CoupTFI Interacts with Retinoic Acid Signaling during
Susan J. Harrison-Uy1, Julie A. Siegenthaler1¤a, Andrea Faedo2¤b, John L. R. Rubenstein2,3,
Samuel J. Pleasure1,3*
1Department of Neurology, University of California San Francisco, San Francisco, California, United States of America, 2Department of Psychiatry, University of California
San Francisco, San Francisco, California, United States of America, 3Programs in Neuroscience and Developmental Biology, Eli and Edythe Broad Center of Regeneration
Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, United States of America
We examined the role of the orphan nuclear hormone receptor CoupTFI in mediating cortical development downstream of
meningeal retinoic acid signaling. CoupTFI is a regulator of cortical development known to collaborate with retinoic acid
(RA) signaling in other systems. To examine the interaction of CoupTFI and cortical RA signaling we utilized Foxc1-mutant
mice in which defects in meningeal development lead to alterations in cortical development due to a reduction of RA
signaling. By analyzing CoupTFI2/2;Foxc1H/Ldouble mutant mice we provide evidence that CoupTFI is required for RA
rescue of the ventricular zone and the neurogenic phenotypes in Foxc1-mutants. We also found that overexpression of
CoupTFI in Foxc1-mutants is sufficient to rescue the Foxc1-mutant cortical phenotype in part. These results suggest that
CoupTFI collaborates with RA signaling to regulate both cortical ventricular zone progenitor cell behavior and cortical
Citation: Harrison-Uy SJ, Siegenthaler JA, Faedo A, Rubenstein JLR, Pleasure SJ (2013) CoupTFI Interacts with Retinoic Acid Signaling during Cortical
Development. PLoS ONE 8(3): e58219. doi:10.1371/journal.pone.0058219
Editor: Makoto Sato, University of Fukui, Japan
Received September 7, 2012; Accepted February 1, 2013; Published March 5, 2013
Copyright: ? 2013 Harrison-Uy 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 work was supported by National Institutes of Health (NIH) National Institute on Drug Abuse (NIDA) R01 DA017627. 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.
* E-mail: firstname.lastname@example.org
¤a Current address: Department of Pediatrics, University of Colorado, Denver, Aurora, Colorado, United States of America
¤b Current address: Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Milano, Italy
During cortical development neurons are produced in a tightly
regulated manner from progenitor cells residing adjacent to the
lateral ventricles. In the developing cortex bipolar radial glia are
the primary neurogenic stem cells. These bipolar cells span the
cortical wall with cell bodies close to the ventricular surface and
primary cilia projecting apically into the ventricle, receiving signals
that promote radial glia proliferation [1,2]. Radial glia also have
basal processes projecting to the pial surface where the radial glia
endfeet interact with the pial extracellular matrix [3,4]. Our
previous studies suggest that meningeally derived retinoic acid
(RA) promotes neurogenesis of radial glia by interacting with the
radial glia basal process . However, radial glia also receive
signaling cues from neighboring progenitor cells and differentiated
neurons [6,7,8,9]. Thus, numerous extrinsic signals from the
surrounding environment during development impact the behav-
ior of radial glia, collaboratively regulating their proliferative
capacity and developmental maturation.
Foxc1 is a transcription factor expressed by cells in the meninges
but not by neural cells in the cortex . Disruption of Foxc1
expression results in a failure of meningeal cells to appropriately
migrate and surround the developing forebrain [5,10]. In addition
to the defects in meningeal development Foxc1 mutants have
major cortical developmental abnormalities, with elongation of the
cortical neuroepithelium, changes in the composition of the
progenitor cell population, and decreased neurogenesis, all
suggesting a failure to transition from symmetric proliferative
divisions to asymmetric neurogenic divisions . Several lines of
evidence support a role for meningeal derived RA in controlling
cortical neurogenesis . RA is a potent neurogenic factor that
regulates areal patterning and neuronal differentiation during
development . Disruption of RA signaling, via loss of retinoic
acid receptor (RAR) or retinoid X receptor (RXR) family
members, results in severe developmental abnormalities across
multiple organ systems . Expression of a dominant negative
RAR construct in the developing forebrain results in altered
proliferation of progenitor cells and increased cell death .
However, how RA regulates the process of neuroepithelial
progenitor division is still very much unclear.
CoupTFI is a nuclear orphan receptor that can function as a
repressor or activator of gene expression via complex, context
dependent interactions with other nuclear orphan receptors and
CoupTFI is expressed in a high-ventral to low-dorsal and high-
caudal to low-rostral gradient, initially in progenitor cells
(including radial glia cells) and during later development also in
neurons . CoupTFI mutant mice have altered areal patterning
of the cerebral cortex [17,18,19] and CoupTFI also regulates
neuronal differentiation and neuron cell type specification
In the developingforebrain
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Several lines of evidence suggest that the transcription factor
CoupTFI may coordinate the response of cortical progenitor cells
to RA. First, CoupTFI binds to RXR and forms DNA
heterodimers with both RAR and RXR family members
[14,15]. CoupTFI also interacts with the ligand-binding domain
of RAR and RXR family members, resulting in transrepression of
retinoid family members [14,15]. Second, CoupTFI binds to
similar DNA binding sites as RAR and RXR, and may function as
a negative regulator of retinoid function [14,15]. Third, CoupTFI-
knockout and CoupTFI-overexpressing mice exhibit cortical
phenotypes consistent with roles for CoupTFI in regulating
neuronal differentiation . Finally, transgenic mice overex-
pressing CoupTFI in cortical progenitor cells exhibit elevated
levels of endogenous RA signaling (See Faedo et al., in their
Supplementary Material: Figure S6E–F9 of ).
In this study we examined the relationship between CoupTFI
and RA signaling in the developing cortex using Foxc1-mutant
mice. We found that CoupTFI is required for RA mediated rescue
of Foxc1-mutants and that overexpression of CoupTFI in cortical
progenitor cells partially rescues the Foxc1-mutant phenotype.
Our studies suggest that CoupTFI interacts with RA signaling to
regulate cortical progenitor cells.
Materials and Methods
Embryos were obtained from matings of Foxc1hith, Foxc1lacZ,
D6-CoupTFI, CoupTFI-null lines and genotyped as previously
described [5,17,21,22]. Noon on the day of vaginal plug was
designated as embryonic day 0.5 (E0.5). For RA treatment,
pregnant mice were fed all-trans (at) RA-enriched food (250mg
atRA/kg food; Harlan Teklab Custom Diets) ab libitum from
E10. Mice consumed on average 20–30 mg atRA/kg body
weight. Embryos were collected via cesarean section, fixed in
4% paraformaldehyde for 5 hours (E12.5) or overnight (E14.5),
processed through a sucrose gradient, and embedded in OCT
for cryosectioning. Embryos were cryosectioned at 12 mm for
immunohistochemistry or 20 mm for in situ hybridization. All
animal protocols were approved by the University of California,
San Francisco Institutional Animal Care and Use Committee.
Immunohistochemistry and cresyl violet staining was performed
as previously described [10,23] using the following antibodies:
mouse anti-BrdU (1:75, BD Biosciences); rat anti-Ctip2 (1:500,
Abcam); rabbit anti-Ki67 (1:400, Thermo Scientific); rabbit anti-
Pax6 (1:500, Abcam); rabbit anti-Tbr1 (1:1000, Abcam); rabbit
anti-Tbr2 (1:1000, Abcam). Primary antibodies were detected
using secondary antibodies conjugated to Alexa fluorophores
(Invitrogen). Six serial 12 mm sections were stained for each
antigen in the following order: CoupTFI double mutant experi-
ment: CV, Tbr2, Tbr1/Ctip2, Pax6. D6-CoupTFI E14.5
experiment: CV, X, Tbr2, X, Pax6, BrdU/Ki67, Tbr1/Ctip2
(X: indicates a skipped slide). Stained sections were visualized on a
Nikon fluorescent microscope and captured with a digital CCD-
cooled camera and QCapture Pro software (QImaging Surrey).
Composite images were prepared in Adobe Photoshop CS4 and
Adobe Illustrator CS4. Contrast, color and brightness were
adjusted in Adobe Photoshop CS4.
In Situ Hybridization
RNA in situ hybridization was performed as previously described
. Probes used were CoupTFI and CoupTFII (gifts of M.J.
Image Analysis, Quantification, and Statistical Analysis
Sections were histologically matched for rostral-caudal level
between genotype. The length of the dorsal forebrain consisted of
measuring the length of the ventricular surface from the cortico-
striatial boundary to the boundary of the cortical hem in sections
from a similar rostral/caudal position in ImageJ. Three matched
sections per animal were quantified and the mean of the three
sections per animal was compared between genotypes. Cell counts
were performed and quantified using a cell counting program, as
previously described . For all cell counts a matched 150 mm
window in the medial-lateral dimension spanning from the
ventricular to pial surface of the dorsal cortex from three sections
per animal were counted, and the mean of the three sections per
animal was compared between genotypes. A minimum of 3 brains
per genotype was analyzed from a minimum of two independent
litters. The 150 mm counting window was chosen based upon the
following criteria. The medial wall of the cerebral cortex, the
hippocampal primordium, has distinct molecular properties 
and in Foxc1-mutants the lateral meninges is intact . In D6-
CoupTFI animals, the D6-promoter drives expression of CoupTFI
in the medial and dorsal cortex [17,27]. Therefore, we chose a
150 mm region in the dorsal cortex (as illustrated in Figure 1) to
examine the role of these signaling pathways in cortical progenitor
cells. We also calculated the total area of the 150 mm counting
window, and the proportion of DAPI+cells in the total area of the
counting region. The Q-fraction was calculated as the number of
BrdU+Ki672cells divided by the total number of BrdU+cells. An
observer blind to the genotype performed the analyses. Statistical
analysis was performed as indicated; using one-way ANOVA or
two-way ANOVA followed by posthoc testing of selected means
with Bonferroni Multiple Comparison Test. A P-value of ,0.05
was considered statistically significant. These results are reported
as the mean +/2 standard error of the mean (SEM) across the
experiments. Statistical analysis and graphs were prepared using
CoupTFI Expression is not Downregulated in Foxc1-
We previously identified a role for meningeal derived RA
signaling in regulating the transition from symmetric prolifera-
tive divisions to asymmetric neurogenic divisions using an allelic
Foxc1H/L) and Foxc1-nulls(Foxc1lacZ/lacZ:
Because CoupTFI overexpression in the cortex increased RA
signaling  and there is evidence that RA signaling may
promote expression of CoupTFI and CoupTFII in other
systems [28,29], we wondered whether dysregulated expression
of CoupTFI in Foxc1-mutants might underlie aspects of the
Foxc1 mutant phenotype.
At E14.5 CoupTFI is expressed in a high-ventral to low-dorsal
pattern in cortical progenitor cells in controls (Figure 2A, C, E). In
Foxc1-mutants, the overall pattern of CoupTFI expression in
cortical progenitor cells was similar, although seemingly slightly
upregulated (Figure 2B, D, F, H), whereas in ventral telencephalon
progenitor cells CoupTFI expression was clearly upregulated (*,
Figure 2B, D, F). In controls, expression of the related family
member CoupTFII is largely restricted to the ventral forebrain
and to Cajal Retzius cells in the cortical marginal zone (Figure 2I,
K, M, O) . In Foxc1-mutants CoupTFII expression was also
upregulated in progenitor cells of the ventral telencephalon (*,
Figure 2L, N) and cortical hem (arrow, Figure 2L, N). Thus,
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downregulation of either CoupTFI or CoupTFII expression in the
cortex does not explain the neocortical phenotype of Foxc1-
CoupTFI is Required for RA Rescue of Foxc1-mutants
Given the evidence that CoupTFI may be a mediator of RA
signaling [14,15] we wondered whether CoupTFI might be a
required component of RA signaling in cortical progenitor cells.
To address this we generated CoupTFI2/2;Foxc1H/Lcompound
Figure 1. Sampling Window Used For Cell Counts. The location of the 150 mm sampling window in the medial-lateral dimension of the cortex
spanning from the ventricular to pial surface of the dorsal cortex is identified by the black box in this coronal forebrain section of an E14.5. Scale
RA Signaling and CoupTFI in Cortical Development
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RA Signaling and CoupTFI in Cortical Development
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mutants, and using our established model  examined the ability
of dietary RA from E10 to E14.5 to rescue cortical development.
We reasoned that if CoupTFI is a component of the RA signaling
pathway, that RA might no longer rescue the Foxc1-mutants in
the absence of CoupTFI.
Previous studies identified that continued symmetric expansion
of the radial glia population at the expense of asymmetric
neurogenic divisions results in elongation of the cortical ventricular
zone [5,31]. We measured the length of the ventricular zone, from
the pallial-subpallial boundary to the dorsal boundary of the
cortical hem, in cresyl violet stained coronal sections at E14.5. In
embryos that were not exposed to dietary RA the cortical
ventricular zone length was elongated in both Foxc1H/Land
CoupTFI2/2;Foxc1H/Lmutants compared to controls (Figure 3C,
D, Figure 4A). As previously reported, in utero RA treatment
restored the ventricular zone length to untreated control levels in
Foxc1H/Lmutants (Figure 3G, Figure 4A). On the other hand, in
CoupTFI2/2;Foxc1H/Lmutants, RA treatment failed to fully
rescue the phenotype to untreated control levels (Figure 3H,
Figure 4A). The modest rescue observed in the CoupTFI2/
2;Foxc1H/Lmutants (Figure 3D, H, Figure 4A), appeared to be
due to shortening of the dorsomedial wall (*, Figure 3D, H).
Next, to determine whether CoupTFI is required for RA-
mediated rescue of cortical progenitor cell properties in Foxc1H/L
mutants, we examined expression of radial glia (Pax6) and
intermediate progenitor cell (Tbr2) markers at E14.5. While RA
rescued the numbers of Pax6+ cells in both Foxc1-mutants and
CoupTFI2/2;Foxc1H/Lanimals (Figure 3I–P, Figure 4B), RA
treatment did not restore the intermediate progenitor cell
population to untreated control levels in CoupTFI2/2;Foxc1H/L
mutants (Figure 3Q–X, Figure 4C).
Finally, to investigate whether CoupTFI is also involved in the
RA-mediated rescue of neuron production, we assayed early born
neuron production by examining expression of Ctip2 and Tbr1 at
E14.5 (Figure 3Y–FF, Figure 4D, E). While RA treatment restored
neuronal differentiation to untreated control levels in Foxc1-
mutants (Figure 3AA, EE, Figure 4D, E), RA treatment no longer
rescued neuron production CoupTFI2/2;Foxc1H/Ldouble mu-
tants (Figure 3BB, FF, Figure 4D, E).
The height of the 150 mm counting region differs across
genotypes. We assessed the area of the counting region and the
density of cells in this area to determine whether proportional
changes in cell numbers account for the observed phenotypes
(Figure 4F, G). The total area of the counting region was
decreased in untreated and RA-treated CoupTFI mutants,
CoupTFI2/2;Foxc1H/Lanimals (Figure 4F). RA-treatment re-
stored the area of the counting region to untreated control levels in
Foxc1H/Lanimals, but not in CoupTF12/2;Foxc1H/Lanimals
(Figure 4F). RA-treatment did not alter the decreased area of the
counting window observed in CoupTFI2/2animals (Figure 4F).
Total cell density (DAPI quantification) within the counting region
was slightly decreased in untreated Foxc1-mutants (Figure 4G).
These findings suggest that changes in cell density do not account
for the observed alterations in Foxc1-mutant mice. The cortical
phenotype observed in Foxc1-mutants reflects alterations in the
radial complexity of the cortex and are rescued by RA-treatment.
We next examined the effect of CoupTFI dose in the RA-
mediated rescue of the Foxc1-mutant phenotype, using RA-
treated CoupTFI+/+;Foxc1H/L(Foxc1H/L-mutants), CoupTFI+/
2;Foxc1H/L, and CoupTFI2/2;Foxc1H/L-mutants. No significant
difference in VZ length or Pax6 cell number was observed across
the genotypes (Figure 5A, B). Tbr2 cell number was decreased in
CoupTFI2/2;Foxc1H/Lbut not in CoupTFI+/2;Foxc1H/Lani-
mals (Figure 5C). Tbr1 and Ctip2 neuron number was signifi-
cantly decreased in both CoupTFI+/2;Foxc1H/Land CoupTFI2/
2Foxc1H/Lanimals (Figure 5D, E). These findings identify a
CoupTFI dose response effect, with respect to the Tbr2+
population, however, the RA mediated production of Ctip2 and
Tbr1 neuron number is affected by loss of even one copy of
Taken together these studies indicate that CoupTFI is required
for RA mediated rescue of many features of the Foxc1-mutant
cortical phenotype. This supports our hypothesis that CoupTFI is
a component of RA signaling during cortical development.
However, because RA-treatment still rescued some of the
CoupTFI2/2;Foxc1H/Lphenotype (i.e. numbers of Pax6+radial
glia cells and partial rescue of ventricular zone length), it seems
likely that RA signaling in the cortex proceeds through other
signaling interactions as well.
Overexpression of CoupTFI in Cortical Progenitor Cells
Partially Rescues Foxc1-mutants
Since CoupTFI is necessary for RA rescue of Foxc1 mutants
(Figure 3, 4), we reasoned that overexpression of CoupTFI might
be sufficient to rescue Foxc1-mutants without RA treatment. One
possibility is that in the normal cortex activation of RAR/RXR by
RA releases CoupTFI to function independently, allowing
CoupTFI to interact with other targets. In this case, increasing
the dosage of CoupTFI might allow this signaling to occur, even in
the presence of low levels of RA that remain in the Foxc1-mutant
To increase CoupTFI dosage, we used mice that express
CoupTFI in the cortical ventricular zone under the control of the
D6-promoter element (D6-CoupTFI); this increased CoupTF1
expression ,4-fold in cortical progenitor cells . We generated
D6-CoupTFI;Foxc1H/Lcompound mutants, to assess whether
increased CoupTFI dosage could rescue Foxc1H/L
phenotypes independent of a RA treatment.
We measured the length of the ventricular zone at E14.5
(Figure 6A–E). One-way ANOVA revealed significant differences
in ventricular zone length between genotypes (Figure 6E). Post-hoc
testing revealed that the ventricular zone length was increased in
Foxc1-mutants (Figure 6C, E), as previously described .
Overexpression of CoupTFI in dorsal cortical progenitor cells
caused a subtle reduction in cortical length (Figure 6B, E).
Strikingly, cortical length was restored to control levels in the D6-
CoupTFI;Foxc1H/Lcompound mutants (Figure 6D, E), providing
strong evidence that CoupTFI overexpression is sufficient to
restore the balance of asymmetric neurogenic divisions in Foxc1-
We next tested whether CoupTFI overexpression could rescue
the deficit in early neurogenesis observed in Foxc1H/Lmice .
To do this we examined expression of Ctip2 and Tbr1 (Figure 6F–
J). D6-CoupTFI mutants showed modest decrease in Ctip2 and
Figure 2. CoupTFI And CoupTFII Expression Is Not Downregulated Or Misexpressed In The Cortex Of Foxc1-Mutants At E14.5. In situ
hybridization of coronal sections of control (A, C, E, G, I, K, M, O) and Foxc1H/L(B, D, F, H, J, L, N, P) at E14.5 with CoupTFI probe (A–H) and CoupTFII
probe (I–P). Higher magnification panels in E, F, K, L correspond to boxed regions in G, H, O, P. Scale bar: 500 mm in AF, I–N, 100 mm in G, H, O, P.
Arrow in L, N points to the cortical hem. * in D, F, L, N indicates the ventral progenitor population. MZ: marginal zone.
RA Signaling and CoupTFI in Cortical Development
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Tbr1 positive cells (Figure 6G, J), probably due to an early
depletion of the progenitor cell population . Foxc1-mutants
had a severe reduction of Ctip2 and Tbr1 neurons (Figure 6H, J).
Despite the fact that both single mutants reduced neurogenesis,
overexpression of CoupTFI in the D6-CoupTFI;Foxc1H/Lcom-
pound mutants increased the production of early born neurons
compared to Foxc1H/Lmutants, although neuron numbers were
not restored to control levels (Figure 6I, J).
Figure 3. CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals. Cresyl Violet staining of E14.5 coronal forebrain
sections of untreated and RA treated (E10–E14.5) embryos (A–H). Asterisk in D, H signifies the medial wall. Pax6 (red) immunohistochemistry of the
dorsal cortex at E14.5 of untreated and RA treated (E10–E14.5 embryos) (I–P). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 of
untreated and RA treated (E10–E14.5) embryos (Q–X). Tbr1 (green) and Ctip2 (red) immunohistochemistry of the dorsal cortex at E14.5 of untreated
and RA treated (E10–E14.5) embryos (Y–FF). Sections are counterstained with DAPI (blue) (I–FF). Scale bar: 500 mm in A–H, 50 mm in I–FF. VZ:
ventricular zone; SVZ: subventricular zone; PC: progenitor cell; CP: cortical plate.
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Figure 4. CoupTFI Is Required For The RA Mediated Rescue Of Foxc1-Mutants Animals. Quantification of dorsal forebrain length at E14.5
(A), Pax6 cell number at E14.5 (B), Tbr2 cell number at E14.5 (C), Ctip2 cell number at E14.5 (D), Tbr1 cell number at E14.5 (E), area of the counting
window (F), density of DAPI+cells in the counting window (G). Error bars represent SEM. A–G were analyzed by two way ANOVA: A: genotype
(F(3,25)=55.86, p,0.001), treatment (F(1,25)=27.84, p,0.001), interaction (F(3,25)=49.98, p,0.001); B: genotype (F(3,26)=9.5, p,0.001), treatment
(F(1,26)=48.66, p,0.01), interaction (F(3,26)=11.67, p,0.001); C: genotype (F(3,27)=19.23, p,0.001), treatment (F(1,27)=8.71, p,0.01), interaction
(F(3,27)=14.55, p,0.001); D: genotype (F(3,26)=40.83, p,0.001), treatment (F(1,26)=18.45, p,0.001), interaction (F(3,26)=14.75, p,0.001); E: genotype
(F(3,26)=47.99, p,0.001), treatment (F(1,26)=48.66, p,0.001), interaction (F(3,26)=14.91, p,0.001); F: genotype (F(3,24)=24.77, p,0.001), treatment
(F(1,24)=8.158, p,0.01), interaction (F(3,24)=13.38, p,0.001); G: genotype (F(3,25)=10.33, p,0.001), treatment (F(1,25)=0.53, p=0.47), interaction
(F(3,25)=1.62, p=0.2). *p,0.05, ***p,0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly
above the bar indicate significance from untreated control; within group differences are indicated by connected lines.
Figure 5. Dose response of CoupTFI in the RA-mediated rescue of Foxc1-mutants. Quantification of dorsal forebrain length at E14.5 (A),
Pax6 cell number at E14.5 (B), Tbr2 cell number at E14.5 (C), Ctip2 cell number at E14.5 (D), and Tbr1 cell number at E14.5 (E) in RA treated animals.
Note: the CoupTFI+/+;Foxc1H/Land CoupTFI2/2;Foxc1H/Ldata are the same as in Figure 4, but are being compared to CoupTFI+/2;Foxc1H/Lfor this
analysis. A–E were analyzed by one-way ANOVA: A: F(2,13)=3.7, p=0.06; B: F(2,12)=1.3, p=0.3; C: F(2,10)=8.5, p,0.01; D: F(2,10)=7.1, p,0.05; E:
F(2,10)=6.9, p,0.05. *p,0.05 and indicates significance for Bonferroni’s Multiple Comparison Test posthoc analysis. Asterisks directly above the bar
indicate significance from CoupTFI+/+Foxc1H/Lgroup.
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Figure 6. Overexpression Of CoupTFI In Cortical Progenitor Cells Partially Rescues The Cortical Phenotype Of Foxc1-Mutants. Cresyl
Violet staining of E14.5 coronal forebrain sections (A–D). Quantification of dorsal forebrain length at E14.5 (E). Tbr1 (green) and Ctip2 (red)
immunohistochemistry of the dorsal cortex at E14.5 (F–I). Quantification of Ctip2 and Tbr1 cell number at E14.5 (J). Pax6 (red) immunohistochemistry
RA Signaling and CoupTFI in Cortical Development
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While increased CoupTFI dosage increased neurogenesis in
Foxc1H/Lmutant, it did not fully restore cortical neurogenesis. To
explore the basis for the partial rescue, we examined the size of the
cortical progenitor cell pools at E14.5, by examining markers of
radial glia (Pax6) and intermediate progenitor cells (Tbr2). In both
D6-CoupTFI and Foxc1-mutants the number of Pax6 cells was
decreased (Figure 6L, M, O) [note: Pax6 expression was
consistently weaker in D6-CoupTFI animals, Figure 6L, N and
]. However, the D6-CoupTFI;Foxc1H/Lcompound mutants
had a further decrease in the Pax6+radial glia population
(Figure 6N, O).
Tbr2+intermediate progenitor cells were slightly decreased in
the D6-CoupTFI mutants (Figure 6Q, T), whereas they were
reduced by greater than 2-fold in the Foxc1-mutants (Figure 6R,
T). Unlike the radial glia cells, there was no further reduction of
intermediate progenitor cells in the D6-CoupTFI;Foxc1H/L
compound mutants (Figure 6S, T).
To assess the cellular basis of the partial rescue of the Foxc1-
mutant phenotype we examined neuronal cell number at an
earlier time point. D6-CoupTFI and D6-CoupTFI;Foxc1H/L
animals showed an increased production of both Ctip2 and
Tbr1 cells at E12.5, compared to controls and Foxc1H/Lanimals
(Figure 7A–E). We also examined the rate of neuronal differen-
tiation by examining the proportion of cells exiting the cell cycle at
E12.5 (BrdU injected E11.5) and E14.5 (BrdU injected E13.5),
termed the Q-fraction. At E12.5 an increased rate of cells exiting
the cell cycle was observed in D6-CoupTFI embryos, although no
significant difference in the Q-fraction was observed in D6-
CoupTFI;Foxc1H/Lembryos (Figure 7F). By E14.5, the Q-fraction
was decreased in D6-CoupTFI, Foxc1H/L, and D6-CoupTFI;-
Foxc1H/Lembryos compared to controls (Figure 7G), likely due to
the depleted progenitor populations present at this age.
Early in neural development D6-CoupTFI promotes neurogen-
esis, increasing the number of neurons in D6-CoupTFI;Foxc1H/L
embryos. By E14.5, D6-CoupTFI;Foxc1H/Lcompound mutants
showed a reduction of Pax6+radial glia progenitors greater than
the single mutants, suggesting that CoupTFI overexpression is able
to partially restore cortical growth and neurogenesis in Foxc1-
mutants through reducing symmetric proliferative divisions, while
maintaining asymmetric neurogenic divisions.
The meninges were traditionally considered to be a protective
covering of the CNS, with roles limited to cerebrospinal fluid and
blood circulation. In recent years several important additional
signaling roles of the meninges have been established. The
meninges produce Cxcl12, which guides neuronal migration
[24,32,33,34] and BMPs, which regulate the formation of the
corpus callosum . The meninges also synthesize RA, which
regulates the onset of asymmetric divisions in the developing
cortical neuroepithelium . During development cortical neu-
rons are born in a gradient from ventral to dorsal and rostral to
caudal. Meningeal fibroblasts, derived from the neural crest,
migrate in a ventral to dorsal wave from E10.5 and surround the
developing forebrain and begin expressing RA synthesizing
enzymes beginning at E12 [5,36]. The migration of the meningeal
fibroblasts and the initiation of expression of the RA synthesizing
enzymes are coincident with the transition from symmetric
proliferative divisions to asymmetric neurogenic divisions across
the neurogenic gradient. The meninges are positioned and
synthesize RA in a manner to temporally and spatially influence
the regulation of radial glia, including controlling cortical
neurogenesis and previous studies from our laboratory using
Foxc1-mutant animals support this hypothesis . This study
supports the hypothesis that meningeal mediated RA signaling in
radial glia involves the transcription factor CoupTFI.
The nuclear orphan receptor CoupTFI is a known modulator of
retinoid responses in other systems [14,15], CoupTFI regulates
neuronal differentiation [17,19], and D6-CoupTFI mice display
elevated cortical RA signaling . Our studies demonstrate a
requirement for CoupTFI to allow RA mediated rescue of
neurogenesis in Foxc1-mutants. We also found that overexpression
of CoupTFI partially rescues the cortical phenotypes in Foxc1-
mutants. Thus, our studies provide evidence that that CoupTFI
interacts with RA signaling in the developing cortex.
A partial rescue of the ventricular zone length and the numbers
of Pax6+radial glia cells was observed in CoupTFI2/2;Foxc1H/L
mutants following RA treatment, whereas, Tbr2 and neuron cell
numbers were not rescued. This suggests that RA regulation of
symmetric proliferation (i.e. the expansion of the radial glia
population) requires other signaling interactions as well. However,
the RA mediated regulation of the production of intermediate
progenitor cells and neurons, which proceed via asymmetric
divisions, is dependent on CoupTFI. Consistent with this
hypothesis, our overexpression studies (Figure 6), in which
ventricular zone length and neurogenesis are rescued but the
number of Pax6+radial glia is further decreased, suggest that
CoupTFI is sufficient to partially restore neurogenesis in Foxc1-
mutants by promoting asymmetric neurogenic divisions at the
expense of symmetric proliferative divisions. The cortical pheno-
type of Foxc1-mutants is evident from E12.5 and, based on
expression of RA-synthesizing enzymes, meningeal RA synthesis
initiates at around E12 . In the D6-CoupTFI rescue model,
CoupTFI is overexpressed in cortical progenitor cells from E10.5
[17,27]. The partial rescue observed in D6-CoupTFI;Foxc1H/L
animals may also reflect a timing issue of expression of CoupTFI
under the control of the D6-promoter and the early depletion of
progenitor cells in D6-CoupTFI mice.
Abnormalities in the ventral forebrain are observed in Foxc1-
mutant animals. The lateral ganglionic eminence (LGE) and
medial ganglionic eminence (MGE) frequently appear smoother
than controls, without a visible LGE/MGE sulcus, as visible in
Figures 3 and 6. Our preliminary data suggest that the LGE/
MGE are specified and patterned (unpublished observations, S
Harrison-Uy and S Pleasure). The structural alterations are not
rescued with RA treatment or overexpression of CoupTFI in
cortical progenitor cells, which suggests that this component of the
Foxc-1 mutant phenotype is not RA-dependent or dependent on
structural changes in the cortex. In Foxc1-mutants the meninges
covering the ventral forebrain is intact and RA-synthesizing
enzymes are present , suggesting that these changes may be
independent of RA signaling and may be dependent upon a
signaling pathway that is directly regulated by Foxc1.
of the dorsal cortex at E14.5 (K–N) Quantification of Pax6 cell number at E14.5 (O). Tbr2 (red) immunohistochemistry of the dorsal cortex at E14.5 (P–
S). Quantification of Tbr2 cell number at E14.5 (T). Sections are counterstained with DAPI (blue) (F–I, K–N, P–S). Scale bar: 500 mm in A–D; 50 mm in F–I,
K–N, P–S. E, J, O, P were analyzed by one way ANOVA: E: F(3,12)=40.87, p,0.001; J: F(3,12)=62.28, p,0.001; F(3,12)=48.8, p,0.001; O: F(3,12)=74.38,
p,0.001; P: F(3,11)=76.71, p,0.001. *p,0.05, **p,0.01; ***p,0.001 and indicate significance for Bonferroni’s Multiple Comparison Test posthoc
analysis. Asterisks directly above the bar indicate significance from untreated control; within group differences are indicated by connected lines.
RA Signaling and CoupTFI in Cortical Development
PLOS ONE | www.plosone.org9 March 2013 | Volume 8 | Issue 3 | e58219
CoupTFI interacts with the retinoid receptors, RAR and RXR
family members, altering the targets of CoupTFI and RA signaling
[14,15]. Our studies indicate that CoupTFI is necessary for the
RA mediated rescue of Foxc1-mutants and is sufficient to rescue,
in part, the cortical phenotype of Foxc1-mutants. Our results are
consistent with a model where meningeal RA signaling alters the
interaction of CoupTFI and RAR/RXR in cortical progenitor
cells. This would allow CoupTFI and RAR/RXR to complex with
additional signaling partners and enables the modification of the
transcriptional targets of CoupTFI and RAR/RXR. We hypoth-
esize that this signaling mechanism is an important regulatory
switch necessary for cortical neurogenesis.
A number of important signaling pathways control cortical
development including Fgf, Wnt, BMP, and Notch [37,38].
Studies of CoupTFI mutants identified changes in the Fgf
regulated pathways, Mapk/Erk kinase and PI3 Kinase/Akt, and
Wnt signaling . In the developing spinal cord Fgf signaling
opposes RA signaling, regulating neural patterning and differen-
tiation  and RA signaling is known to interact with Wnt
signaling [40,41,42]. BMP and Notch signaling did not appear to
be altered in the developing cortex of CoupTFI mutants ,
although CoupTFI has been implicated in Notch signaling during
hair cell differentiation  and BMP signaling in bone
development . Interestingly, retinoic acid signaling has
recently been shown to repress BMP signaling  and the
retinol binding protein CRBP1 is a direct target of Notch1 .
Thus, CoupTFI and RA signaling may function to coordinate
some of the major signaling pathways that control cortical
We thank Trung Huynh and Katelin Patterson for technical assistance. We
thank Tom Kume for the Foxc1lacZmouse line.
Conceived and designed the experiments: SH-U JAS AF JLR SJP.
Performed the experiments: SH-U JAS AF. Analyzed the data: SH-U JLR
SJP. Contributed reagents/materials/analysis tools: SH-U JAS AF JLR
SJP. Wrote the paper: SH-U JAS AF JLR SJP.
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RA Signaling and CoupTFI in Cortical Development
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