Prox1 Is Required for Granule Cell Maturation and
Intermediate Progenitor Maintenance During Brain
Alfonso Lavado1, Oleg V. Lagutin1, Lionel M. L. Chow2, Suzanne J. Baker2, Guillermo Oliver1*
1Department of Genetics & Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2Department of Developmental
Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America
The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the
dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate
gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of
granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult
neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell
self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory
feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the
ectopic expression of Prox1 induces premature differentiation of neural stem cells.
Citation: Lavado A, Lagutin OV, Chow LML, Baker SJ, Oliver G (2010) Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance
During Brain Neurogenesis. PLoS Biol 8(8): e1000460. doi:10.1371/journal.pbio.1000460
Academic Editor: Theo D. Palmer, Stanford University, United States of America
Received March 9, 2010; Accepted July 9, 2010; Published August 17, 2010
Copyright: ? 2010 Lavado et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported in part by National Institutes of Health grants R01-HL073402, P01- CA096832, Cancer Center Support CA-21765, and the
American Lebanese Syrian Associated Charities (ALSAC).The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: DG, dentate gyrus; DNE, dentate neuroepithelium; MS, migratory stream; SGZ, subgranular zone; TM, tamoxifen
* E-mail: email@example.com
In the brain, the dentate gyrus (DG) is the primary afferent
pathway into the hippocampus. The DG has a crucial role in
learning and memory [1,2,3]. In mammals, neurogenesis occurs in
the subgranular zone (SGZ) of the DG throughout adulthood
[4,5,6,7]; this activity is thought to be the basis for the acquisition
of new memories [3,8,9].
The formation of the DG is a complex process that involves cell
migration and neuronal differentiation [10,11]. Factors that
regulate DG development are thought to have a similar function
during adult neurogenesis. In the SGZ, astrocyte-like adult neural
stem cells (NSCs) give rise to a series of intermediate progenitors
that eventually differentiate into neurons . Several signaling
molecules, including Wnt, Noggin/BMP, Shh, and Notch,
regulate adult NSC self-maintenance, proliferation, and progen-
itor differentiation [13,14]. However, little is known about how the
generation of the proper number of descendants is controlled. It
has been proposed that once generated, NSC descendants can
trigger some type of feedback mechanism to stop stem cell
differentiation . In this context, Notch signaling has been
considered a candidate to regulate such a feedback mechanism
during adult neurogenesis .
The homeobox gene Prox1 is expressed in several brain regions
(i.e., cortex, DG, thalamus, hypothalamus, cerebellum) during
prenatal and postnatal stages of development [16,17,18]. Inter-
estingly, Prox1 is expressed throughout all stages of DG
development and in adult granule cells; therefore, Prox1 is
commonly used as a specific marker for these cells [15,19].
However, no data are yet available on the functional role(s) of
Prox1 during brain development.
We have now determined that functional inactivation of Prox1
during DG development results in defective granule cell
maturation and the loss of this cell population. We also report
that conditional inactivation of Prox1 in the SGZ during adult
neurogenesis leads to the lack of intermediate progenitors, and as a
consequence, the disruption of the mechanism involved in NSC
self-maintenance. Therefore, we have identified a previously
unknown non-cell autonomous regulatory feedback mechanism
that links adult NSC self-maintenance with the generation of the
proper number of descendants in the SGZ. Finally, we show that
ectopic expression of Prox1 in NSCs promotes premature
differentiation during DG development and adult neurogenesis
in the SGZ.
Prox1 Activity Is Required for DG Formation
Standard Prox1-null embryos die during midgestation ;
therefore, to evaluate the possible functional roles of Prox1 in the
mammalian brain, we used a conditional-inactivation approach.
An available Prox1-floxed strain  was initially bred with Nestin-
Cre mice in which constitutively active Cre recombinase is
expressed in neural progenitors from embryonic day (E) 10.5
PLoS Biology | www.plosbiology.org1August 2010 | Volume 8 | Issue 8 | e1000460
. Adult Nestin-Cre;Prox1F/Fmice were viable but had only a few
scattered Prox1+/NeuN+wild-type granule cells in their hippo-
campi (Figure 1B,D). During embryonic development, Prox1
expression is detected in both the dentate neuroepithelium
(DNE) and the DG [17,23]. Therefore, we performed a detailed
characterization of the development of the DG in Nestin-
At E14.5, the DG of Nestin-Cre;Prox1F/Fembryos showed normal
Ammon’s horn formation (Figure 2A–L). At E16.5, and as shown
by an anti-C-Prox1 antibody that recognizes only the wild-type
form of Prox1 (see Figure S1A,B for more details), only a few cells
escaped Cre-mediated deletion in the DG of Nestin-Cre;Prox1F/F
embryos (Figure 1F). However, as indicated by an anti-N-Prox1
antibody that recognizes both wild-type and conditional-mutant
forms of Prox1 (Figure S1A,B), the number of N-Prox1+cells was
reduced in the DG of Nestin-Cre;Prox1F/Fembryos at this stage
(Figure 1H; Figure S1C,D). Reduced numbers of Notch1+, Ngn2+,
and NeuroD1+cells were also observed by ISH in the mutant DG
(Figure 1J,L,N); however, we found no obvious alterations in the
expression of Wnt3a in the FNE (Figure S2A,B) , or lef1 in the
migratory stream (MS) (Figure S2C,D) . We also did not find
obvious anomalies in radial glia scaffolding  in the mutant DG
at E16.5 (Figure S2E–H). These results indicate that in the Nestin-
Cre;Prox1F/Fembryos, the N-Prox1+cells are capable of migrating
out of the DNE and reach the DG.
Prox1 Controls Cell Proliferation in the DNE
Prox1 absence leads to cell cycle alterations in the developing
neuroretina . To evaluate possible alterations in cell
proliferation during DG development, we compared the number
of cycling cells in the DNE of wild-type and Nestin-Cre;Prox1F/F
embryos by using a 1-h BrdU pulse starting at E14.5. Significantly
fewer BrdU+cells were observed in the Nestin-Cre;Prox1F/FDNE at
E16.5 (Figure 3D,G) and E18.5 (Figure 3F,G). Similar results were
obtained with Ki67 (Figure 3E,H). Results using BrdU/Ki67
double immunostaining (Figure 3I) determined that cells in the
DNE were cycling more slowly in Nestin-Cre;Prox1F/Fembryos at
E16.5 and E18.5. We also found that CyclinE expression was
reduced in the Nestin-Cre;Prox1F/FDNE at E16.5 (Figure 3K). To
confirm that the reduced proliferation of cells in the DNE was
caused by the lack of Prox1 in that region at around E16.5, we
used a tamoxifen (TM)-inducible Nestin-CreERT2strain  to
induce Prox1 deletion later during development. Following TM
administration at E16.5 (see Figure S3A for details), most C-
Prox1+cells were missing in the DNE and MS of E18.5 Nestin-
CreERT2;Prox1F/Fembryos (Figure 3M). Fewer Ki67+cells were
also observed in the DNE of these mutant embryos (Figure 3N).
Thus, Prox1 is required to regulate cell proliferation in the DNE
during early stages of DG development.
Granule Cell Maturation Is Arrested in the Absence of
During DG formation, Prox1 expression is upregulated in
intermediate progenitors and immature granule cells, and
throughout adulthood, its expression is maintained in mature
granule cells [17,23]. Therefore, we analyzed whether Prox1 is
necessary for the acquisition of granule cell identity by monitoring
the D-Prox1 cells. Using this approach, we observed an increase in
the number of D-Prox1 cells (cells recognized by the anti-N-Prox1
antibody but not by the anti-C-Prox1 antibody; see Figure S1A,B
for details) in the DG region of Nestin-Cre;Prox1F/Fembryos until
E18.5 (Figure S1D,F and Figure 4E); however, at later stages, the
number of D-Prox1 cells in the DG decreased. This reduction was
particularly apparent at postnatal stages; by postnatal day (P) 15,
most Prox1+cells in the mutant DG corresponded to those that
escaped deletion (Figure 4B,D,E). As revealed by TUNEL assay,
starting at E16.5 the number of TUNEL+cells increased in the
DG of Nestin-Cre;Prox1F/Fembryos (Figure 4F), a finding suggesting
that Prox1 is necessary for the survival of intermediate progenitors
and immature granule cells.
In the DG, terminal differentiation of granule cells occurs
during early postnatal stages. Therefore, we analyzed whether the
observed increase in the number of TUNEL+cells was due to
defective differentiation of D-Prox1 granule cells. Previous studies
have shown that the bHLH protein NeuroD1 is required for the
maturation of granule cells [15,29,30]. We found that at P10, N-
Prox1+cells in the mutant DG were also NeuroD1+(Figure 4H);
however, they did not co-express Dcx+ or Calretinin+
(Figure 4J,L), and only the few C-Prox1+granule cells that escaped
Cre deletion co-expressed NeuN (Figure 4N) . These results
suggest that lack of Prox1 activity arrested granule cell
Next, we used an in vitro assay to determine whether the
functional inactivation of Prox1 affects neuronal differentiation.
isolated from E16.5 hippocampal regions. Nestin-Cre;Prox1F/F
neurospheres produced cells that were positive for the early
neuronal marker b-tubulin-III at a relatively similar rate (119 of
135) compared to that of wild-type neurospheres (159 of 175)
(Figure S4A–C). However, fewer Nestin-Cre;Prox1F/Fneurospheres
produced Dcx+cells (8 of 190), as compared with wild-type
controls (199 of 216) (Figure S4D–F).
To determine whether Prox1 is necessary for Dcx expression, we
cotransfected Nestin-Cre;Prox1F/Fneurospheres with GFP-express-
ing and full-length Prox1 cDNA-expressing plasmids. We found
that Nestin-Cre;Prox1F/Fneurospheres transfected with Prox1 cDNA
produced Dcx+cells (20 of 76 GFP+neurospheres), but those
transfected only with the GFP-expressing plasmid did not (0 of 73
In the brain, the hippocampus has a crucial role in learning
and memory. In mammals, neurogenesis (the birth of new
neurons) occurs in the dentate gyrus region of the
hippocampus throughout adulthood, and this activity is
thought to be the basis for the acquisition of new
memories. In this study we describe for the first time the
functional roles of the transcription factor Prox1 during
brain development and adult neurogenesis. We demon-
strate that in mammals, Prox1 is required for the
differentiation of granule cells during dentate gyrus
development. We also show that conditional inactivation
of Prox1 results in the absence of specific intermediate
progenitors in the subgranular zone of the dentate gyrus,
which prevents adult neurogenesis from occurring. This is
the first report showing blockade of adult neurogenesis at
the level of progenitor cells. Next, we demonstrate that in
the absence of Prox1-expressing intermediate progenitors,
the stem cell population of the subgranular zone becomes
depleted. Further, we show that Prox1-expressing inter-
mediate progenitors are required for adult neural stem cell
self-maintenance in the subgranular zone. Finally, we
demonstrate that Prox1 ectopic expression induces
premature granule cell differentiation in the subgranular
zone. Therefore, our results identify a previously unknown
non-cell autonomous feedback mechanism that links adult
stem cell self-maintenance with neuronal differentiation in
the dentate gyrus and could have important implications
for neurogenesis in other brain regions.
Prox1 Function in Brain Neurogenesis
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Figure 1. Lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin (HE) staining of coronal sections of adult hippocampus of (A)
control and (B) Nestin-Cre;Prox1F/Fadult brains shows the absence of a dentate gyrus (DG) in the mutant mice. (C) Mature granule cells are C-Prox1+
and NeuN+. (D) In the Nestin-Cre;Prox1F/Fbrain, the few remaining Prox1+cells present in the hippocampal region are C-Prox1+NeuN+mature granule
cells that escaped deletion (insert in D). (E, F) Only a few wild-type C-Prox1+granule cells remain in the Nestin-Cre;Prox1F/Fbrain at E16.5. (G, H) There
is a reduction in the number of N-Prox1+cells in the Nestin-Cre;Prox1F/Fbrain at this stage, although these cells are still able to migrate outside the
dentate neuroepithelium (DNE) (arrows). Although reduced, Notch1 (I, J) and Ngn2 (K, L) expression remains in the DNE and DG of Nestin-Cre;Prox1F/F
embryos as shown by ISH. Moreover, Notch1 and Ngn2 progenitors (arrows) migrate outside of the mutant DNE. (M, N) The NeuroD1 expression
domain, although reduced, remains in the migratory stream (MS) and the DG of Nestin-Cre;Prox1F/Fembryos as shown by ISH. Scale bar: 100 mm. CA,
Pyramidal cell layer; T, Thalamus. Scale bar: 100 mm.
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GFP+neurospheres) (Figure S4G,H). Thus, as seen in vivo, Prox1
is not necessary to induce neuronal differentiation in vitro but is
required for the expression of later neuronal markers such as Dcx.
Prox1 Is Required for Granule Cell Maturation at
To confirm that the conditional inactivation of Prox1 at
postnatal stages is directly responsible for the defective granule
cell maturation phenotype observed in Nestin-Cre;Prox1F/Fmice, we
next deleted Prox1 during postnatal stages by using the Nestin-
CreERT2transgenic mouse strain . TM was administered to
Nestin-CreERT2;Prox1F/Fpups at early postnatal stages (see Figure
S3B for details); at these stages, there are no Prox1+NSCs in the
hilus (Figure S5A–E). At 2 mo of age, TM-treated Nestin-
CreERT2;Prox1F/Fmice had smaller DGs than their control
littermates (Figure 5B). The number of Tbr2+intermediate
progenitors  was reduced in both P5 (Figure S5R,S) and
P10 (Figure 5P,Q) pups. They also exhibited fewer Dcx+cells at P5
(Figure S5T,U) and P10 (Figure 5R,S). Moreover, the ratio of
Figure 2. Patterning of the Ammon’s horn is normal in the dentate gyrus of E14.5 Nestin-Cre;Prox1F/Fembryos. (A) Prox1 is expressed in
the dentate gyrus neuroepithelium (DNE) at E14.5. (B) Nestin-Cre successfully removed most Prox1 expression from the DNE at this stage. However,
the expression pattern of Notch1 (C, D) and Ngn2 (E, F) is not changed in the DNE of Nestin-Cre;Prox1F/Flittermates. (G, H) Lhx2 is expressed in the
developing dentate gyrus (DG) but not in the fimbria neuroepithelium (FNE, arrows) of control and Nestin-Cre;Prox1F/Fbrains. (I, J) Wnt3a expression is
detected in the DG and FNE of control and conditional mutant brains at this stage (arrows). (K, L) Calretinin expression in the marginal zone (MZ) is
also normal in the Nestin-Cre;Prox1F/Fbrain. Cx, cortex; T, thalamus; GE, ganglionic eminence. Scale bar in (A, B): 50 mm. Scale bar in (C–L): 100 mm.
Prox1 Function in Brain Neurogenesis
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Tbr2+:Calretinin+cells was increased in the TM-treated Nestin-
CreERT2;Prox1F/F(1.5560.25:1; N=3) pups at P10 (control:
1.0960.28:1; N=3; p,0.1). These results suggest that the reduced
size of the DG was caused by a reduction in the number of
intermediate progenitors and by an arrest in granule cell
differentiation. Moreover, as indicated by TUNEL (Figure S5V–
Y and Figure 4T–V) and active Caspase-3 assays (Figure S6A,B),
an increase in cell death was observed in the mutant DG at these
stages. On the other hand, cell proliferation was reduced at P5 but
not at P10 in the DG of Nestin-CreERT2;Prox1F/Fpups (Figure S5P;
Figure 5N). The number of Nestin+, Sox2+, or Id1+NSCs was
normal at P5 and P10 (Figure S5J,M; Figure 5C–K). These data
suggest that the lack of Prox1 during postnatal stages is directly
responsible for the reduction in the number of intermediate
progenitors and the defect in granule cell differentiation that
secondarily promotes an increase in cell death and ultimately a
reduction in the size of the DG.
Prox1 Is Required for the Maintenance of Intermediate
Progenitors in the SGZ During Adult Neurogenesis
In the SGZ, Prox1 expression is initially detected in Tbr2+
(Figure 6E)  and Dcx+(Figure 6F) Type-IIb intermed-
iate progenitors  and is absent from adult NSCs
(Figure 6A,B,C) [37,38] and Ascl1+intermediate progenitors
(Figure 6D) . As neurogenesis progresses, Prox1 is detected
in Calretinin+cells (Figure 6H). Analysis of the SGZ of Nestin-
CreERT2;Prox1F/Fpups treated with TM daily from P0 to P15
(see Figure S3B for the details of the TM treatment) identified
a reduced number of Tbr2+(Figure 6K) and Dcx+(Figure
6N) cells at P20; these cells were nearly absent at 4 and 8
mo of age (Figure 6J,K,M,N). As indicated by C-Prox1
immunostaining, the few remaining Dcx+cells were those
that escaped deletion (Figure S7). Fewer Calretinin+immature
neuron cells were also observed in the mutant brains
Figure 3. Reduced proliferation in the dentate gyrus neuroepithelium of Nestin-Cre;Prox1F/Fembryos. (A, D) BrdU (after 1-h pulse) and Ki67 (B,
E) immunostaining revealed reduced proliferation in the dentate gyrus neuroepithelium (DNE) of Nestin-Cre;Prox1F/Fbrains starting at E16.5. Similar results
control brains at E18.5. Following TM administration at E16.5, there are fewer C-Prox1+cells in the DNE and MS (M) of Nestin-CreERT2;Prox1F/Fbrains. (N) As
indicated by Ki67 counting, the number of cycling cells is reduced in the DNE of Nestin-CreERT2;Prox1F/Fembryos. Data represent the mean number of
positive cells per DG section 6 SD (N=3 embryos). Paired t test. * p,0.1; ** p,0.01; **** p,0.0001. Scale bar in (A–M): 50 mm.
Prox1 Function in Brain Neurogenesis
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To determine whether the lack of intermediate progenitors in
adult stages is a direct or indirect result of previously identified
alterations in postnatal DG development, we treated 8-wk-old
(adult) Nestin-CreERT2;Prox1F/Fmice with TM 3 d/week (see Figure
S3C for the details of the TM treatment). Four weeks after the
initial TM induction, the number of Tbr2+(Figure 7A–C) and
Dcx+(Figure 7D–F) cells was reduced in the SGZ of 12-wk-old
Nestin-CreERT2;Prox1F/Fmice. Accordingly, the number of Calre-
tinin+cells was also reduced (Figure 7G–I). Similar results were
observed8 wkafter induction
To evaluate whether the lack of Prox1 activity leads to an
increase in cell death, as it does during developmental stages, we
performed double TUNEL immunohistochemistry in 12- and 16-
wk-old mice after TM induction. At both time points, we observed
an increased number of TUNEL+Tbr2+(Figure 7J,K,M) and
TUNEL+Dcx+(Figure 7J–L) cells in the Nestin-CreERT2;Prox1F/F
SGZ, a result indicating that Prox1 is required for the survival of
To evaluate whether the lack of Tbr2+intermediate progenitors
and Dcx+cells affects adult neurogenesis in the SGZ, we
performed BrdU labeling 15 d prior to collection of the brains
for analysis. Fewer BrdU+newborn cells were observed in the
SGZ of 12-wk-old (Figure 7N–P) and 16-wk-old (Figure 7Q–S)
Nestin-CreERT2;Prox1F/Fmice. This result suggests that the lack of
Prox1 in the SGZ affects adult neurogenesis.
Next, to determine whether the defective neurogenesis in the
SGZ is solely because of a reduction in the number of intermediate
progenitors or it is also because of an alteration in immature
neurons, we analyzed the ratio of Tbr2+:Calretinin+cells in the
SGZ of 12-wk-old and 16-wk-old Nestin-CreERT2;Prox1F/Fmice. A
consistent increase in the ratio of Tbr2+:Calretinin+cells was
observed in 12-wk-old (N=3) (2.3960.58:1) and 16-wk-old (N=3)
(4.3961.92:1) TM-treated Nestin-CreERT2;Prox1F/Fmice (12-wk-
old control: 1.7760.34:1; 16-wk-old control: 1.5160.17:1; p value
for both cases was p,0.1). This result suggests that granule cell
maturation is also affected by the lack of Prox1 in the SGZ during
We next examined whether the induction of neurogenesis
rescues the reduction in the number of progenitor cells observed in
the SGZ of adult Nestin-CreERT2;Prox1F/Fmice. At 12 wk of age,
TM-induced and control mice were treated with kainic acid (KA),
a compound that induces adult hippocampal neurogenesis (see
Figure S3D for the details of the TM/KA treatment) [39,40].
Eight days after KA administration, the brains of control mice
treated with TM and KA or only with KA showed an increased
Figure 4. Granule cell maturation is affected in D-Prox1 cells. (A–E) As shown by double-staining with anti-N-Prox1 (red) and anti-C-Prox1
(green), the number of D-Prox1 cells (red bars in E) is reduced in the Nestin-Cre;Prox1F/Fdentate gyrus (DG) at postnatal stages of development (blue
bars in E represent double N-Prox1/C-Prox1+wild-type cells). (F) There is an increase in the number of TUNEL+cells in the mutant DG as granule cell
maturation progresses. IHC using anti-N-Prox1 and anti-NeuroD1 antibodies revealed that all granule cells are NeuroD1+both in P10 wild-type (G)
and conditional mutant (H) DG. However, in the conditional mutant hippocampal region most of the N-Prox1+cells are Dcx2(J) and Calretinin2(L).
The only NeuN+cells left in the Nestin-Cre;Prox1F/Fhippocampal region are the C-Prox1+cells that have escaped Cre deletion (N). Panels I, K, and M
correspond to controls. Data represent the mean number of positive cells per DG section 6 SD. N=3 brains. Paired t test. ** p,0.01; *** p,0.001;
**** p,0.0001. CA, Pyramidal cell layer. Scale bar: 100 mm.
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number of Tbr2+(Figure S8A,B), Dcx+(Figure S8D,E), or
Calretinin+(Figure S8G,H) cells in the SGZ. However, similar
to that in the TM-treated Nestin-CreERT2;Prox1F/Fmice, the SGZ
of Nestin-CreERT2;Prox1F/Fmice treated with TM and KA had
fewer Tbr2+intermediate progenitors (Figure S8A,C) and fewer
Dcx+(Figure S8D,F) and Calretinin+(Figure S8G,I) cells. These
results show that KA-mediated induction of neurogenesis cannot
rescue the reduction in the number of Tbr2+, Dcx+, or Calretinin+
cells in the SGZ of TM-treated adult Nestin-CreERT2;Prox1F/Fmice.
Lack of Intermediate Progenitors Results in Defective
Self-Maintenance of Adult Neural Stem Cells
It has been proposed that upon their differentiation, interme-
diate progenitors trigger a feedback mechanism necessary to stop
NSC differentiation and support NSC maintenance [13,15].
Therefore, the lack of intermediate progenitors observed in the
SGZ of Nestin-CreERT2;Prox1F/Fmice may impinge on this
feedback mechanism and ultimately affect the number of adult
NSCs produced in this region.
To assess whether the reduced number of intermediate
progenitors in the SGZ of Nestin-CreERT2;Prox1F/Fmice treated
postnatally with TM affected that of NSCs, we compared the
number of Type-I NSCs in wild-type and Prox1 conditional-
mutant brains. We detected no difference in the number of
Nestin+, Sox2+, or Id1+adult NSCs at P20 (Figure 8A,D,G).
However, we unexpectedly observed fewer Nestin+, Sox2+, or Id1+
NSCs in the SGZ of Nestin-CreERT2;Prox1F/Fmice older than 2 mo
(Figure 8A–I). We also observed a similar reduction in the number
of Nestin+Gfap+Blbp+NSCs in the SGZ of 4-mo-old Nestin-
CreERT2;Prox1F/Fmice (Figure S9). This result argued that the
absence of intermediate progenitors observed in the SGZ of Nestin-
CreERT2;Prox1F/Fbrains leads to a non-cell autonomous reduction
in the number of adult NSCs.
To determine whether defective maintenance of adult NSCs is a
direct result of the lack of intermediate progenitors or an indirect
result because of alterations in DG development, we treated 8-wk-
old control and Nestin-CreERT2;Prox1F/Fmice with TM (see Figure
S3C for the details of the TM treatment) and counted the number
of adult NSCs. Four weeks after the beginning of the treatment,
the number of Nestin+, Sox2+, or Id1+adult NSCs was higher in
the SGZ of Nestin-CreERT2;Prox1F/Fmice than in wild-type
littermates (Figure 8J,M,P); however, 8 wk after TM treatment,
we detected fewer Nestin+, Sox2+, or Id1+adult NSCs in the SGZ
of Nestin-CreERT2;Prox1F/Fmice (Figure 8J–R).
To examine whether Prox1 has a direct role in this defective
maintenance of adult NSCs, we generated Nestin-CreERT2;Prox1F/
F;R26R mice and treated them with TM (3 d/wk) starting at 8 wk
of age (see Figure S3C for the details of the TM treatment). After
4 wk of treatment, the ratios of Nestin+b-gal+cells and Sox2+b-
gal+cells were the same in wild-type and Prox1 conditional-mutant
mice (Figure 8S). This result indicates that the absence of Prox1
did not directly affect the adult NSC population. Similar results
were seen in mice analyzed 8 wk after the beginning of TM
administration (Figure 8T). Only the Tbr2+b-gal+and the Dcx+b-
gal+cell populations were significantly reduced in the SGZ of
Nestin-CreERT2,Prox1F/F;R26R mice at 4 and 8 wk after the
beginning of TM treatment (Figure 8S,T). These results, together
with the absence of increased apoptosis in adult NSCs
Figure 5. Post-natal lack of Prox1 affects dentate gyrus formation. Hematoxylin and eosin staining (A, B) of coronal sections of the
hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreERT2;Prox1F/Fmice treated with TM from P0 to P15 (B). There are no changes
in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreERT2;Prox1F/FDG. BrdU staining
after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreERT2;Prox1F/Fbrains (M, N). The number of Tbr2+
intermediate progenitors (O–Q) and Dcx+(R, S) cells is reduced in the Nestin-CreERT2;Prox1F/FDG at P10. We also observed an increase in the number
of TUNEL+cells (T–V) in the DG area of the Nestin-CreERT2;Prox1F/Fbrains at P10. Data represent the mean number of positive cells per DG section 6
SD (N=3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreERT2;Prox1F/Fbrains. Paired t test. * p,0.1; ** p,0.01. CA, pyramidal cell
layer; T, thalamus; H, Hilus. Scale bar: 100 mm.
Prox1 Function in Brain Neurogenesis
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Figure 6. Prox1 is required in the subgranular zone during adult neurogenesis. During adult neurogenesis, Prox1 is not expressed in
Nestin+(A), Sox2+(B), or Id1+(C) adult neural stem cells or in Acsl+Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2+(E) and
Dcx+Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin+immature neurons. Graphs compare the number of
Tbr2+(K) Dcx+(N) and Calretinin+(Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreERT2;Prox1F/F(red) mice. A reduced
number of Tbr2+(J, K), Dcx+(M, N), and Calretinin+(P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreERT2;Prox1F/Fmice. Data represent the
mean number of positive cells per DG section 6 SD. N=3 brains. Paired t test. * p,0.1; ** p,0.01; *** p,0.001; **** p,0.0001. GCL, granule cell layer;
IML, inner molecular layer; M, month. Scale bar in (A–H): 25 mm. Scale bar in (I, J, L, M, O, P): 50 mm.
Prox1 Function in Brain Neurogenesis
PLoS Biology | www.plosbiology.org8 August 2010 | Volume 8 | Issue 8 | e1000460
(Figure 7J,K), suggest that the defective maintenance of adult
NSCs in the mutant SGZ is indirectly caused by the absence of
We next investigated the possible mechanisms of this interme-
diate progenitor-dependent control of adult neurogenesis in the
SGZ. In the developing telencephalon  and in the SGZ ,
Notch signaling is necessary for NSC maintenance, and the
expression of Jagged1, a Notch receptor ligand, is restricted to
intermediate progenitors in the SGZ . We found a reduced
number of Jagged1+cells in the SGZ of Nestin-CreERT2;Prox1F/F
mice treated with TM starting at 8 wk of age and analyzed 4 wk
later (Figure 9C,D). To examine the relationship between the
number of Nestin+Type-I cells in the SGZ and active Notch
signaling, we performed Hes1 immunohistochemistry. In the SGZ
of control mice, only 17% of the Type-I cells were Nestin+Hes1–
(Figure 9F); however, in the Nestin-CreERT2;Prox1F/Fmice, this
number increased to 55% (Figure 9F). The number was also
higher in the SGZ of Nestin-CreERT2;Prox1F/Fmice (59%) when
compared with wild-type controls (25%) at 16 wk of age
(Figure 9G). A reduced number of Hes5-expressing cells was also
observed in the SGZ of Nestin-CreERT2;Prox1F/Fmice at 16 wk of
age (Figure S10). These results show that in the absence of
intermediate progenitors, the proportion of adult NSCs with active
Notch signaling at 12 and 16 wk is reduced. This mechanism may
explain why the self-maintenance of adult NSCs is eventually
overruled at 16 wk of age.
Ectopic Expression of Prox1 in NSCs Induces Their
Our results showed that Prox1 is necessary for granule cell
differentiation and intermediate progenitor maintenance. There-
fore, we next determined whether Prox1 activity is sufficient to
induce granule cell differentiation. To do this, we generated a new
mouse transgenic line in which Prox1 expression was under the
control of the ubiquitous CMV promoter (CMV-CAG-loxP-eGFP-
Stop-loxP-Prox1-Ires-b Gal; JoJo-Prox1 for brevity) . Crossing this
line with any available Cre strain will lead to the release of the
Stop-signal cassette and the transcription of Prox1 in a tissue-
specific manner. We used this novel strain to generate adult Nestin-
Cre;JoJo-Prox1 mice that ectopically expressed Prox1 in several
brain regions (Figure S11). Importantly, we found that the number
of granule cells was reduced in the DG of these mice
Prox1 was ectopically expressed in the ventricular, subventri-
cular, and mantle zones of several brain regions of Nestin-Cre;JoJo-
Prox1 embryos (Figure S11), including the hippocampal neuroep-
ithelium and the hippocampal field (Figure 10D). However, we
observed no change in the number of Prox1+(Figure 10E), Sox2+
(Figure 10F), or Ki67+cells (Figure 10G) in the DNE or DG.
These results suggest that ectopic expression of Prox1 in
embryonic Nestin-expressing neuroepithelium is not sufficient to
affect embryonic NSC differentiation.
To perform a similar analysis during postnatal stages, we
administered TM to Nestin-CreERT2;JoJo-Prox1 pups daily starting
at P0 (see Figure S3B for the details of the TM treatment). At P8,
the DG of Nestin-CreERT2;JoJo-Prox1 pups exhibited an increased
number of Dcx+(Figure 10I) and NeuN+(Figure 10L) cells and a
reduced number of Nestin+cells (Figure 10N). At this stage,
proliferation was already reduced in the DG of Nestin-CreERT2;-
JoJo-Prox1 mice, as shown by Ki67 staining (Figure S12G).
To determine the fate of cells that ectopically expressed Prox1,
we performed double immunohistochemistry using antibodies
against b-gal and Nestin, Dcx, NeuN, Gfap, or NG2 (Figure S12I–
M). We determined that 2.11%60.38% of the b-gal+cells in the
64.57%612.99% were Dcx+; 34.52%66.32% were NeuN+; and
less than 0.1% were Gfap+or NG2+(Figure 10O). No increase in
Figure 7. Prox1 is required during SGZ neurogenesis for the
maintenance of intermediate progenitors. A reduced number of
Tbr2+intermediate progenitors (A–C) and of Dcx+(D–F) and Calretinin+
(G–I) cells was observed in the SGZ of Nestin-CreERT2;Prox1F/Fmice. An
increase in the number of TUNEL+/Tbr2+(J, M) and TUNEL+/Dcx+(J, L)
cells was observed in 12-wk-old and 16-wk-old (K) Nestin-CreERT2;
Prox1F/Fmice. As a consequence of the reduction in the number of
intermediate progenitors, fewer BrdU+cells are found in the DG of
Nestin-CreERT2;Prox1F/Fmice 4 (N–P) and 8 (Q–S) wk after the beginning
of TM treatment. Data represent the mean number of positive cells per
DG section 6 SD. (N=4 mice). Blue bars are controls. Red bars are TM-
treated Nestin CreERT2;Prox1F/Fbrains. Paired t test. * p,0.1; ** p,0.01;
*** p,0.001; **** p,0.0001. W, weeks. Scale bar: 100 mm. Scale bar in
(L, M): 10 mm.
Prox1 Function in Brain Neurogenesis
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the number of TUNEL+cells was observed in the DG of these
mice (Figure S12H). These results suggest that there is premature
neuronal differentiation in the DG of Nestin-CreERT2;JoJo-Prox1
mice during postnatal stages of brain development.
Finally, we addressed the consequences of ectopic expression of
Prox1 in the SGZ during adult neurogenesis. Analysis of the brains
of P30 Nestin-CreERT2;JoJo-Prox1 mice revealed the presence of
Nestin+;Prox1+adult NSCs (Figure S12N). As a consequence of
Prox1 ectopic expression, the numbers of Nestin+(Figure 10Q,T)
and Sox2+(Figure S12P) cells were reduced at this stage.
Accordingly, the numbers of Dcx+(Figure 10S,U) and Calretinin+
(Figure S12R) intermediate progenitors were also reduced. These
results indicate that the ectopic expression of Prox1 depleted the
adult NSC population.
In this article, we report for the first time the functional role for
Prox1 in mammalian brain development. We determined that in
the mouse, Prox1 is required for the maturation of granule cells
during DG development. Prox1 is expressed through all the stages
of DG formation; therefore, it is possible that the defective granule
cell maturation observed in Nestin-Cre;Prox1F/Fmice is an indirect
consequence of the earlier absence of Prox1 at the intermediate
progenitor level. However, it is also possible that Prox1 plays
additional functional roles during granule cell formation. More-
over, the granule cells in the DG are one of the few types of brain
cells that express Prox1 throughout adulthood. This suggests that
Prox1 might be necessary not only for the maturation of granule
cells but also for the regulation of other aspects of granule cell
function. NeuroD1 is also required for granule cell maturation
[15,29,30]. During embryogenesis and in the absence of Prox1 or
NeuroD1, mutant granule cells express some neuronal markers
but fail to fully differentiate and undergo apoptosis . As a
consequence, Nestin-Cre;Prox1F/Fand NeuroD1–/–mice have very
few granule cells (Figure 1) [15,30]. Few Prox1+granule cells are
present in the DG of NeuroD1–/–mice , and NeuroD1 is
expressed in the DG of Prox1-conditional mutants. These results
suggest that although NeuroD1 and Prox1 might control similar or
parallel pathways of granule differentiation, they might not
necessarily induce each other’s expression.
We also showed that during adult neurogenesis, Prox1 is
necessary for the survival of Tbr2+intermediate progenitors in the
SGZ. In this case and similarly to DG development, our data
suggest that the lack of Prox1 results in defects in granule cell
maturation that could also be an indirect consequence of the
earlier lack of Prox1 at the progenitor level. Nevertheless, the lack
of Prox1 leads to an increase in apoptosis in Tbr+and Dcx+cells
and the absence of adult neurogenesis. Also in this case, similar
results have been reported for NeuroD1-mutant mice during adult
Our results provide the strongest evidence so far about a
feedback mechanism involved in the regulation of adult neuro-
genesis and progenitor cell numbers in the adult SGZ. We also
provide evidence supporting the proposal that Tbr2+intermediate
progenitors and Dcx+cells are required to maintain the adult NSC
population in the SGZ niche. We showed that in the absence of
Tbr2+and Dcx+cells and as this feedback regulation becomes
Figure 8. Adult NSCs in the SGZ do not self-maintain in the
absence of intermediate progenitors. Nestin (B, C), Sox2 (E, F), and
Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-
mo-old controls (B, E, H) and Nestin-CreERT2;Prox1F/F(C, F, I) mice treated
with TM from P0 to P15. Graphs compare the number of Nestin+(A),
Sox2+(D), and Id1+PECAM2(G) cells in P20, 2-mo-old, 4-mo-old, and 8-
mo-old control (blue) and Nestin-CreERT2;Prox1F/F(red) mice treated with
TM from P0 to P15. At P20, the number of Nestin+, Sox2+, or
Id1+PECAM2NSCs is similar in controls and Nestin-CreERT2;Prox1F/F
brains (A, D, G). However, after P20, the number of Nestin+(A–C), Sox2+
(D–F), or Id1+PECAM2(G–I) cells is reduced in Nestin-CreERT2;Prox1F/F
mice. Graphs compare the number of Nestin+(J), Sox2+(M), and Id1+
PECAM2(P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-
CreERT2;Prox1F/F(red) mice treated with TM for 4 wk starting at 8 wk of
age. The number of Nestin+, Sox2+, and Id1+PECAM2adult NSCs was
increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the
number of Nestin+(J, L), Sox2+(M, O), and Id1+PECAM-(P, R) cells is
reduced in the SGZ of conditional-mutant mice. The percentage of
Nestin+/b-Gal+and Sox2+/b-Gal+cells is similar in 12-wk-old (S) and 16-
wk-old (T) control (blue) and Nestin-CreERT2;Prox1F/F(red) brains that
carry the ROSA allele. However, the percentage of Tbr2+/b-Gal+and
Dcx+/b-Gal+cells is reduced at both stages in the Nestin-CreERT2;
>Prox1; SGZ (S, T).<?FIGTITLE 0 Data represent the mean number of
positive cells per DG section 6 SD. N=4 brains. Paired t test. * p,0.1; **
p,0.01; *** p,0.001; **** p,0.0001. GCL, Ganglion cell layer. Scale bar in
(B, C, E, F, H, I): 25 mm. Scale bar in (K, L, N, O): 100 mm. M, month; W,
weeks. Scale bar in (Q, R): 50 mm.
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defective, adult NSCs continue generating new progeny such that
the adult NSC population in the SGZ of Prox1-conditional
mutants transiently expands but is ultimately depleted. We do not
yet know whether this depletion of NSCs is due to their premature
differentiation or their exhaustion resulting from increased
How is this feedback mechanism regulated? We showed that in
the SGZ of Prox1 conditional-mutant mice the lack of intermediate
progenitors leads to a reduction in Jagged1 expression. Moreover,
we determined that in these conditional-mutant mice the lack of
intermediate progenitors leads to the absence of active Notch
signaling in adult NSCs; active Notch signaling is necessary for the
maintenance of adult stem cells in the brain, bone marrow, and
gut [42,47–51]. Previous work has shown that during embryonic
development, the activity of mind bomb homolog 1 (Mib1) is required
for Jagged and Delta-like-mediated Notch signaling. In the
absence of Mib1, Notch signaling is not activated in radial glia
cells, which results in their premature differentiation [41,52]. In
the SGZ, the absence of Notch1 is sufficient to induce neuronal
differentiation and reduce the proportion of adult NSCs and
intermediate progenitors, which suggests that this receptor is
involved in NSC self-renewal in the SGZ . Therefore, the role
of other Notch receptors during adult neurogenesis could be either
redundant or restricted to a subpopulation of cells in the SGZ
Together, these and others results  suggest that in the SGZ,
the Notch1/Jagged1 pathway may regulate NSC self-maintenance.
However, we cannot exclude the possibility that Notch activation
in adult NSCs is also mediated by other Notch ligands expressed
by other cell types (e.g., vascular endothelial cells) . Also, we
cannot rule out the possibility that other signaling molecules or
trophic factors produced by Prox1-expressing intermediate pro-
genitors may be involved in the feedback mechanism that controls
adult neurogenesis in the SGZ.
In summary, we have provided evidence demonstrating that
Prox1 plays a key role as a neurogenic factor during granule cell
formation. In the absence of Prox1, granule cell maturation is
affected at later stages of differentiation; ectopic expression of
Prox1 in the brain is sufficient to overrule the mechanisms that
control NSC self-maintenance and induce premature differentia-
tion in the SGZ during postnatal stages of brain development and
Materials and Methods
Prox1F/F mice, Nestin-Cre  mice, and Nestin-CreERT2
mice were previously described. The JoJo-Prox1 construct was
generated by the introduction of a 2.2 kb Prox1 cDNA into a
CMV-CAG-loxP-eGFP-Stop-loxP-IRES-bGal expression vector .
Mice were kept in the NMRI background. TM (Sigma) was
dissolved in safflower oil at 20 mg/ml. For prenatal induction,
time-mated females were treated with TM by gavage at E16.5 and
embryos were harvested at E18.5. To induce Cre recombination
postnatally, pups were fed TM (4 mg/20 g body weight) daily
from P0 until the collection day. As a consequence of postnatal
TM administration, body weight was reduced in TM-treated
versus non-TM-treated pups. This reduction was temporary; 2-
Figure 9. Lack of intermediate progenitors disrupts Notch signaling in the SGZ of Nestin-CreERT2;Prox1F/Fmice. (A) Prox1+/Jagged1+cells
in the SGZ of adult mice. (B) TM administration schedule. (C, D) Jagged1+cells were not detected in 12-wk-old Nestin-CreERT2;Prox1F/Fmice after 4 wk
of TM treatment. (E) Nestin+Hes1+cells are present in the SGZ of control and Nestin-CreERT2;Prox1F/Fmice. However, the number of double positive
cells is reduced in TM-treated Nestin-CreERT2;Prox1F/Fin 12-wk-old (F) and 16-wk-old mice (G). Data represent the mean number of positive cells per
DG section 6 SD. N=3 brains. Paired t test. *** p,0.001. Scale bar in (A, E): 25 mm. Scale bar in (C, D): 100 mm.
Prox1 Function in Brain Neurogenesis
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mo-old postnatally TM-treated Nestin-CreERT2;Prox1F/+and Nestin-
CreERT2;Prox1F/Fmice had a similar weight than non-TM-treated
animals. In adults (8-wk-old), TM (4 mg/20 g body weight) was
administered by gavage 3 times/wk for 4 wk. No obvious weight
differences were observed in the TM-treated animals at the time of
sample collection. Genotypes were determined by PCR analysis.
Figure 10. Prox1 ectopic-expression promotes NSC premature differentiation. Anti-C-Prox1 staining (A, control) shows a reduced dentate
gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1+cells in the DG region of control (C, E) and Nestin-
Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2+(F) or Ki67+(G) cells in the dentate neuroepithelium (DNE) and the
DG at E16.5. At P8, there is an increase in the number of Dcx+(H, I) and NeuN+(J, L) cells in TM-treated Nestin-CreERT2;Prox1F/Fbrains. However, the
number of Nestin+(M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly
differentiate towards a neuronal lineage. The number of Nestin+adult NSCs (P, Q, T) and Dcx+intermediate progenitors (R, S, U) is reduced in the SGZ
of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section 6 SD. N=3 brains. Paired t test. *** p,0.001.
Blue bars are controls. Red bars are Nestin-Cre;JoJo-Prox1. MS, Migratory stream; HF, Hippocampal field. Scale bar: 100 mm.
Prox1 Function in Brain Neurogenesis
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Nestin-Cre;Prox1F/+, Nestin-CreERT2;Prox1F/+, and JoJo-Prox1 mice
were used as controls.
For Hes1 staining during adult stages, brains were perfused with
4% PFA and embedded in paraffin. Antigen retrieval was
performed for 7 min at 105uC in a decloaking chamber with
Antigen Retrieval Solution (Dako). The ABC Kit (Vector
Laboratories) and TSA Fluorescein System (Perkin Elmer) were
used for signal amplification. Jagged1 staining at adult stages was
performed on brains perfused with 4% PFA and cryoprotected in
30% sucrose. Signal amplification was obtained with the ABC Kit
and the TSA Fluorescein System. Immunohistochemistry for the
rest of the antibodies was performed as described . The
following antibodies and dilutions were used: rabbit anti-C-Prox1
(1:1000; Millipore), guinea pig anti-C-Prox1 (1:100; our own),
rabbit anti-N-Prox1 (1:1000; a gift from B. Sosa-Pineda), goat anti-
N-Prox1 (1:100; R&D systems), goat anti-Nestin (1:100; R&D
systems), rabbit anti Id-1 (1:200; Biocheck), rat anti-PECAM
(1:500; Pharmingen), mouse anti-Gfap (1:500; Sigma), rabbit anti
Blbp (1:200; Chemicon), rabbit anti-Sox2 (1:1000; Milipore),
rabbit anti-Sox2 (1:500; Invitrogen), mouse anti-Sox2 (1:50,
Milipore), mouse anti-Ascl1 (1:100; Milipore), mouse anti-bTubIII
(1:500; BabCO), rabbit anti-NeuroD1 (1:500; Chemicon), rabbit
anti-Dcx (1:100; Abcam), rabbit anti-Dcx (1:1000; Chemicon),
rabbit anti-Dcx (1:500; Cell Signaling), rabbit anti-Calretinin
(1:5000; Millipore), mouse anti-NeuN (1:100; Millipore), rabbit
anti-active caspase-3 (1:100; BD-Pharmingen), rabbit anti-Hes1
(1:50, Santa Cruz), goat anti-Jagged1 (1:50, Santa Cruz), rabbit
anti-GFP (1:1000; Molecular Probes), rabbit anti-bGal (1:1000,
ICN), and chicken anti-bGal (1:1000, Abcam). The following
secondary antibodies were used: anti-rabbit, anti-mouse, anti-
guinea pig, anti-chicken, or anti-goat Alexa 488, Alexa 594
(Molecular Probes), Cy3, or Cy5 (Jackson Immunoresearch). Low-
magnification images were obtained with a Leica MZFLIII
stereomicroscope equipped with a Hanamatsu C5810 camera
and a Zeiss Axiovert 1.0 microscope equipped with a ProgRes C14
camera. The remaining images were obtained with a Leica SP1
confocal microscope or a Zeiss LSM 510 NLO Meta confocal
In Situ Hybridization
In situ hybridization of sections was performed as previously
described . The following probes were obtained from: Ngn2
(Q. Ma), Wnt3a (A. McMahon), Lhx2 (H. Westphal), Notch1 (G.
Weinmaster), Lef1 (G. Kardon). Double–in situ hybridization/
immunohistochemistry was performed as described .
TUNEL and Proliferation Assays
TUNEL assay was performed on tissue sections as previously
described . For proliferation assays at embryonic stages, time-
mated female mice were injected with BrdU (100 mg/g body
weight, intraperitoneally), and embryos were harvested 1 h later.
Embryos were fixed o/n in 4% PFA and cryoprotected in 30%
sucrose. For proliferation assays at early postnatal stages, P5 and
P10 pups were injected with BrdU (100 mg/g body weight,
intraperitoneally) 1 h before harvest. Brains were perfused with
4%PFA and cryoprotected in 30% sucrose. For proliferation
assays at adult stages, animals were injected with BrdU (100 mg/g
body weight, intraperitoneally) 15 d before harvest. Brains were
perfused in 4% PFA and cryoprotected in 30% sucrose. BrdU
incorporation was exposed after 20-min treatment in 2N HCl.
Mouse anti-BrdU (1:10; BD Biosciences) antibody was used.
Sections were counterstained with DAPI.
Neurosphere Cultures and Immunocytochemistry
Neurosphere cultures were established as described, with
modifications . Briefly, E16.5 hippocampi were dissected,
disaggregated in trypsin, and maintained in culture in neurosphere
culture medium (Neurobasal medium with GlutaMAX, Pen/
Strep, B27, and N2) (Gibco) supplemented with 20 ng/ml EGF
(Upstate) and 20 ng/ml FGF (Millipore). After the fourth passage,
neurospheres were differentiated for 4 d in neurosphere culture
medium with 10% fetal calf serum (FCS) without supplements, in
Lab-Tek II CC2chamber slides (Nunc). Cells were fixed in 2%
PFA for 15 min at room temperature (RT). Cells were blocked in
10% FCS for 30 min at RT and incubated with the appropriate
primary and secondary antibodies (see above) in 2% FCS for 2 h.
For the transfection of the neurospheres, a Mouse NSC
NucleofectorH Kit (Amaxa) was used according to the manufac-
turer’s specifications. For Prox1 expression, a Prox1 cDNA was
cloned in an expression plasmid under the PGK promoter.
Kainic Acid Assay
Adult neurogenesis was induced by KA as described  with
minor modifications. Briefly, TM (4 mg/20 g body weight) was
administered by gavage 3 times/wk for 4 wk to 8-wk-old Nestin-
CreER;Prox1F/Fand Nestin-CreER;Prox1F/+mice. At the end of TM
treatment, animals were injected intraperitoneally with KA
(30 mg/kg body weight; Sigma) dissolved in PBS. Approximately
40 min after KA injection, all mice displayed status epilepticus for
2 to 3 h. Approximately 15% of control and conditional mutant
mice died 12 h after KA administration. Eight days after KA
injection, the surviving animals were euthanized and their brains
perfused with 4% PFA and cryoprotected in 30% sucrose.
al mutant mice. (A, B) Schematic representation of the different
Prox1 alleles described in this article and the recognition domains
of the anti-C-Prox1 and anti-N-Prox1 antibodies. (A) In the Prox1
floxed allele, loxP sites flank part of the prospero domain (purple)
and the homeodomain (green). (B) Following Cre-mediated
excision (D form), only a nonfunctional N-terminal part of Prox1
remains, without most of the prospero domain and without the
entire homeodomain. Anti-N-Prox1 antibodies recognize both the
wild-type and D forms of the Prox1 protein, but anti-C-Prox1
antibodies recognize only the wild-type full-length protein. For
more details, see Hervey et al. (2005). (C–F) Coronal sections of
control (C, E) and Nestin-Cre;Prox1F/F(D, F) dentate gyrus at
different developmental stages show the presence of the D-Prox1+
cell population in the conditional-mutant mice. Sections were
immunolabeled with antibodies against N-Prox1+(red) and C-
Found at: doi:10.1371/journal.pbio.1000460.s001 (0.64 MB TIF)
N-Prox1+cells migrate from the dentate
neuroepithelium to the region of the dentate gyrus in
Nestin-Cre;Prox1F/Fmice. (A, B) Wnt3a is expressed in the
fimbria neuroepithelium (FNE) (arrows) of E16.5 control and
Nestin-Cre;Prox1F/Fbrains. (C, D) In response to Wnt3a signaling
from the FNE, Lef1 is expressed in N-Prox1+migrating cells
(arrows) in E16.5 control and mutant embryos. The radial glia
scaffolding is normal in the Nestin-Cre;Prox1F/Fdentate gyrus (DG),
as shown by Gfap (E, F) and Nestin (G, H) immunostaining. DNE,
Dentate neuroepithelium; MS, Migratory stream. Scale bar in (M–
T): 50 mm.
Found at: doi:10.1371/journal.pbio.1000460.s002 (6.42 MB TIF)
Detection of D-Prox1 cells in Prox1 condition-
Prox1 Function in Brain Neurogenesis
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istration protocol. (A) For prenatal induction, TM was
administered to time-mated female mice at E16.5 and embryos
were harvested at E18.5. (B) For postnatal induction, pups were
fed daily with TM until collection day or P15. (C) For adult
induction, 8-wk-old mice were treated with TM for 4 wk, 3 d a
week. Samples were collected 4 wk or 8 wk after the beginning of
TM treatment. (D) At the end of the TM treatment, kainic acid
was administered to control and conditional mutant mice. The
samples were collected 8 d after KA administration.
Found at: doi:10.1371/journal.pbio.1000460.s003 (0.28 MB TIF)
Schematic representation of the TM admin-
tion in vitro. (A–C) Neurospheres generated from the hippo-
campus of E16.5 control and Nestin-Cre;Prox1F/Fmice are capable
of producing bTub-III+neurons (N=3 experiments; 175 control
and 135 Nestin-Cre;Prox1F/Fneurospheres were analyzed). (D–F)
However, most of the neurospheres obtained from Nestin-
Cre;Prox1F/Fmice failed to generate Dcx+cells (N=3 experiments;
216 control and 190 Nestin-Cre;Prox1F/F
analyzed). (G, H) 3 d after co-transfection with GFP- and Prox1-
expressing plasmids, around 20% of the Nestin-Cre;Prox1F/FGFP+
neurospheres generated Dcx+neurons (N=3 experiments; a total
of 76 Nestin-Cre;Prox1F/FGFP+neurospheres and 73 GFP+Nestin-
Cre;Prox1F/Fneurospheres transfected with Prox1 were analyzed).
Data represent the mean number of the percentage of neuro-
spheres per experiment 6 SD. Paired t test. *** p,0.001;
**** p,0.0001. Scale bar in (A, B, D): 50 mm. Scale bar in (E):
100 mm. Scale bar in (G): 25 mm.
Found at: doi:10.1371/journal.pbio.1000460.s004 (1.52 MB TIF)
Prox1 is necessary for neuronal differentia-
maturing granule cells is reduced in the dentate gyrus of
P5 Nestin-Cre;Prox1F/Fpups treated with TM from P0 to
P5. (A–E) Sox2+Prox1+cells are not detected in the wild-type
dentate gyrus (DG) at P10. (F) C-Prox1+cells are present in the
control DG and the hilus (H) at P5. (G) TM administration from
P0 to P5 reduces the number of C-Prox1+cells in the hilus of P5
Nestin-CreERT2;Prox1F/Fpups. However, no alterations in the
number of Sox2+(H–J) or Id1+PECAM2(K–M) NSCs were
detected in Nestin-CreERT2;Prox1F/Fmutant brains. (N–P) Follow-
ing a 1-h pulse, BrdU immunostaining revealed a small reduction
in proliferation in the DG of Nestin-CreERT2;Prox1F/Fbrains. (Q–S)
As shown by Tbr2 staining, the number of intermediate
progenitors is reduced in the DG of the conditional mutant mice
at this stage. (T, U) The number of Dcx+cells is also reduced in the
DG of Nestin-CreERT2;Prox1F/Fpups at P5. An increase in the
number of TUNEL+cells (V–Y) is observed in the DG of Nestin-
CreERT2;Prox1F/Fbrains at P5. Data represent the mean number of
positive cells per DG section 6 SD (N=3 mice). Paired t test.
* p,0.1; **** p,0.0001. GCL, granule cell layer; SGZ,
Subgranular zone. Scale bar: 100 mm.
Found at: doi:10.1371/journal.pbio.1000460.s005 (2.97 MB TIF)
The number of intermediate progenitors and
CreERT2;Prox1F/Fpups exhibits an increased number
of active caspase-3+cells at P5 and P10. The number of
active caspase-3+cells is increased in the dentate gyrus of P5 (A)
and P10 (B) TM-treated Nestin-CreERT2;Prox1F/Fpups. Data
represent the mean number of positive cells per DG section 6
SD (N=3 mice). Paired t test. ** p,0.01; **** p,0.0001.
Found at: doi:10.1371/journal.pbio.1000460.s006 (0.12 MB TIF)
Dcx+cells remaining in TM-treated Nestin-
CreERT2;Prox1F/Fmice are those that have escaped Cre-
mediated deletion. Double C-Prox1/Dcx immunostaining
The dentate gyrus of TM-treated Nestin-
shows that in 2-mo-old Nestin-CreERT2;Prox1F/Fmice treated with
TM from P0 to P15 the remaining Dcx+cells were also C-Prox1+.
Found at: doi:10.1371/journal.pbio.1000460.s007 (1.97 MB TIF)
in the SGZ of TM-treated Nestin-CreERT2;Prox1F/Fmice.
(A, B) The number of Tbr2 intermediate progenitors is increased
in the dentate gyrus (DG) of control mice treated with TM or with
TM and Kainic Acid (KA). However, KA is not able to increase
the number of Tbr2+cells in 12-wk-old TM-treated Nestin-
CreERT2;Prox1F/Fmice (A, C). Similar results were observed with
Dcx+(D–F) and Calretinin+(G–I) cells. Data represent the mean
number of positive cells per DG section 6 SD (N=3 mice). Paired
t test. * p,0.1; ** p,0.01; *** p,0.001. Scale bar: 100 mm.
Found at: doi:10.1371/journal.pbio.1000460.s008 (3.77 MB TIF)
Kainic Acid do not induce adult neurogenesis
Nestin-CreERT2;Prox1F/Fmice. (A) Radial type I adult NSCs
are Nestin+Gfap+Blbp+. The number of triple Nestin+Gfap+
Blbp+cells observed in a 4-mo-old control (C) is reduced in Nestin-
CreERT2;Prox1F/Flittermates treated with TM from P0 to P15 (B,
D). These results are similar to the ones shown in Figure 8A when
counting radial-glia-like cells using only Nestin+. Data represent
the mean number of positive cells per DG section 6 SD. (N=3
mice). Paired t test. *** p,0.001.
Found at: doi:10.1371/journal.pbio.1000460.s009 (3.55 MB TIF)
Analysis of the NSC population in TM-treated
observed in the SGZ of 16-wk-old control mice. (B) Hes5
expression was barely detected in the SGZ of 16-wk-old Nestin-
Found at: doi:10.1371/journal.pbio.1000460.s010 (2.70 MB
Hes5 expression is downregulated in the
SGZ. (A) Hes5 expression was
regions of Nestin-Cre;Jojo-Prox1 embryos. Prox1 is ectop-
ically expressed in the ventricular, subventricular, and mantle
zones of the brain of Nestin-Cre;Jojo-Prox1 embryos like the olfactory
bulb (A), lateral ventricle (F), and cortex (K). Prox1 ectopic
expression in these regions does not induce premature differen-
tiation as shown by Nestin (B, B9, G, G9, L, L9), Sox9 (C, C9, H,
H9, M, M9), and b-TubIII (E, E9, J, J9, O, O9) IHC. As shown by
Ki67 staining, no changes in proliferation were observed (D, D9, I,
I9, N, N9). OV, Olfactory ventricle; NE, Neuroepithelium; IIIV,
Third ventricle; ST, Striatum; SP, Septum; Cortex, Cx.
Found at: doi:10.1371/journal.pbio.1000460.s011 (2.86 MB TIF)
Prox1 ectopic expression in several brain
differentiation of NSCs. Anti-C-Prox1 immunostaining shows
a smaller dentate gyrus (DG) in P8 Nestin-CreERT2;JoJo-Prox1 mice
(B, E). There is no difference in the number of PH3+cells in the
DG region of control and Nestin-CreERT2;JoJo-Prox1 (F) mice at P8.
However, the number of Ki67+cells is reduced in the Nestin-
CreERT2;JoJo-Prox1 DG at this stage (G). There are no differences
in the number of TUNEL+cells at this stage (H). Double b-Gal
(red) and Nestin (I), Dcx (J), NeuN (K), Gfap (L), and NG2 (M)
(blue) IHC on the DG of P8 Nestin-CreERT2;JoJo-Prox1 pups shows
that Prox1-misexpression induces neuronal differentiation. (N)
Prox1 is ectopically expressed in a Type I Nestin+cell (arrow) of
adult Nestin-Cre;JoJo-Prox1 mice. As a consequence of Prox1
ectopic expression, the number of Sox2+(P) and Calretinin+(R)
cells is reduced in the SGZ of adult Nestin-Cre;JoJo-Prox1 mice.
Data represent the mean number of positive cells per DG section
6 SD. N=3 brains. Paired t test. ** p,0.01; *** p,0.001. Blue
bars are controls. Red bars are Nestin-Cre;JoJo-Prox1. Scale bar:
Prox1 mis-expression promotes premature
Prox1 Function in Brain Neurogenesis
PLoS Biology | www.plosbiology.org14 August 2010 | Volume 8 | Issue 8 | e1000460
Found at: doi:10.1371/journal.pbio.1000460.s012 (3.11 MB TIF) Download full-text
We thank A. McArthur for editing of this manuscript and M. Dillard for
technical support. We thank A. Mansouri, A. Stoykova, and J. Berger for
the CMV-CAG-loxP-eGFP-Stop-loxP-Ires- bGal construct. L.M.L.C. is a
recipient of the Jean-Franc ¸ois St.-Denis Fellowship in Cancer Research
from the Canadian Institutes of Health Research.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: AL GO.
Performed the experiments: AL. Analyzed the data: AL. Contributed
reagents/materials/analysis tools: OVL LMLC SJB. Wrote the paper: AL
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PLoS Biology | www.plosbiology.org 15August 2010 | Volume 8 | Issue 8 | e1000460