There is tremendous interest in adult neurogenesis in the mammalian
brain, both from the perspective of the normal role of new neurons
in circuit plasticity and of the potential for brain repair implied by
the persistence of adult neurogenesis (Gage, 2000). There are two
well-established regions of adult neurogenesis in the rodent brain –
the subventricular zone (SVZ) and the dentate gyrus (DG). The SVZ
generates new olfactory interneurons involved in plasticity in the
adult olfactory system; and in the DG, new granule neurons are
believed to be involved in learning and memory.
Interestingly, there are stark contrasts in the developmental plan
used to form these two adult neurogenic niches. Many studies
have indicated that the SVZ and the rostral migratory stream are
remnants of the embryonic SVZ (Merkle and Alvarez-Buylla,
2006; Merkle et al., 2004). By contrast, the DG uses a very
distinct developmental plan to produce a durable neurogenic
niche (Li and Pleasure, 2005). The multi-potential neural
precursors seed the developing dentate gyrus beginning around
mid-gestation from their origin in the medial cortical
neuroepithelium. As the scaffolding of the DG forms around the
first postnatal week, the neurogenic precursors settle at the border
between the granule cell layer (GCL) and the hilus, also called the
subgranular zone (SGZ). Migration of multi-potential precursors
from one neurogenic zone (the dentate ventricular zone) to a
nascent neurogenic zone (SGZ) has long been recognized to be a
unique reorganization for the cortex, sharing some features with
the migration of granule cell precursors in the cerebellum.
Previous studies have implicated the Wnt and Shh pathways as
regulators of precursor behavior in the embryonic ventricular
zone and SVZ, as well as the perinatal conversion of the SVZ to
its adult state (Galceran et al., 2000; Machold et al., 2003; Pozniak
and Pleasure, 2006; Zhou et al., 2004). In addition, both of these
pathways are involved in the maintenance of precursors in the
postnatal and adult DG (Lai et al., 2003; Lie et al., 2005).
However, it is not clear what factors regulate the behaviors of
precursors during transit to the DG. In this study, we provide
evidence that en route to the DG, many precursors are localized
in a specialized temporary neurogenic zone before they occupy
the nascent DG. Interestingly, the organization of this zone is
controlled by Cajal-Retzius cell-derived reelin and meningeally
produced Cxcl12. By genetic fate-mapping analysis, we also
show that subpial precursors may contribute to the SGZ
MATERIALS AND METHODS
Most mouse strains (Cxcr4, reeler, Emx1ires-cre, Rosa-lacZ, Rosa-YFP and
Z/EG) were obtained from the Jackson Laboratory (Bar Harbor, Maine).
Cxcr4-flox line was kindly provided by Dr Dan Littman (New York
University), Nestin-GFP transgenic line by Dr Masahiro Yamaguchi (Japan),
Gli1CreERT2line by Dr Alexandra L. Joyner (Sloan-Kettering) and Rosa26-
PTX was generated in Dr Shaun R. Coughlin’s laboratory (UCSF). All the
lines were maintained in the C57/B6 genetic background. The day of vaginal
plug was considered to be embryonic day 0.5 (E0.5). Mouse colonies were
housed at the University of California, San Francisco, in accordance with
National Institutes of Health and UCSF guidelines.
Details can be provided on request.
In situ hybridization (ISH)
In situ hybridization protocol and probes (Cxcl12 and Cxcr4) were used as
described (Li et al., 2008a).
In utero electroporation and DiO Injection
Details can be provided on request.
Glial process tracing
A series of overlapping thin section confocal images were taken with LSM
510 meta two-photon microscope (Carl Zeiss, Inc.). BLBP glial processes
were traced with NIH ImageJ program.
Identification of a transient subpial neurogenic zone in the
developing dentate gyrus and its regulation by Cxcl12 and
Guangnan Li1,*, Hiroshi Kataoka2,3, Shaun R. Coughlin2,3,4and Samuel J. Pleasure1,4,5,*
One striking feature of dentate gyrus development, distinct from the other cortical structures, is the relocation of neural precursors
from the ventricular zone to the forming dentate pole to produce a lifelong neurogenic subgranular zone (SGZ). In this study, we
demonstrate that dentate progenitors first dwell for up to 1 week in a previously unrecognized neurogenic zone intimately
associated with the pial meningeal surface lining the outer edge of the forming dentate. This zone also serves as the organizational
matrix for the initial formation of the dentate glial scaffolding. Timely clearance of neural precursors from their transient location
depends on reelin, whereas initial formation of this transient stem cell niche requires Cxcl12-Cxcr4 signaling. The final settlement of
the neural precursors at the subgranular zone relies on a pertussis toxin-sensitive pathway independent of Cxcl12-Cxcr4 signaling.
Furthermore, genetic fate-mapping analysis suggests that subpial precursors contribute to the SGZ formation. These results
demonstrate that the relocation of neural precursors in the dentate gyrus consists of discrete steps regulated by multiple pathways.
KEY WORDS: Dentate gyrus, Meninges, Neurogenesis
Development 136, 327-335 (2009) doi:10.1242/dev.025742
1Department of Neurology, 2Cardiovascular Research Institute, 3Department of
Medicine, 4Program in Developmental Biology and 5Program in Neuroscience, UCSF
School of Medicine, San Francisco, CA 94158, USA.
*Authors for correspondence (e-mail: email@example.com; firstname.lastname@example.org)
Accepted 12 November 2008
Timed pregnant mice were intraperitoneally (i.p.) injected with BrdU
(Roche) dissolved in 1?PBS (10 mg/ml) at the dose of 100 mg/kg animal
for acute labeling or 50 mg/kg animal for birthdating analysis.
Tamoxifen induction and lacZ staining
Tamoxifen (TM, Sigma) was dissolved in corn oil (Sigma) at 20 mg/ml.
Pregnant females were dosed i.p. with 3 mg of tamoxifen/40 g animal.
Embryos were collected at the specified ages and lacZ staining was carried
out as described (Li et al., 2008b).
Details can be provided on request.
A transient subpial neurogenic zone in the
Pioneering studies by Altman and Bayer (Altman and Bayer, 1990a;
Altman and Bayer, 1990b) showed that proliferative dentate
precursors leave the dentate neuroepithelium and form a migratory
stream into the forming dentate gyrus during late embryonic
development. To better characterize the spatiotemporal dynamics of
the precursors in the dentate migratory stream in mice, we decided
to re-examine this framework with new molecular markers. Using
Nestin-GFP transgene and Tbr2 to label the dentate precursors and
neurogenic transit amplifying cells, respectively (Englund et al.,
2005; Yamaguchi et al., 2000), we revealed the sequential
distribution of these cells during initial dentate gyrus formation.
By E14.5, the domain adjacent to the cortical hem in the
ventricular zone (VZ) representing the dentate primordium was
marked by strong Nestin-GFP expression (Fig. 1A,A1).
Concurrently, Tbr2+ cells were abundant in the subventricular zone
(SVZ) and a stream of Tbr2+ cells stretched from the SVZ above the
forming fimbria to form a line of cells along the pial surface (Fig.
1A2,A4). A day later at E15.5, Nestin-GFP+ cells emanating from
the dentate notch formed a narrow stream oriented toward the
subpial region at the junction between the fimbria and forming
dentate gyrus (Fig. 1B); we have termed this the fimbriodentate
junction (FDJ). At high magnification, Tbr2+ cells were also found
Development 136 (2)
Fig. 1. Progressive development of the dentate
neurogenic zones is revealed by Nestin-GFP transgene
and Tbr2. (A)At E14.5, Nestin-GFP is upregulated in the
dentate primordium abutting the cortical hem, which is
shown in higher magnification in (A1), with a few Nestin-
GFP+ cells leaving the dentate VZ. (A2)Tbr2+ cells, which
represent neurogenic precursors, have migrated away from
the SVZ (arrowheads). (A3)The fimbria has started to form in
the relatively cell-free region of the cortical hem (shown by
nuclei staining with DAPI). (A4)The organization of the
medial wall with all the markers. Scale bar: 200μm in A;
50μm in A1-A4. (B)At E15.5, Nestin-GFP+ cells (green)
formed a new neurogenic zone in the subpial region of the
fimbriodentate junction (FDJ). (B1-B4) The distribution of
Nestin-GFP+ and Tbr2+ cells in the subpial region of the FDJ.
Some of the Nestin-GFP+ cells (arrowheads in B1 and B2)
were Tbr2– (arrowheads in B3 and B4). Scale bar: 100μm in
B; 20μm in B1-B4. (C)At E17.5, Nestin-GFP+ cells were
distributed across the hilus. (C1-C3) The distribution of
Nestin-GFP+ cells (C1,C3), Tbr2+ cells (red, C2,C3) and DAPI
(blue, C3) at higher magnification. Scale bar: 200μm in C;
50μm in C1-C3. (D)At P0, the subpial neurogenic zone was
established around the edge of the dentate pole. (D1-D3)
Both Nestin-GFP+ and Tbr2+ cells were largely subpially
localized in the forming dentate pole (arrowheads). Scale
bar: 200μm in D; 100μm in D1-D3. (E)At P2, the
subgranular zone (SGZ) started to take shape. (E1-E3)
Nestin-GFP+ cells began to seed the nascent SGZ with the
largest population being adjacent to the pia, whereas most
Tbr2+ cells were distributed in the ML. Scale bar: 200μm in
E; 50μm in E1-E3. (F)By P7, the permanent neurogenic
niche was formed in the subgranular zone. (F1-F3) Most
Nestin-GFP+ and some Tbr2+ cells were present in the SGZ.
Scale bar: 200μm in F; 50μm in F1-F3. (G1-G4) A schematic
representation of the two stages involved in neurogenic
zone relocation during the development of the dentate
gyrus. Green dots indicate the location of stem/progenitor
cells as labeled by Nestin-GFP transgene. D, dentate; DN,
dentate notch; F, fimbria; FDJ, fimbriodentate junction; GCL,
granule cell layer; Hem, cortical hem; Hip, hippocampus; HF,
hippocampal fissure; ML, molecular layer; SGZ, subgranular
zone; SVZ, subventricular zone; VZ, ventricular zone.
in the FDJ region (Fig. 1B, B3-4) and, in fact, this structure appeared
to have formed as a continuation of the subpial collection of Tbr2+
cells seen at E14.5. Interestingly, in the FDJ, the strongly stained
Nestin-GFP+ cells did not overlap with the Tbr2 staining, implying
that these were largely non-overlapping cell populations
(arrowheads in Fig. 1B1-B4).
At E17.5, Nestin-GFP+ cells had fanned out into the hilus (Fig.
1C,C1) and lined the hippocampal fissure (HF) (Fig. 1C1). Tbr2+
cells were also distributed across the hilus and along the HF (Fig.
1C2,C3). At P0, Nestin-GFP+ cells completely covered the dentate
side of the HF and the subpial region of the future lower blade,
whereas few Nestin-GFP+ cells were located in the hilus (Fig.
1D,D1,D3). Tbr2+ cells continued to mirror this distribution pattern
By P2, Nestin-GFP+ cells and processes appeared to spread
toward the hilus from the marginal zone (MZ) of the granule cell
layer (GCL) (Fig. 1E,E1), but by contrast, most Tbr2+ cells were
still restricted to the emerging molecular layer at this stage (Fig.
1E2,E3). At the end of the first postnatal week, most Nestin-GFP+
cells populated the hilus and the subgranular zone (SGZ), and had
exited the molecular layer (Fig. 1F,F1). The Tbr2+ population in the
molecular layer was reduced and now found largely in the SGZ
(arrowhead in Fig. 1F2,F3).
Consistent with work in the adult dentate (Yamaguchi et al.,
2000), perinatal analysis of Nestin-GFP+ cells with acute BrdU
labeling showed that the subpial Nestin-GFP+ cells close to the HF
were actively dividing with little overlap with Prox1 (see Fig.
S1A,A?,A?in the supplementary material). Similarly, subpial Tbr2+
cells were proliferating (see Fig. S1B in the supplementary material)
with clear distinction from the reelin+ Cajal-Retzius cells (see Fig.
S1C in the supplementary material). In agreement with their
neurogenic nature in the developing cortex (Arnold et al., 2008;
Englund et al., 2005; Sessa et al., 2008), the weak Tbr2+ cells also
showed low Prox1 expression in both MZ and the newly formed
SGZ at P5 (see Fig. S1D-F in the supplementary material). Taken
together, the spatiotemporal distribution of Nestin-GFP+ and Tbr2+
cells reveal two phases of neurogenic zone transitions (Fig. 1G1-
G4): the dentate VZ-to-subpial transition and the subpial-to-
Formation of the transient neurogenic zone
coincides with the appearance of transhilar glial
Since previous studies indicate that radial glial cells regulate the
morphogenesis of the dentate gyrus (Eckenhoff and Rakic, 1984;
Rickmann et al., 1987), we further investigated the distribution of
Dentate transient niche
Fig. 2. Development of the transhilar
glial scaffolding proceeds without
evidence of VZ connection. (A)By
E18.5, a distinct upper blade was
formed as labeled by granule cell marker
Prox1. The GFAP+ glial scaffolding was
highly concentrated at the border of the
fimbria and extended into FDJ.
(B,C)GFAP+ glial orientation was
correlated with the granule cell
arrangement in the forming dentate
plate. At the entrance of the hilus (red
arrow in B), GFAP+ radial glia radiated
out into HF, whereas most Prox1+
granule cells were gathered towards HF.
Scale bar: 50μm in B and C. (D)Labeling
radial glia in the ventricular zone by DiO
injected into the ventricle at E18.5
revealed that no radial glial fibers directly
projected from the VZ into the forming
dentate. (E-H)The dynamic changes of
radial glial projection in the developing
dentate by in utero electroporation (EP)
of GFP expression vector targeting the
medial wall. (E)Examined at E14.5 after
EP at E13.5, GFP labeled radial glia at
the dentate VZ showed the association
of the endfeet with pia (arrowheads).
(F)Examined at E15.5 after EP at E14.5,
GFP labeled radial glia no longer
extended into the field of the dentate
gyrus, in contrast to the GFP labeled glial
fibers radially oriented elsewhere in the hippocampal fields. A few cells (arrows) migrated toward the dentate gyrus. (G)Forty-eight hours after EP at
E17.5, GFP marked the VZ of the whole medial wall. Radial glial processes in the hippocampal field were clearly labeled but not in the dentate
gyrus. GFP labeled a stream of cells along the edge of the fimbria (arrows). Some of them already reached the entrance of hilus, which are shown
at higher power in H. (I-K)The ongoing relocation of BLBP+ glia at E18.5. BLBP+ glial processes were revealed by thin section confocal imaging (I).
BLBP+ cell bodies (arrow) were seen in the FDJ (inset in I). z-projection of image stacks revealed the long processes spanning from FDJ to HF. The
location of individual cell bodies (red and yellow arrows in J) was identified through tracing serial sections. A few glial cell bodies already arrived at
the HF (white arrows in J). (K) The two long processes and corresponding cell bodies (arrows), in addition to the glial cell bodies (in green) localized
in the FDJ and HF. FDJ, fimbriodentate junction; HF, hippocampal fissure; Hip, hippocampus.
the radial glial scaffolding by GFAP staining as the Nestin-GFP+
precursors migrate from the dentate primordium to the subpial
neurogenic zone. By E18.5, Prox1+ granule cells already occupied
the forming upper blade, whereas GFAP+ glial fibers were enriched
at the border of the fimbria (Fig. 2A). These GFAP+ fibers appeared
to spread out at the entrance of the hilus and project to the pia all
around the forming dentate (Fig. 2B), whereas Prox1+ granule cells
were arranged in parallel to this glial scaffolding in the hilus (Fig.
2C). Previous studies assumed that these glial fibers are projecting
from radial glial cells with their cell bodies located in the VZ
(Eckenhoff and Rakic, 1984). To determine whether these hilar
fibers directly project from the dentate VZ or the new organizing
center at the FDJ (Fig. 2B, red arrow), we injected DiO solution into
the ventricle of E18.5 embryos and allowed these to survive for only
3 hours to physically label all the fibers projecting from the VZ. This
labeling prominently marked all the radial glial fibers spanning the
whole hippocampal fields except the dentate field (Fig. 2D). It
suggests that radial glial fibers in the forming dentate during this
migratory phase do not directly project to the dentate from the
dentate ventricular zone.
To further examine which cells contribute to this scaffolding in
the developing dentate, a GFP expression construct was
electroporated in utero into the medial wall, including the dentate
VZ, at different ages followed by various survival times. In brains
of mice electroporated at E13.5 and examined at E14.5, cells in the
dentate VZ projected to the pia or cells themselves appeared to be
migrating in the same trajectory (Fig. 2E, arrowheads). However,
when electroporated at E14.5 and examined at E15.5, the dentate VZ
showed no direct radial projection into the emerging dentate at all,
despite the prominent radial glial scaffolding in the hippocampus
proper (Fig. 2F). Instead, apparently migrating GFP+ cells were seen
at the hilar entrance (Fig. 2F, arrow). Therefore, although it appeared
that there was a direct VZ to dentate cellular connection at E14.5,
this was no longer apparent by E15.5. To further address this at later
stages, the medial cortex was electroporated at E17.5. Two days
later, extensive labeling of radial fibers was seen in the hippocampal
fields, but the dentate was completely devoid of radial fibers from
the VZ (Fig. 2F). Instead, GFP+ cells formed a distinct stream from
the dentate VZ along the fimbria (Fig. 2F, arrows) and some of these
were again visible at the entry of the hilus (Fig. 2H).
Staining with a radial glial marker, brain lipid binding protein
(BLBP), labeled a prominent subset of glial processes in the forming
dentate at E18.5 (Fig. 2I). Strikingly, unlike with GFAP staining,
glial cell bodies were clearly identifiable with BLBP in the FDJ
(arrow in Fig. 2I inset) and some of them had already reached the
HF (arrows in Fig. 2J). A collapsed z-projection of the serial sections
is shown in Fig. 2J, allowing the reconstruction of two whole
BLBP+ cells. The somata of the BLBP+ processes spanning the
hilus were pinpointed by tracing the overlapping thin sections (Fig.
2K). Interestingly, somata were found near the FDJ pia or in the hilus
(red and yellow arrows in Fig. 2J,K), suggesting that some glia were
in the process of migration from the FDJ towards the HF.
Reelin is dispensable for the formation of but
controls exit from the transient zone
Numerous studies have shown that reelin secreted from Cajal-
Retzius cells is essential for the development of the neocortex and
hippocampus by controlling the proper lamination of projection
Development 136 (2)
Fig. 3. Sustained subpial neurogenic
zone in the reeler mice. (A-C,A? ?-C? ?)
The formation of subpial neurogenic
zone proceeded in reeler mutants. By P0,
the Nestin-GFP+ (A,A?) and Tbr2+ (B,B?)
cells were localized in the subpial region
in both control and reeler mutant
(arrowheads in A,B,A?,B?). Merged
images for Nestin-GFP and Tbr2 in
control and mutant are shown in C and
C?, respectively. (D,D? ?) Granule cells
migration relied on reelin signaling. At
P0, Prox1+ granule cells were collected
as a well-defined upper blade in the
control (D), whereas they were evenly
scattered in the dentate plate of the
Reeler mutant (D?). (E-F,E? ?-F? ?) The
prolonged presence of neurogenic
precursors (both Nestin-GFP+ and Tbr2+
cells) in the subpial neurogenic zone in
the reeler mutant at P4. By P4, as most
of the Nestin-GFP+ cells left the subpial
region in the control (E), many cells still
persisted subpially in the reeler mutant
(E?). Tbr2+ cells were loosely distributed
in the molecular layer in control (F and
inset). By contrast, they formed a
compact subpial layer in the reeler
mutant (F? and inset). (G)The regional
schema for quantification of Tbr2+ cells
in the dorsal half of the dentate gyrus.
The distribution of Tbr2+ cells at P4 was
quantified in the MZ (in red) and the area (in blue) including granule cell layer and hilus. (H)The percentage of Tbr2+ cells in the control
marginal zone (28.6±1%) was significantly lower than reeler mutant (75.4±0.9%, n=6, *P<0.001, χ2-test). Ctr, control; Rlr, reeler; MZ,
marginal zone; GCL, granule cell layer; SGZ, subgranular zone. Scale bar: 100μm in A-D and A’-D’; 200μm in E-F,E’-F’.
neurons (Rice and Curran, 2001). As the dentate is also quite
abnormal in reelin mutants (Forster et al., 2002), we wondered what
role reelin plays in the migration of dentate precursors to the newly
identified transient subpial zone. To tackle this issue, we examined
the distribution of Nestin-GFP+ cells at birth in reeler mice. In both
the controls and reeler mice, Nestin-GFP+ or Tbr2+ cells were
properly localized to the subpial zone (arrowheads in Fig. 3A,B and
Fig. 3A?,B?). Consistent with the known role of reelin in neuronal
migration, Prox1+ granule cells were abnormally distributed across
the dentate field in the mutants (arrow in Fig. 3D?) instead of
forming a relatively compact upper blade as in the controls (Fig.
3D). Thus, reelin signaling is required for proper granule cell
migration but not for the subpial localization of Nestin-GFP+ cells.
In control mice at P4, most of the Nestin-GFP+ cells were in the
SGZ and hilus, whereas the subpial zone was essentially depleted of
Nestin-GFP+ cells (Fig. 3E). Strikingly, in Reeler mutant mice, the
subpial zone was still packed with Nestin-GFP+ cells (Fig. 3E?). As
the upper blade was well-defined at this age, we quantified the
distribution of Tbr2+ cells in the dorsal half of the dentate (Fig. 3H)
with the regional schema shown in Fig. 3G. Only a small proportion
of Tbr2+ cells (28.6±1%) were localized in the control marginal
zone, with the majority (71.4±1%) scattered in the GCL, SGZ and
hilus (arrow in Fig. 3F, inset), whereas in the mutants the Tbr2+ cells
(75.4±0.9%) were clustered in the marginal zone (arrow in Fig. 3F?,
inset) with a small number (24.6±0.9%, n=6, *P<0.001, χ2-test, Fig.
3H) in the region outside the marginal zone. Therefore, the dentate
neurogenic niche failed to undergo the subpial-to-subgranular
transition in the absence of functional reelin. Although previous
studies (Forster et al., 2002) suggest that reelin signaling may cell-
autonomously regulate the behavior of radial glial cells, it is also
possible that this role for reelin could be due to a non-autonomous
role for reelin in organizing other cellular components in the dentate.
However, what is clear is that reelin is dispensable for the original
positioning of radial glial and transit-amplifying cells in the subpial
zone but is indispensable for the later reorganization of this zone.
Formation of the subpial zone requires
Previous studies indicate that the chemokine Cxcl12 and its cognate
receptor Cxcr4 regulate the morphogenesis of the dentate gyrus
(Bagri et al., 2002; Lu et al., 2002),but the mechanistic basis of this
defect is not well characterized. More recent studies suggest the
Cxcl12/Cxcr4 signaling plays a crucial role in regulating the
positioning of neurons adjacent to the pia(Borrell and Marin, 2006;
Li et al., 2008a; Lopez-Bendito et al., 2008; Paredes et al., 2006;
Tiveron et al., 2006). Owing to the expression of Cxcr4 in the
migratory stream and subpial zone and expression of Cxcl12 by the
pial meninges (see Fig. S2 in the supplementary material) (Berger
et al., 2007), we sought to determine whether Cxcl12/Cxcr4
signaling controls the concentration of Nestin-GFP+ precursors in
the subpial region of the developing dentate. At E18.5 in control
animals, Nestin-GFP+ cells occupied the subpial region around the
Dentate transient niche
Fig. 4. Defective subpial neurogenic
zone, abnormal radial glial scaffolding
and premature granule cell production
in Cxcr4 null. (A-D)Defective subpial
neurogenic zone in Cxcr4 mutant. By
E18.5, Nestin-GFP+ and Tbr2+ cells were
scarce in the subpial region in the Cxcr4
mutant compared with the control (white
arrows in A-D). Tbr2+ cells were ectopically
packed in the hilus in the mutant (yellow
arrow in D). (E-H)Aberrant development of
the transhilar glial fibers in the Cxcr4
mutant. At E18.5, GFAP+ processes were
more enriched in the HF in the controls
compared with the mutants (arrows in E,F).
A subset of transhilar glial fibers labeled by
BLBP at E17.5 in the controls (G) were
almost lost in the mutants (H). BLBP+
somas seen in the HF and hilus of controls
were absent in the mutants (arrows in
G,H). (I-O)Premature production of granule
cells in the Cxcr4 mutant. By E18.5, acute
BrdU labeling was decreased in the forming
dentate plate in the mutant (J) when
compared with the control (I). However,
Prox1+ granule cells were not
overwhelmingly affected in the mutant
despite the abnormal distribution on the
migratory stream (J). By E15.5, Nestin-GFP+
precursors were able to leave the dentate
primordium and form a migratory stream in
the mutant (arrow in L), although it was
not as robust as in the control (arrow in K).
At this stage, there were more Prox1+
granule cells in the mutant (N) compared with the control (M). Boxed areas in M and N are shown at higher power in M? and N?. (O) Birthdating
analysis with BrdU pulses (at E15.5 and E16.5) and quantification at E18.5 showed that the density of BrdU+/Prox1+ cells produced at E15.5 was
much higher in the mutants (227±21%), but much lower at E16.5 (51.7±7.5%) compared with the controls (n=4, *P<0.01, Student’s t-test).
entire profile of the forming dentate gyrus from hippocampal fissure
superficial to the nascent upper blade and ventrally to the future
lower blade (arrows in Fig. 4A). In stark contrast, in Cxcr4–/–mice
the Nestin-GFP+ cells were largely scarce in the subpial region
(arrows in Fig. 4B). Consistent with these findings, Tbr2+ cells no
longer formed a compact subpial zone along the FDJ and HF in the
Cxcr4 mutants as they did in controls (white arrows in Fig. 4C,D).
Instead, Tbr2+ cells were widely dispersed in the dentate (yellow
arrow in Fig. 4D). Taken together, these findings indicate that Cxcr4
is required for the proper formation of the subpial neurogenic zone.
Subpial organization of precursors correlates with
the transhilar glial scaffolding and neuronal
The displacement of Nestin-GFP+ progenitors from their subpial
location prompted us to ask whether this defect affects the
development of the transhilar glial scaffolding. Staining for GFAP
showed dense glial processes across the hilus and enriched fiber
plexus at the HF in the controls (Fig. 4E). By contrast, Cxcr4
mutants showed reduced hilar GFAP+ fibers and decreased fiber
plexus in the HF (Fig. 4F). In the controls, BLBP staining revealed
a subset of transhilar glial fibers emanating from the FDJ and
crossing the hilus (Fig. 4G). BLBP+ somata were clearly identified
in three locations: FDJ, hilus and HF. However, both BLBP+
processes and somata were almost absent in the hilus and HF in
mutants (arrows in Fig. 4G,H).
To determine whether the disruption of the subpial zone and the
radial glial progenitors leads to any dynamic consequences for the
cellular output of the stem/progenitor cells, we analyzed acute BrdU
labeling at E18.5. In agreement with previous studies (Bagri et al.,
2002; Lu et al., 2002), we found that the number of BrdU+ cells was
significantly decreased in the dentate of Cxcr4 mutants compared
with the controls (Fig. 4I,J). However, this finding cannot be simply
explained by an increase in cell death or a migration defect resulting
from the loss of Cxcr4, as the production of granule cells did not
seem to have drastically declined despite their abnormal distribution
(Fig. 4I,J). We noticed that in association with the decrease in BrdU+
cell numbers in the Cxcr4 mutants at E18.5, there was not only an
overall decrease in the number of Nestin-GFP+ cells (Fig. 4A,B) but
also a corresponding increase in the number of Tbr2+ cells in the
dentate field of the Cxcr4 mutants compared with the controls (Fig.
4C,D). This led us to look into the possibility that dentate precursors
prematurely differentiate into granule cells when they were
displaced from the subpial zone. At E15.5, a robust stream of Nestin-
GFP+ cells was present in the controls but it was diminished in the
Cxcr4 mutants (arrows in Fig. 4K,L). Conversely, the mutants had
a larger patch of Prox1+ granule cells around the FDJ than the
controls. This loss of progenitors and the excess of granule cells at
this early developmental stage suggested the premature
differentiation of dentate progenitors upon displacement from the
To test this more directly, we birthdated granule cells by injecting
BrdU at E15.5 and counted the number of BrdU+/Prox1+ cells at
E18.5. Interestingly, the density of double-labeled cells was
dramatically higher in the mutants (227±21%) compared with
controls (n=4, *P<0.05, Student’s t-test). However, when BrdU was
administered at E16.5, the density of BrdU+/Prox1+ cells was
significantly lower in mutants Cxcr4 mutants (51.7±7.5%) than
controls (n=4, *P<0.01, Student’s t-test) (Fig. 4O). This indicates
that mutant mice have an early excessive burst of production of
Development 136 (2)
Fig. 5. Compromised organization of
the subgranular niche in Emx1-PTX
mice. (A-H)Abnormal SGZ organization
at early postnatal ages in Emx1-PTX mice.
By P4, Nestin-GFP+ cells started to
populate the SGZ in the controls (A),
whereas they showed patchy distribution
in the Emx1-PTX animals (red outlines in
B). Compared with the control (C and E),
the Ki67+ proliferating cells tended to
cluster in the hilus and Tbr2+ cells were
more widely spread in the Emx1-PTX
animal (D,F). In contrast to the controls
(G), the granule cell layer revealed by
Prox1 staining was very poorly organized
in the Emx1-PTX animal, with ectopic cells
in the migratory stream (arrow in H).
(I-R)Persistent defects in the organization
of subgranular zone in Emx1-PTX mice at
P10. Compared with the controls
(I,K,M,O), the transgranular radial glial
scaffolding labeled by BLBP failed to form
properly (J) and the SGZ (shown by BrdU,
Ki67 and Tbr2 staining in L, N and P) was
dramatically disorganized in the Emx1-
PTX animals. In sharp contrast to the
control (Q), the Prox1+ GCL did not form
a distinct boundary with the hilar region
and there were numerous ectopic granule
cells in the hilus and in the migratory
route in the Emx1-PTX animals (R). Scale
bar: 100μm in A,B; 200μm in C-H;
200μm in I,J; 200μm in K-R.
granule neurons but fail to produce the appropriate number of
granule cells only a day later. Taken together, these data indicate that
progenitors displaced from the subpial zone prematurely
differentiate and the localization to the subpial transient zone may
be required to maintain dentate precursors in an undifferentiated
state during late embryonic stages.
SGZ formation is not affected in Emx1-Cxcr4 cKO
but severely compromised in Emx1-PTX mice
To examine the SGZ formation, we used a conditional knockout
model by crossing the floxed Cxcr4 allele with Emx1ires-cre(Gorski
et al., 2002) (Emx1-Cxcr4 cKO thereafter) to bypass the prenatal
lethality of null Cxcr4. The prenatal development of the dentate
gyrus in Emx1-Cxcr4 cKO resembled the null mutants with Tbr2
and Prox1 staining (see Fig. S3 in the supplementary material).
By P5, there was distinct reorganization of the radial glial
scaffolding in control mice shown by the Nestin-GFP+ cells at the
SGZ, whereas in the cKO mice the Nestin-GFP+ cells were
scattered throughout the dentate formation (see Fig. S4A,B in the
supplementary material). However, the distribution patterns of
Tbr2+ or Ki67+ cells showed subtle differences between controls
and cKOs (see Fig. S4C-F in the supplementary material).
Surprisingly, despite the prenatal abnormalities in the early GCL of
the Cxcr4–/–and the Emx1-Cxcr4 cKO, Prox1+ cells formed distinct
upper and lower blades of GCLs in the cKO mice (see Fig. S4G,H
in the supplementary material). Our data suggests the granule cells
are largely able to adopt appropriate layer positioning in Emx1-
Cxcr4 cKO despite the early defects in SPZ.
At P14, Emx1-Cxcr4 cKOs showed almost normal organization
of the SGZ with BLBP, Tbr2 and BrdU (see Fig. S4I-N in the
supplementary material) and GCL with Prox1 (see Fig. S4O-P in the
supplementary material). This recovery was sustained into
adulthood (see Fig. S5 in the supplementary material). As the cre
activity of Emx1ires-crecompletely covered dentate primordium at
E14.5 and showed complete recombination at P14 (see Fig. S6A-F
in the supplementary material), low penetrance of Emx1ires-creis
unlikely to explain the developmental recovery in the cKO mice.
One possible explanation for the SGZ recovery in the Emx1-
Cxcr4 cKOs is that other ligand-receptor systems may compensate
for the loss of Cxcr4. To test this, pertussis toxin (PTX) expression
(Regard et al., 2007) was conditionally activated by Emx1ires-cre
(Emx1-PTX thereafter), potently blocking all the trimeric Gi/o
signaling including Cxcr4. As expected, the perinatal subpial
neurogenic zone did not properly form in the Emx1-PTX animals
(see Fig. S7 in the supplementary material). By P4, Nestin-GFP+
cells were distributed as patches scattered throughout the dentate
(Fig. 5A,B) and both Ki67+ and Tbr2+ cells were chaotically
dispersed throughout the whole dentate (Fig. 5C-F). Prox1+ granule
cells also failed to assume their distinct layered organization and
many were ectopically located in the subpial region of FDJ (Fig.
5G,H). Emx1-PTX animals died in the second postnatal week, so we
chose P10 animals for further analysis. Compared with the controls
(Fig. 5I,K,M,O), Emx1-PTX animals almost complete lost
organization of the BLBP+ scaffolding in the SGZ (Fig. 5J);
furthermore, Tbr2+ neurogenic precursors and the BrdU+ or Ki67+
cycling cells in the SGZ were also ectopically localized in the MZ,
GCL and hilus (Fig. 5L,N,P). In addition, the border between the
hilus and Prox1+ GCL was obscured owing to ectopic dispersion of
granule cells into the hilus and granule cell heterotopias were visible
in the remnant of the migratory stream to the dentate (Fig. 5Q,R).
Therefore, the formation of SGZ appears to rely on a PTX-sensitive
Contribution of subpial progenitors to the
formation of the subgranular zone
Previous genetic fate-mapping analysis with the Gli1CreERT2line
revealed that the self-renewing stem cells in the dentate gyrus first
appear at the late embryogenesis (Ahn and Joyner, 2005). In order
Dentate transient niche
Fig. 6. Contribution of subpial precursors to
the SGZ. (A)Tamoxifen (TM) was administered at
E17.5 into Gli1CreERT2line to mark the Shh-
responding cells with Rosa-lacZ reporter. Twenty-
four hours later, the labeled cells were found in
the marginal zone (inset 1) and the outer edge of
the upper blade (inset 2). (B)Forty-eight hours
later, the labeled cells spread into the granule cell
layer and appeared in the hilus (inset). (C)When
tamoxifen was administered at E18.5, most
labeled cells appeared 24 hours later in the upper
blade (inset) and the future lower blade.
(D-F)RosaYFP reporter was used to mark the
Shh-responding cells when tamoxifen was
administered at E17.5 into Gli1CreERT2line. Only
very few cells were GFP+. One GFP+ cell in the
upper blade did not show Prox1 expression (E),
whereas one of the GFP+ doublet started to have
weak Prox1 expression (arrows; F,F?). (G-I)The
Shh-responding cells marked at E17.5 gave rise to
Prox1+ granule cells at P14 (G and inset). Some of
them were BLBP+ (H,H?) or GFAP+ (I,I?). Boxed
areas in H and I are shown at higher power in
H?,I?, respectively. GCL, granule cell layer; MZ,
marginal zone; SGZ, subgranular zone. Scale bar:
100μm in A-D,G-I; 10μm in E,F,F’; 20μm in H’-I’.
to test whether the SPZ progenitors may contribute to the neural
stem cells settled in the SGZ, we reasoned that when tamoxifen
(TM) was injected at E17.5 into the Gli1CreERT2line in the presence
of Rosa-lacZreporter, the labeled cells would initially emerge from
the subpial zone and then spread toward the GCL from there over
time. If the hilar progenitors exclusively contribute to the SGZ, we
would expect the opposite. Interestingly, after 24 hours, lacZ+ cells
were first detected in the MZ (inset 1 in Fig. 6A) and the edge
between MZ and GCL (inset 2 in Fig. 6A) in the upper blade. After
48 hours, lacZ+ spread across the GCL and SGZ in the upper blade
(Fig. 6B and inset). When tamoxifen was administered at E18.5 and
lacZ expression was analyzed 24 hours later, we found most lacZ+
cells were restricted in the GCL of the upper blade (Fig. 6C and
inset) and others were observed in the future lower blade (Fig. 6C).
To further analyze the cellular identity of cells produced after
recombination induced at E17.5, we turned to the RosaYFP reporter
line. The earliest GFP+ cells were detected at P0 (Fig. 6D) and did
not express Prox1 (Fig. 6E). In other cases, it appeared that a cell
might have just divided and Prox1 could be detected in one of the
GFP+ doublet cells (arrows in Fig. 6F,F?). When cell fates were
mapped in animals at P14, most recombined cells were Prox1+
granule cells (Fig. 6G, inset), and a few of them showed radial glial
morphology (Fig. 6H,I) and were co-labeled with BLBP (Fig.
6H,H?) or GFAP (Fig. 6I,I?). Taken together, these findings support
the idea that perinatal subpial progenitors contribute to the neural
stem cells that eventually settle in the SGZ.
In this study, we described the progressive development of distinct
neurogenic zones in the dentate gyrus, from the dentate VZ to the
subpial zone (SPZ) and then to the subgranular zone (SGZ). We found
that reelin signaling is dispensable for the formation of the SPZ but is
required for the timely transition of dentate precursors from the SPZ
to the SGZ. Cxcl12/Cxcr4 signaling is essential for the initial
organization of the SPZ. When subpial localization of precursors is
impaired, the transhilar glial scaffolding and the undifferentiated state
of the dentate precursors are altered. In postnatal mice with loss of
Cxcr4 signaling, the SGZ initially fails to form properly but shows
gradual recovery so that it appears normal by the second week of life.
A conditional PTX reporter mouse demonstrates that this recovery is
likely to be due to the developmental onset of compensatory
mechanisms. Fate-mapping analysis shows that the subpial precursors
contribute to the neural stem cells in the SGZ. Taken together, these
data provide a novel framework for understanding the development
of the subgranular zone in the dentate gyrus where neurogenesis
persists throughout adulthood.
A novel, temporary neurogenic zone in the
developing dentate gyrus adjacent to the
The most distinct feature of dentate development compared with
other forebrain areas is the extended migration of neural precursors
from the VZ to a newly formed region in the SGZ. The SGZ is a
long-lived durable niche that allows survival and self-renewal of
neural stem cells. But how do neural precursors manage to traverse
the territories from VZ to ultimately form SGZ? This is not a small
hurdle, because at the stage that precursors begin to exit the VZ there
is no formed dentate gyrus or SGZ for them to occupy.
Our analysis of the Nestin-GFP transgene in combination with
transit amplifying cell marker Tbr2 shows that neurogenic
precursors initially follow a subpial migratory route to
fimbriodentate junction, some then move across the hilus and take
residence in the subpial region of the hippocampal fissure leaving a
population of progenitors adjacent to the pia around the entire pole
of the dentate (Fig. 7). From this base of operations, neurogenic
precursors generate Tbr2+ transient amplifying cells and then
granule neurons that form the initial structure of the dentate granule
cell layer. Nestin-GFP+ and Tbr2+ cells gradually disappear from
the subpial region in the first postnatal week, both cell types
accordingly increase in the SGZ and hilus, suggesting there is a
subpial-to-hilar transition. In agreement with our findings, Ngn2
mutant mice were recently shown to have severe neurogenic defects
in the developing dentate gyrus and using a Ngn2-GFP mouse line
the same authors found a similar group of neurogenic precursors was
in close proximity to the subpial zone (Galichet et al., 2008).
Complex organization of the transient neurogenic
zone in the developing dentate gyrus
The transient subpial zone is regulated by Cxcl12 secreted from
meningeal fibroblasts and reelin from the Cajal-Retzius cells in the
dentate marginal zone. It is interesting to consider what other factors
may be important in the constitution of this zone and their ability to
maintain dentate progenitors in an undifferentiated state (Fig. 7B).
The analysis of the mutants in the canonical Wnt signaling pathway
suggests that Wnts play a crucial role in the expansion and
maintenance of the dentate precursor pool during this same
developmental period(Galceran et al., 2000; Grove et al., 1998; Lee
et al., 2000; Zhou et al., 2004).Also shown to be active and required
for the formation of dentate stem cell niches is sonic hedgehog (Shh)
(Machold et al., 2003). However, it is not clear which cells express
Shh or Wnts in the subpial zone. There is evidence that the
subgranular zone niche is intimately associated with blood vessels
(Palmer et al., 2000) and that endothelial cells may provide
Development 136 (2)
Fig. 7. Schematic representation of the
progression of dentate stem cells to form the
SGZ. This figure shows our conception of the
events leading to the formation of the SGZ.
(A)Canonical Wnts are embryonically expressed in
the fimbria/cortical hem and support expansion of
dentate progenitors. (B)During their migration, the
dentate progenitors are located in the transient
neurogenic zone, where their position is
maintained by Cxcl12 signaling. (C)By the first
postnatal week they relocate to the SGZ where
they are regulated by Wnts from the hilus and Shh
(not shown) from unknown sources. DN, dentate
notch; F, fimbria; FDJ, fimbriodentate junction;
HF, hippocampal fissure.
important regulators to stem cell behaviors (Shen et al., 2004). These
findings may have relevance to the timing and importance of the
The roles of Gi/o signaling pathway in stem cell
migration and maintenance
The loss of Cxcr4 only transiently affects the formation of the SGZ
despite the disorganization of the transient SPZ. Prominent roles for
other chemokines and their receptors have been postulated in the
dentate gyrus (Tran et al., 2007). The redundancy of signaling
pathways is evidenced by our use of the recently developed Cre-
mediated PTX expression line (Regard et al., 2007). These mice
have a very dramatic developmental dentate phenotype, which
suggests the search for other Gi/o-coupled receptor/ligand
combinations in the dentate gyrus is likely to result in both other
important developmental regulators of dentate morphogenesis but
also perhaps potent molecular targets to design reagents to regulate
We thank Drs Shi-Bing Yang in Lily Jan’s laboratory, Yonghua Pan and Jyothi
Arikkath in Louis Reichardt’s laboratory for helping with the confocal imaging,
and Mojgan Khodadoust in the Pleasure laboratory for mouse genotyping. This
work was supported by funding from NIMH and Autism Speaks to S.J.P.
Deposited in PMC for release after 12 months.
Supplementary material for this article is available at
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Dentate transient niche