production of
late-born matrix neurons in Foxg1Cre; N1 cKOs, as
these cell types are severely impaired in Foxg1Cre;
N1; N3 DKOs. In addition, the generation of patch neurons is also
further compromised, revealing that Notch3 also plays a role early in
neurogenesis in the absence of Notch1. These results raise two
interesting points. First, they show that Notch3 is capable of
functioning in place of Notch1 to regulate stritatal neurogenesis.
Second, the defects observed in the early-born cell types in the
Foxg1Cre; N1 cKOs suggest that Notch3
cannot perfectly replace the activity of Notch1, a point that will be
discussed in more detail below. We interpret these results as indicating that
the loss of Notch1 alone results in an early temporal window of
severely compromised Notch signaling that in turn leads to specific defects in
the patch compartment. However, by E14.5 the overall forebrain morphology and
birthdating data suggests that neurogenesis is occurring normally in the
absence of Notch1. This contrasts sharply with other regions of the
developing CNS, such as the cerebellum and the eye, in which Notch1
removal alone results in severe, progressive and permanent defects
(Lutolf et al., 2002)
(Fig. 2).
Unlike later phases of neurogenesis, Notch3 alone is insufficient
for the normal development of patch neurons in the absence of Notch1.
One simple explanation may be that the levels of Notch3 are too low
early in neurogenesis to provide effective Notch signaling. Although it
remains a possibility that the selective expression of Notch3 in
matrix progenitors and not in patch progenitors underlies this difference, the
expression of Notch3 appears uniform in VZ progenitors. We therefore
favor a model in which progenitor cells giving rise to early- and late-born
neurons arise from distinct progenitor pools that require Notch signaling at
sequential times during development. One intriguing possibility is that the
mode of cell division is linked to the requirement of a progenitor cell for
Notch signaling. According to our data, the progenitors that are likely to be
dependent on Notch signaling are the ones in a neurogenic mode of division at
the time Notch1 is removed in Foxg1Cre;
N1 cKOs, which are the progenitors that are producing patch neurons.
Progenitors that give rise to late-born neurons appear to be in a
Notch-independent mode of division, most likely undergoing self-renewing
divisions that produce additional progenitor cells rather than post-mitotic
neurons. The progenitors that give rise to late-born neurons ultimately become
dependent on Notch signaling to regulate their differentiation (most likely
when they initiate neurogenic divisions) because matrix development is
severely impaired in Foxg1Cre; N1; N3 DKOs
(Fig. 6). This model is
consistent with the evidence that Foxg1Cre; N1
cKOs do not display any obvious defects during the initial phases of neural
development (between E9.5 and E10.5; Fig.
1B), a period characterized primarily by symmetric cell divisions
that amplify the progenitor population rather than neurogenic divisions. A
growing number of genes, including Numb and lethal giant larvae
1 (Lgl1) also appear to be required at the onset of neurogenesis
(Klezovitch et al., 2004;
Li et al., 2003
;
Petersen et al., 2002
;
Petersen et al., 2004
;
Shen et al., 2002
). These
genes may function to promote asymmetric divisions through interactions with
the Notch pathway.
The selective effect of Notch1 on early-born cells in the striatum
in Foxg1Cre; N1 cKOs, in conjunction with
previous studies that demonstrated that Notch activity prevents
differentiation and maintains a progenitor state
(Hitoshi et al., 2002;
Ohtsuka et al., 2001
),
suggested to us that Notch signaling is critical in neural progenitors that
reside in the VZ. However, Notch1 and Notch3 gene function
in Foxg1Cre; N1; N3 DKOs is also absent during
all subsequent development stages, including neuronal migration and
differentiation. It is therefore impossible to know from this analysis whether
Notch signaling is used iteratively for a variety of developmental steps. To
address the potential role of Notch signaling in later stages of neuronal
development, we used the Dlx5/6Cre driver line to remove
Notch signaling after cells have exited the VZ. In
Dlx5/6Cre; N1; N3 DKOs, both the patch and matrix
compartments develop normally (Fig.
7). These results suggest that Notch signaling is not required for
proper striatal patterning once cells have exited the VZ. Recent reports have
suggested that Notch activity is important for regulating neurite morphology
in postmitic neurons in the cortex
(Berezovska et al., 1999
;
Franklin et al., 1999
;
Redmond et al., 2000
;
Sestan et al., 1999
). The
present study did not examine axonal or dendrite morphology although it will
be interesting to address this question in future studies. The neuronal
migration defects in presenilin 1 mutants raised the possibility that
Notch signaling might be involved in migration, as Notch receptors require
cleavage by presenilins to be activated. However, our data supports the idea
the presenilin 1 exerts its effects on migration by acting on other proteins,
such as cytoskeletal proteins (Louvi et
al., 2004
). Therefore, Notch1 and Notch3 are not
necessary for the subsequent phases of neuronal development that ultimately
form the characteristic striatal mosaic, such as neuronal migration, the
segregating of SCS, patch and matrix neurons, and their ultimate
differentiation and expression of specific cellular and molecular markers. In
these mutants, it remains possible that Notch2 could be compensating
for the absence of Notch1 and Notch3. However, several
observations do not support this possibility. First, the fact that the
morphology of the Foxg1Cre; N1; N3 DKOs is
severely compromised (Fig. 6)
suggests that Notch2 activity is not sufficient to mediate normal
striatal development. Second, we do not observe Notch2 upregulation
in either Foxg1Cre or Dlx5/6Cre double
knockouts (data not shown). Third, we see no indication of Notch2
expression outside of the VZ at any time-point (data not shown).
Apart from the role of Notch in regulating neurogenesis in the VZ, the only
other developmental process we found to be affected when Notch signaling was
removed later (in neurons after they exited the ventricular zone) was for cell
survival. Specifically, we observed elevated levels of cell death in both
Foxg1Cre (Mason et
al., 2005) and Dlx5/6Cre double knockouts
(data not shown). These results suggest that Notch signaling plays a
generalized role in cell survival, and that in the absence of Notch1
and Notch3, cells have a higher probability of undergoing programmed
cell death during embryonic development. However, this increased apoptosis
appears to affect all types of neurons equally, as we could not find a
selective effect on either the patch or the matrix neurons in either of our
conditional knockouts.
In conclusion, our data indicates that early-born neuronal fates are selectively altered in the striatum of Foxg1Cre; N1 cKOs, whereas later born cell types are generated normally. Ectopic innervation of the cortex from midbrain dopaminergic fibers is observed in these mutants, most likely as a consequence of this defect. We further show that Notch3 can compensate for the loss of Notch1 in the generation of late-born matrix neurons in the striatum. Finally, we demonstrate that both the patch and matrix compartments develop normally when Notch1 and Notch3 are removed after cells have exited the VZ. The results pinpoint the critical window of Notch activity in progenitor cells in the VZ, and suggest that neurons can migrate and differentiate in the absence of additional Notch signaling.
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angevine, J. B. J. and Sidman, R. L. (1961). Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192,766 -768.[Medline]
Anthony, T. E., Klein, C., Fishell, G. and Heintz, N. (2004). Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 41,881 -890.[CrossRef][Medline]
Anthony, T. E., Mason, H. A., Fishell, G. and Heintz, N.
(2005). Brain lipid-binding protein is a direct target of Notch
signaling in radial glial cells. Genes Dev.
19,1028
-1033.
Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D. J., Honjo, T., Hrabe de Angelis, M., Lendahl, U. and Edlund, H. (1999). Notch signalling controls pancreatic cell differentiation. Nature 400,877 -881.[CrossRef][Medline]
Beatus, P., Lundkvist, J., Oberg, C. and Lendahl, U.
(1999). The notch 3 intracellular domain represses notch
1-mediated activation through Hairy/Enhancer of split (HES) promoters.
Development 126,3925
-3935.
Beatus, P., Lundkvist, J., Oberg, C., Pedersen, K. and Lendahl, U. (2001). The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity. Mech. Dev. 104,3 -20.[CrossRef][Medline]
Beckstead, R. M. and Kersey, K. S. (1985). Immunohistochemical demonstration of differential substance P-, met-enkephalin-, and glutamicacid-decarboxylase-containing cell body and axon distributions in the corpus striatum of the cat. J. Comp. Neurol. 232,481 -498.[CrossRef][Medline]
Berezovska, O., McLean, P., Knowles, R., Frosh, M., Lu, F. M., Lux, S. E. and Hyman, B. T. (1999). Notch1 inhibits neurite outgrowth in postmitotic primary neurons. Neuroscience 93,433 -439.[CrossRef][Medline]
Cai, L., Hayes, N. L., Takahashi, T., Caviness, V. S., Jr and Nowakowski, R. S. (2002). Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior. J. Neurosci. Res. 69,731 -744.[CrossRef][Medline]
Casarosa, S., Fode, C. and Guillemot, F.
(1999). Mash1 regulates neurogenesis in the ventral
telencephalon. Development
126,525
-534.
Conlon, R. A., Reaume, A. G. and Rossant, J.
(1995). Notch1 is required for the coordinate segmentation of
somites. Development
121,1533
-1545.
de la Pompa, J. L., Wakeham, A., Correia, K. M., Samper, E.,
Brown, S., Aguilera, R. J., Nakano, T., Honjo, T., Mak, T. W., Rossant, J. et
al. (1997). Conservation of the Notch signalling pathway in
mammalian neurogenesis. Development
124,1139
-1148.
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J. et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398,518 -522.[CrossRef][Medline]
Domenga, V., Fardoux, P., Lacombe, P., Monet, M., Maciazek, J.,
Krebs, L. T., Klonjkowski, B., Berrou, E., Mericskay, M., Li, Z. et al.
(2004). Notch3 is required for arterial identity and maturation
of vascular smooth muscle cells. Genes Dev.
18,2730
-2735.
Foster, G. A., Schultzberg, M., Hokfelt, T., Goldstein, M., Hemmings, H. C., Jr, Ouimet, C. C., Walaas, S. I. and Greengard, P. (1987). Development of a dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP-32) in the prenatal rat central nervous system, and its relationship to the arrival of presumptive dopaminergic innervation. J. Neurosci. 7,1994 -2018.[Abstract]
Franklin, J. L., Berechid, B. E., Cutting, F. B., Presente, A., Chambers, C. B., Foltz, D. R., Ferreira, A. and Nye, J. S. (1999). Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1. Curr. Biol. 9,1448 -1457.[CrossRef][Medline]
Frantz, G. D. and McConnell, S. K. (1996). Restriction of late cerebral cortical progenitors to an upper-layer fate. Neuron 17,55 -61.[CrossRef][Medline]
Fuccillo, M., Rallu, M., McMahon, A. P. and Fishell, G.
(2004). Temporal requirement for hedgehog signaling in ventral
telencephalic patterning. Development
131,5031
-5040.
Furukawa, T., Mukherjee, S., Bao, Z. Z., Morrow, E. M. and Cepko, C. L. (2000). rax, Hes1, and notch1 promote the formation of Muller glia by postnatal retinal progenitor cells. Neuron 26,383 -394.[CrossRef][Medline]
Gaiano, N., Nye, J. S. and Fishell, G. (2000). Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26,395 -404.[CrossRef][Medline]
Garel, S., Marin, F., Grosschedl, R. and Charnay, P.
(1999). Ebf1 controls early cell differentiation in the embryonic
striatum. Development
126,5285
-5294.
Gerfen, C. R. (1984). The neostriatal mosaic: compartmentalization of corticostriatal input and striatonigral output systems. Nature 311,461 -464.[CrossRef][Medline]
Gerfen, C. R. (1992). The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci. 15,133 -139.[CrossRef][Medline]
Graybiel, A. M. (1984). Correspondence between the dopamine islands and striosomes of the mammalian striatum. Neuroscience 13,1157 -1187.[CrossRef][Medline]
Graybiel, A. M. and Chesselet, M. F. (1984).
Compartmental distribution of striatal cell bodies expressing
[Met]enkephalin-like immunoreactivity. Proc. Natl. Acad. Sci.
USA 81,7980
-7984.
Graybiel, A. M., Ragsdale, C. W., Jr, Yoneoka, E. S. and Elde, R. P. (1981). An immunohistochemical study of enkephalins and other neuropeptides in the striatum of the cat with evidence that the opiate peptides are arranged to form mosaic patterns in register with the striosomal compartments visible by acetylcholinesterase staining. Neuroscience 6,377 -397.[CrossRef][Medline]
Halliday, A. L. and Cepko, C. L. (1992). Generation and migration of cells in the developing striatum. Neuron 9,15 -26.[CrossRef][Medline]
Hebert, J. M. and McConnell, S. K. (2000). Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev. Biol. 222,296 -306.[CrossRef][Medline]
Henrique, D., Hirsinger, E., Adam, J., Le Roux, I., Pourquie, O., Ish-Horowicz, D. and Lewis, J. (1997). Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina. Curr. Biol. 7, 661-670.[CrossRef][Medline]
Hitoshi, S., Alexson, T., Tropepe, V., Donoviel, D., Elia, A.
J., Nye, J. S., Conlon, R. A., Mak, T. W., Bernstein, A. and van der Kooy,
D. (2002). Notch pathway molecules are essential for the
maintenance, but not the generation, of mammalian neural stem cells.
Genes Dev. 16,846
-858.
Ishibashi, M., Ang, S. L., Shiota, K., Nakanishi, S., Kageyama, R. and Guillemot, F. (1995). Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects. Genes Dev. 9,3136 -3148.[Abstract]
Janis, L. S., Cassidy, R. M. and Kromer, L. F.
(1999). Ephrin-A binding and EphA receptor expression delineate
the matrix compartment of the striatum. J. Neurosci.
19,4962
-4971.
Klezovitch, O., Fernandez, T. E., Tapscott, S. J. and
Vasioukhin, V. (2004). Loss of cell polarity causes severe
brain dysplasia in Lgl1 knockout mice. Genes Dev.
18,559
-571.
Krebs, L. T., Xue, Y., Norton, C. R., Sundberg, J. P., Beatus, P., Lendahl, U., Joutel, A. and Gridley, T. (2003). Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation. Genesis 37,139 -143.[CrossRef][Medline]
Li, H. S., Wang, D., Shen, Q., Schonemann, M. D., Gorski, J. A., Jones, K. R., Temple, S., Jan, L. Y. and Jan, Y. N. (2003). Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis. Neuron 40,1105 -1118.[CrossRef][Medline]
Lindsell, C. E., Boulter, J., diSibio, G., Gossler, A. and Weinmaster, G. (1996). Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development. Mol. Cell Neurosci. 8, 14-27.[CrossRef][Medline]
Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: lessons from the retina. Nat. Rev. Neurosci. 2,109 -118.[CrossRef][Medline]
Loizou, L. A. (1972). The postnatal ontogeny of monoamine-containing neurones in the central nervous system of the albino rat. Brain Res.. 40,395 -418.[CrossRef][Medline]
Louvi, A., Sisodia, S. S. and Grove, E. A.
(2004). Presenilin 1 in migration and morphogenesis in the
central nervous system. Development
131,3093
-3105.
Luskin, M. B. and Shatz, C. J. (1985). Neurogenesis of the cat's primary visual cortex. J. Comp. Neurol. 242,611 -631.[CrossRef][Medline]
Lutolf, S., Radtke, F., Aguet, M., Suter, U. and Taylor, V. (2002). Notch1 is required for neuronal and glial differentiation in the cerebellum. Development 129,373 -385.[Medline]
Malatesta, P., Hartfuss, E. and Gotz, M.
(2000). Isolation of radial glial cells by fluorescent-activated
cell sorting reveals a neuronal lineage. Development
127,5253
-5263.
Mason, H. A., Rakowiecki, S. M., Gridley, T. and Fishell, G. (2005). Loss of Notch activity in the developing central nervous system leads to increased cell death. Dev. Neurosci. (in press).
McConnell, S. K. (1995). Strategies for the generation of neuronal diversity in the developing central nervous system. J. Neurosci. 15,6987 -6998.[Abstract]
McConnell, S. K. and Kaznowski, C. E. (1991). Cell cycle dependence of laminar determination in developing neocortex. Science 254,282 -285.[Medline]
Mizutani, K. and Saito, T. (2005). Progenitors
resume generating neurons after temporary inhibition of neurogenesis by Notch
activation in the mammalian cerebral cortex.
Development 132,1295
-1304.
Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster, G. and Anderson, D. J. (2000). Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101,499 -510.[CrossRef][Medline]
Murrin, L. C. and Ferrer, J. R. (1984). Ontogeny of the rat striatum: correspondence of dopamine terminals, opiate receptors and acetylcholinesterase. Neurosci. Lett. 47,155 -160.[CrossRef][Medline]
Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. and Kriegstein, A. R. (2001). Neurons derived from radial glial cells establish radial units in neocortex. Nature 409,714 -720.[CrossRef][Medline]
Noctor, S. C., Martinez-Cerdeno, V., Ivic, L. and Kriegstein, A. R. (2004). Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. 7,136 -144.[CrossRef][Medline]
Novak, A., Guo, C., Yang, W., Nagy, A. and Lobe, C. G. (2000). Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28,147 -155.[CrossRef][Medline]
Ohtsuka, T., Sakamoto, M., Guillemot, F. and Kageyama, R.
(2001). Roles of the basic helix-loop-helix genes Hes1 and Hes5
in expansion of neural stem cells of the developing brain. J. Biol.
Chem. 276,30467
-30474.
Oishi, K., Kamakura, S., Isazawa, Y., Yoshimatsu, T., Kuida, K., Nakafuku, M., Masuyama, N. and Gotoh, Y. (2004). Notch promotes survival of neural precursor cells via mechanisms distinct from those regulating neurogenesis. Dev. Biol. 276,172 -184.[CrossRef][Medline]
Olson, L., Seiger, A. and Fuxe, K. (1972). Heterogeneity of striatal and limbic dopamine innervation: highly fluorescent islands in developing and adult rats. Brain Res. 44,283 -288.[CrossRef][Medline]
Olsson, M., Bjorklund, A. and Campbell, K. (1998). Early specification of striatal projection neurons and interneuronal subtypes in the lateral and medial ganglionic eminence. Neuroscience 84,867 -876.[CrossRef][Medline]
Petersen, P. H., Zou, K., Hwang, J. K., Jan, Y. N. and Zhong, W. (2002). Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 419,929 -934.[CrossRef][Medline]
Petersen, P. H., Zou, K., Krauss, S. and Zhong, W. (2004). Continuing role for mouse Numb and Numbl in maintaining progenitor cells during cortical neurogenesis. Nat. Neurosci. 25,25 .
Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R. and Aguet, M. (1999). Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10,547 -558.[CrossRef][Medline]
Ragsdale, C. W., Jr and Graybiel, A. M. (1990).
A simple ordering of neocortical areas established by the compartmental
organization of their striatal projections. Proc. Natl. Acad. Sci.
USA 87,6196
-6199.
Rakic, P. (1974). Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183,425 -427.[Medline]
Redmond, L., Oh, S. R., Hicks, C., Weinmaster, G. and Ghosh, A. (2000). Nuclear Notch1 signaling and the regulation of dendritic development. Nat. Neurosci. 3, 30-40.[CrossRef][Medline]
Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R. and Nakanishi, S. (1992). Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6,2620 -2634.[Abstract]
Sauer, B. and Henderson, N. (1988).
Site-specific DNA recombination in mammalian cells by the Cre recombinase of
bacteriophage P1. Proc. Natl. Acad. Sci. USA
85,5166
-5170.
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100,431 -440.[CrossRef][Medline]
Schuurmans, C. and Guillemot, F. (2002). Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr. Opin. Neurobiol. 12, 26-34.[CrossRef][Medline]
Sestan, N., Artavanis-Tsakonas, S. and Rakic, P.
(1999). Contact-dependent inhibition of cortical neurite growth
mediated by notch signaling. Science
286,741
-746.
Shen, Q., Zhong, W., Jan, Y. N. and Temple, S. (2002). Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development 129,4843 -4853.[Medline]
Snyder-Keller, A. and Costantini, L. C. (1996). Glutamate receptor subtypes localize to patches in the developing striatum. Brain Res. Dev. Brain Res. 94,246 -250.[Medline]
Song, D. D. and Harlan, R. E. (1994). Genesis and migration patterns of neurons forming the patch and matrix compartments of the rat striatum. Brain Res. Dev. Brain Res. 83,233 -245.[Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stenman, J., Toresson, H. and Campbell, K.
(2003). Identification of two distinct progenitor populations in
the lateral ganglionic eminence: implications for striatal and olfactory bulb
neurogenesis. J. Neurosci.
23,167
-174.
Struhl, G. and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398,522 -525.[CrossRef][Medline]
Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G. and Gridley, T. (1994). Notch1 is essential for postimplantation development in mice. Genes Dev. 8, 707-719.[Abstract]
Takahashi, T., Nowakowski, R. S. and Caviness, V. S., Jr
(1996). The leaving or Q fraction of the murine cerebral
proliferative epithelium: a general model of neocortical neuronogenesis.
J. Neurosci. 16,6183
-6196.
Takebayashi, K., Akazawa, C., Nakanishi, S. and Kageyama, R.
(1995). Structure and promoter analysis of the gene encoding the
mouse helix-loop-helix factor HES-5. Identification of the neural precursor
cell-specific promoter element. J. Biol. Chem.
270,1342
-1349.
van der Kooy, D. and Fishell, G. (1987). Neuronal birthdate underlies the development of striatal compartments. Brain Res. 401,155 -161.[CrossRef][Medline]
Wang, S., Sdrulla, A. D., diSibio, G., Bush, G., Nofziger, D., Hicks, C., Weinmaster, G. and Barres, B. A. (1998). Notch receptor activation inhibits oligodendrocyte differentiation. Neuron 21,63 -75.[CrossRef][Medline]
Wichterle, H., Turnbull, D. H., Nery, S., Fishell, G. and
Alvarez-Buylla, A. (2001). In utero fate mapping reveals
distinct migratory pathways and fates of neurons born in the mammalian basal
forebrain. Development
128,3759
-3771.
Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225,361 -373.[Medline]
Yoon, K. and Gaiano, N. (2005). Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat. Neurosci. 8,709 -715.[CrossRef][Medline]
Yoon, K., Nery, S., Rutlin, M. L., Radtke, F., Fishell, G. and
Gaiano, N. (2004). Fibroblast growth factor receptor
signaling promotes radial glial identity and interacts with Notch1 signaling
in telencephalic progenitors. J. Neurosci.
24,9497
-9506.