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First published online 18 March 2009
doi: 10.1242/dev.034967
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1 Department of Anatomy and Neurobiology and the Center for Complex Biological
Systems, University of California, Irvine, CA 92697, USA.
2 Department of Developmental and Cell Biology and the Center for Complex
Biological Systems, University of California, Irvine, CA 92697, USA.
Author for correspondence (e-mail:
alcalof{at}uci.edu)
Accepted 17 February 2009
 |
SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mouse, Olfactory epithelium, TGFβ, Follistatin, p21Cip1, Fgf8, Neuronal progenitor, Neurogenesis, Mash1, Ngn1, Sox2, Olfactory receptor neuron, Cerebral cortex, Gene dosage
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Foxg1 is highly expressed in anterior neural structures, and promotes
their development; neural structures whose development is adversely affected
in Foxg1-/- mice include the cerebral cortex, ventral
telencephalon, ear, retina and olfactory epithelium (OE)
(Duggan et al., 2008;
Hanashima et al., 2007;
Hanashima et al., 2004;
Hebert and McConnell, 2000;
Martynoga et al., 2005;
Pauley et al., 2006;
Pratt et al., 2004;
Xuan et al., 1995). In mice
that are null for Foxg1, the cerebral hemispheres are dramatically
reduced in size, ventral telencephalic structures are lacking, and the animals
die at birth (Xuan et al.,
1995). Foxg1 is also expressed in the OE from an early
age (Hatini et al., 1999), and
Foxg1-/- mice lack an OE and most of the nasal cavity
(Xuan et al., 1995). For these
reasons, Foxg1 has been described as a general positive regulator of anterior
nervous system development.
It has been proposed that positive effects of Foxg1 on neurogenesis are
closely linked to the effects of fibroblast growth factors (FGFs) (reviewed by
Hebert and Fishell, 2008). In
the telencephalon, Foxg1 positively regulates expression of
Fgf8 (Martynoga et al.,
2005), which plays a central role in neurogenesis not only in the
telencephalon, but also in the OE
(Kawauchi et al., 2005).
Although these data raise the possibility that Foxg1 promotes neurogenesis by
inducing Fgf8, other studies indicate that FGFs such as FGF8 act
upstream of Foxg1 to control Foxg1 expression and function
(Regad et al., 2007;
Shimamura and Rubenstein,
1997; Storm et al.,
2006).
An alternative mechanism by which Foxg1 could influence neural development
is through its effects on the transforming growth factor beta (TGFβ)
pathway (Dou et al., 2000;
Rodriguez et al., 2001;
Seoane et al., 2004).
TGFβ family ligands signal primarily by triggering the phosphorylation of
receptor-regulated Smads, which translocate to the nucleus and interact with
diverse DNA-binding proteins to influence the transcription of target genes
(Massague, 2000;
Moustakas et al., 2001).
Experiments using cultured neuroepithelial cells and cell lines have
demonstrated that, upon treatment with TGFβ1, Foxg1 binds to a
Smad3-containing complex and prevents it from inducing the expression of
p21Cip1 (Cdkn1a - Mouse Genome Informatics), which encodes a
cyclin-dependent kinase inhibitor (CKI) that is both a Smad3 target gene and
an effector of TGFβ-mediated cell cycle arrest
(Dou et al., 2000;
Massague and Gomis, 2006;
Rodriguez et al., 2001;
Seoane et al., 2004). These
findings indicate that, in cells that express Foxg1, Foxg1 can interact
directly with Smad-containing transcriptional complexes to block the
expression of TGFβ target genes.
Recently, we discovered that growth differentiation factor 11 (Gdf11), a
member of the TGFβ superfamily, is an important component of an autocrine
negative-feedback loop that regulates neurogenesis in the OE
(Kawauchi et al., 2004;
Kawauchi et al., 2005;
Wu et al., 2003).
Gdf11 is made by olfactory receptor neurons (ORNs) and late-stage
neuronal progenitors (immediate neuronal precursors, or INPs) within the OE
proper, and is present there as early as embryonic day 10.5 (E10.5)
(Nakashima et al., 1999;
Wu et al., 2003) (also see
Results). Tissue culture studies show that Gdf11 can both arrest the division
of INPs and promote the differentiation of INP progeny, effects that are
accompanied by increased expression of the CKI p27Kip1
(Lander et al., 2009;
Wu et al., 2003). Moreover,
Gdf11-null mice show increased OE neurogenesis in vivo, with
increased numbers of proliferating INPs and an extra layer of ORNs
(Wu et al., 2003).
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;
Newfeld et al., 1999;
Schneyer et al., 2008), Gdf11
signals via the same receptor-activated Smads (Smad2 and Smad3) as Tgfβ1
(Nomura et al., 2008;
Tsuchida et al., 2007). This
raises the possibility that some of the effects of Foxg1 on neurogenesis in
the OE are due to antagonism of Gdf11 signaling. To test this, we analyzed OE
development in Foxg1;Gdf11 compound mutant mice. We observed that
deficits in neurogenesis in the Foxg1-/- OE, which are
apparent from the earliest times in OE development, are substantially rescued
in Foxg1-/-;Gdf11-/- mice, and even in
Foxg1-/-;Gdf11+/- mice. Alterations in the
expression of follistatin (Fst), which encodes a secreted Gdf11
antagonist, appear to account for part of this rescue. Overall, our results
imply that the pro-neurogenic effects of Foxg1 in the OE are
mediated, to a large degree, by antagonism of Gdf11. Interestingly,
analysis of the same animals indicates that, in the cerebral cortex,
Foxg1 acts through different targets. |
MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
obtained by intercrossing Gdf11+/tm2 mice maintained on a
C57bl/6J background (Jackson Labs, Bar Harbor, ME, USA).
Foxg1cre/+ mice, in which the Foxg1 coding
sequence is replaced by the gene encoding Cre-recombinase (Cre)
(Hebert and McConnell, 2000),
were maintained on an outbred Swiss Webster background (Harlan, Indianapolis,
IN, USA). Although expression of Cre recombinase in the Foxg1 locus
has been shown to have some effects on telencephalon development when the
allele is maintained on a congenic C57bl/6 background
(Eagleson et al., 2007), when
maintained on an outbred background, Foxg1cre/cre mice
have been shown to have nervous system phenotypes identical to those observed
in another Foxg1 null strain, Foxg1lacZ/lacZ
(Duggan et al., 2008;
Eagleson et al., 2007;
Hanashima et al., 2007;
Martynoga et al., 2005;
Muzio and Mallamaci, 2005;
Pratt et al., 2004).
Therefore, we used Foxg1cre as a null allele and
Foxg1cre/cre mice are designated
Foxg1-/- hereafter.
Foxg1+/-;Gdf11+/- mice were obtained by
crossing Gdf11+/- females with
Foxg1+/- male animals. Double knockouts
(Foxg1-/-;Gdf11-/-) and compound mutants
(Foxg-/-;Gdf11+/-) were obtained by
intercrossing the resulting Foxg1+/-;Gdf11+/-
mice. Mid-day of the day of vaginal plug detection was designated embryonic
day 0.5 (E0.5). All protocols for animal use were approved by the
Institutional Animal Care and Use Committee of the University of California,
Irvine.
Tissue culture, in situ hybridization, immunofluorescence, histology and TUNEL staining
Dissected tissues were fixed, cryoprotected, embedded and cryosectioned
(12-20 µm) as described (Murray et al.,
2003). Hematoxylin staining was performed using Mayer's
Hematoxylin solution (Sigma MHS 16-500, St Louis, MO, USA). In situ
hybridization (ISH) was performed according to published protocols
(Murray et al., 2003). cRNA
probes used in this study were generated from: 1.5 kb mouse Foxg1
partial cDNA (Calof et al.,
2002); 1.2 kb mouse Gdf11 partial cDNA
(Wu et al., 2003); 1.0 kb
mouse follistatin full-length cDNA (generous gift of M. M. Matzuk, Baylor
College of Medicine, Houston, TX, USA); 0.6 kb mouse Sox2 partial
cDNA (Kawauchi et al., 2004);
2.0 kb mouse Mash1 full-length cDNA
(Guillemot and Joyner, 1993);
2.0 kb mouse Ngn1 cDNA (Ma et
al., 1999); 391 bp mouse Ncam cDNA
(Barthels et al., 1987); 687 bp
mouse Otx2 partial cDNA (bp 894-1581 of GenBank accession number
NM144841); full-length mouse Fgf8 ORF
(Kawauchi et al., 2005);
full-length of P1 bacteriophage Cre recombinase cDNA
(Lewandoski et al., 1997); 675
bp mouse p21Cip1 cDNA (bp 380-1055 of GenBank accession number
NM007669).
For pulse-fix analysis of bromodeoxyuridine (BrdU) incorporation, BrdU
(Sigma) was injected intraperitoneally into pregnant dams (50 µg/gm body
weight) and embryos collected 30 minutes later. Tissue (12 µm cryosections)
was processed for anti-BrdU immunoreactivity as described
(Kawauchi et al., 2005;
Kim et al., 2005). TUNEL
(deoxynucleotidyl Transferase-mediated dUTP Nick End Label) staining to detect
DNA fragmentation in apoptotic cells was performed as described
(Kawauchi et al., 2005),
except that 20 µm cryosections were used.
Explant cultures from E14.5-E15.5 CD-1 mice (Charles River, Wilmington, MA,
USA) were prepared as described (DeHamer
et al., 1994; Wu et al.,
2003). Purified recombinant human GDF11 (20 ng/ml; obtained by
agreement with Wyeth Pharmaceuticals, Cambridge, MA, USA) was added at the
beginning of the culture period. After 14 hours in vitro, explants were fixed
as described (Wu et al., 2003)
and processed for p21Cip1 immunoreactivity using monoclonal mouse anti-p21Cip1
(1:1000; Neomarker, Fremont, CA, USA: clone # AB-6 [HJ-21]), detected with
unlabeled goat anti-mouse IgG (1:300; Southern Biotech, Birmingham, AL, USA),
followed by Cy2-conjugated donkey anti-goat-IgG (1:100; Jackson
ImmunoResearch, West Grove, PA, USA). Cell nuclei were counterstained with
Hoechst 33342 (10 µg/ml, Sigma). For quantification, total migratory cells
in a minimum number of 10 randomly chosen fields were counted for each
condition (n=500-1000 cells per condition). The mean fluorescence
intensity for each cell was quantified as the mean pixel density over the
total area of the cell, measured using Zeiss AxioVision software (Carl Zeiss,
Thornwood, NY, USA). The percentage of cells with mean fluorescence
intensities of >6500 (`P21+ cells') was plotted for each condition (mean
fluorescence intensity for >95% of cells in both control and
no-first-antibody conditions was <6500).
|
). Possible artefacts from amplification of genomic DNA
were controlled for by processing samples prepared without reverse
transcriptase; in all cases these proved negative. All PCR reactions were
performed on an iQ5 iCycler (BioRad). Cycling conditions were 95°C for 3
minutes followed by 35 cycles of 95°C for 30 seconds, 58°C for 30
seconds and 72°C for 45 seconds. Primers used were: mouse Gdf11
exon3 (final concentration 200 nM),
5'-CTAAGCGCTACAAGGCCAAC-3' and
5'-AGCATGTTGATTGGGGACAT-3'; mouse Sox2
3'UTR (100 nM), 5'-AAGGGTTCTTGCTGGGTTTT-3' and
5'-AGACCACGAAAACGGTCTTG-3'; mouse Gapdh (150 nM),
5'-TTCACCACCATGGAGAAGGC-3' and
5'-GGCATGGACTGTGGTCATGA-3'. PCR product sizes were 151 bp, 150 bp
and 237 bp, respectively. Delta cycle time (dCT) values were obtained for each reaction by subtracting the CT value of Gapdh for that reaction from the CT value of the tested transcript (Gdf11 or Sox2) run in parallel. Means and standard errors (s.e.m.) were calculated for dCTs obtained from duplicate or triplicate reactions for each biological sample. To obtain ddCT values, the mean dCT for a given transcript in wild type was subtracted from the mean dCT for that same transcript obtained from a given experimental sample. Fold-change relative to the wild-type value was calculated as 2-(ddCT). Errors in fold-change were propagated from errors of dCT values, as the square root of the sum of the squares of the error (s.e.m.) for the dCT value of each transcript.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), cells
at all stages of the OE neuronal lineage are evident
(Fig. 1A)
(Beites et al., 2005;
Kawauchi et al., 2004): neural
stem cells can be identified by expression of Sox2; early committed
neuronal progenitors that derive from these cells express the proneural gene
Mash1 (Ascl1 - Mouse Genome Informatics); cells of the next
lineage stage - referred to as immediate neuronal precursors (INPs) - express
neurogenin 1 (Ngn1; Neurog1 - Mouse Genome Informatics); and
differentiated ORNs that derive from INPs are identified by expression of
Ncam (Ncam1 - Mouse Genome Informatics).
In Foxg1-/- embryos at E11, cells expressing these
lineage markers were present, but were greatly reduced in number
(Fig. 1A). Even at this early
age, OPs were greatly reduced in size; the domain of Sox2-expressing
neural stem cells was correspondingly reduced compared with that of wild-type
littermates. In addition, the concentric arrangement of gene expression
domains in the OP, reflective of cells at different states of neuronal
differentiation (Cau et al.,
1997; Kawauchi et al.,
2005), was altered in Foxg1-/- embryos.
Although some cells in the dorsal recess of Foxg1-/- OPs
do express neuronal markers, the number of these cells was dramatically
reduced compared with wild type, even when the relative decrease in OP size of
the mutant was taken into account: only a few Mash1+ early
progenitors could be detected in a restricted dorsomedial domain, and the
decrease in Ngn1+ INPs and Ncam+ ORNs
cells was even more dramatic. By contrast, wild-type OPs at this stage
displayed a clear concentric arrangement of neuronal cells:
Mash1-expressing progenitors were present near the rim, with
Ngn1-expressing INPs and Ncam-expressing ORNs at
progressively more central zones. To assess whether the deficits in
Foxg1-/- OE reflected an increase in cell death, TUNEL
staining was performed. As shown in Fig.
1B, we found no significant difference in relative numbers or
density of apoptotic cells in Foxg1-/- versus wild-type OE
at E11. Together, these observations indicate that, in
Foxg1-/- OE, development and differentiation of neuronal
cells begins at the normal time. However, because there was no marked increase
in apoptosis in Foxg1 nulls, some other process must result in the
markedly hypoplastic structure of Foxg1-/- OE.
|
). Fgf8 is also essential for morphogenesis of the
nasal cavity, and for neurogenesis within the OE
(Kawauchi et al., 2005), but
we saw no obvious change in Fgf8 expression in the rim of
Foxg1-/- OPs (Fig.
1C). In fact, the size of the Fgf8-expression domain
appeared to be essentially normal in Foxg1 mutants, despite a radical
difference in OP size (Fig.
1C). We next compared the patterns of expression of Fgf8
and Foxg1 in the OPs of wild-type and Foxg1 mutant embryos,
in the latter case using a probe for Cre to detect cells with
endogenous Foxg1 promoter activity (see Materials and methods). In
both wild-type and mutant embryos, we observed no overlap between the
expression domains of Foxg1 and Fgf8
(Fig. 1C), which provides a
probable explanation for the lack of a direct transcriptional
relationship.
Although these data do not rule out the possibility that Foxg1
acts by influencing Fgf8 signaling, rather than Fgf8
expression, this too seems unlikely given that we observe no increase in
apoptosis in the Foxg1-/- OE (see above and
Fig. 1B), whereas a marked
increase in apoptosis of Fgf8+/Sox2+ primary
neural stem cells is a hallmark phenotype when Fgf8 is inactivated in
and around the OE (Kawauchi et al.,
2005). We thus infer that the disruption of nasal cavity
morphogenesis and OE histogenesis (neurogenesis) in the Foxg1 mutant
is likely to occur through a mechanism that is distinct from a disruption in
Fgf8 expression and/or signaling.
Expression of Gdf11 and Foxg1 overlap in developing OE
As the first step toward testing the hypothesis that Foxg1 promotes
neurogenesis via antagonism of Gdf11 signaling, we examined whether
Foxg1 and Gdf11 are expressed at appropriate times and
locations to interact in this way. At E10.5, widespread expression of
Foxg1 mRNA was observed in the neuroepithelium of the invaginating
OP, as well as in the developing forebrain
(Fig. 2A). At E11.5,
Foxg1 expression was absent from the distal rim of the OP, and became
restricted to the central region, where neuronal differentiation is taking
place (Fig. 2B)
(Kawauchi et al., 2005). As
development proceeds (E12.5 to E17.5), Foxg1 expression is maintained
in the OE, but becomes progressively restricted to the basal compartment of
the epithelium, where stem and neuronal progenitor cells are located
(Fig. 2C-E)
(Beites et al., 2005).
Gdf11 expression is first evident at E10.5 in the epithelium of
the OP, and is also observed outside of the OE proper, in what are probably
the migrating olfactory pioneer neurons that demarcate the pathway of the
developing olfactory nerve (Fig.
2A) (Astic et al.,
2002). By E12.5, Gdf11 expression expands to include the
entire sensory neuroepithelium of the OE, and this pattern is maintained
throughout development (Fig.
2C-E) (Nakashima et al.,
1999; Wu et al.,
2003).
Overall, the expression domain of Gdf11 overlaps substantially
with that of Foxg1 throughout pre-natal development. As
Gdf11 is known to both be expressed by, and act upon, OE neuronal
progenitor cells (Lander et al.,
2009; Wu et al.,
2003), Foxg1-expressing cells are in the correct
locations and at the right times to be targets of Gdf11.
Interestingly, the expression of Foxg1 throughout the lateral extent of the OE is not uniform. By E12.5, there are clear regional differences: Foxg1 expression is highest at locations such as the recesses of the developing turbinates and the posterior recess of the nasal cavity (at the junction of the septum and the turbinates). By comparing such patterns over time, it can be seen that the locations of high Foxg1 expression represent the sites where the OE is most actively expanding into the nasal mesenchyme. By contrast, Gdf11 expression is rather uniformly expressed wherever OE is present (Fig. 2C-E, and insets).
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;
Gomis et al., 2006;
Hanashima et al., 2002;
Martynoga et al., 2005), and
it has been proposed that the major role of Foxg1 in cortical development is
to promote neural precursor proliferation
(Xuan et al., 1995). To
quantify stem/progenitor cell proliferation in the OE of
Foxg1-/- mice, we performed pulse-fix experiments at E11
and E12, using BrdU to label cells in S-phase
(Fig. 3A). At both ages, the
number of BrdU-immunopositive cells in any given section through the OE was
much lower in Foxg1-/- mice, as was the overall size of
the OE. Even if the discrepancy in size was corrected for by normalizing the
number of BrdU-immunopositive cells to the linear distance along the basal
lamina of the epithelium, 44% less labeling was still observed in
Foxg1-/- OE than in wild type (at E11.0: wild type,
207±4 BrdU+ cells/mm OE; Foxg1-/-,
121±6 BrdU+ cells/mm OE).
Interestingly, this change in BrdU labeling in Foxg1-/-
OE was accompanied by an expansion of expression of the CKI p21Cip1
(Fig. 3B). At E10.5, expression
of p21Cip1, which at later stages of OE development correlates with
neuronal differentiation (Kastner et al.,
2000; Legrier et al.,
2001), was normally confined to the rim of the invaginating nasal
pit (Fig. 3B). In
Foxg1-/- OE, however, p21Cip1 expression was
expanded to include both the rim and the central region of the OE. This
finding suggests that increased p21Cip1 expression in
Foxg1-/- OE might contribute to the alterations in the
number of cycling cells and the deficits in neuronal cell differentiation
observed in these mutants.
In cultured neural cells and cell lines, it has been shown that Foxg1 can
repress p21Cip1 induction effected by TGFβ signaling
(Dou et al., 2000;
Rodriguez et al., 2001;
Seoane et al., 2004). Because
Gdf11 acts through the same intracellular effectors as TGFβ, we
investigated whether Gdf11 controls the expression of p21Cip1 in the
OE. Two types of experiments were done. First, we performed ISH for
p21Cip1 in Gdf11 nulls and their wild-type littermates. As
shown in Fig. 3C,
p21Cip1 levels were greatly reduced in the OE of E13.5
Gdf11-/- animals, implying that Gdf11 is a
positive regulator of p21Cip1 expression in vivo. Second, we cultured
OE explants from wild-type embryos at a similar age (E15.5), and examined
expression of p21Cip1 by immunofluorescence after 14 hours' growth in the
presence or absence of recombinant Gdf11. The results are shown in
Fig. 3D. As described
previously, neuronal cells (neuronal progenitors and immature ORNs) comprise
virtually all of the migratory cells in explant cultures of OE purified from
mouse embryos at this age (Calof and
Chikaraishi, 1989; DeHamer et
al., 1994; Mumm et al.,
1996). Figure 3D
shows that only a small percentage (2.5%) of neuronal cells in untreated
control cultures showed significant p21Cip1 immunoreactivity. By contrast, the
percentage of p21Cip1-immunoreactive neuronal cells was more than fivefold
greater in GDF11-treated cultures (14.3%). Thus, GDF11 treatment causes an
increase in p21Cip1 expression in OE neuronal progenitors and/or immature
ORNs.
Inactivation of Gdf11 rescues neurogenesis in Foxg1-/- OE
The presence of Gdf11 and Foxg1 at similar times and
locations in the OE, the known ability of Foxg1 to inhibit the induction of
TGFβ pathway target genes that are also induced by Gdf11, and the
oppositely directed effects of Gdf11 and Foxg1 mutations on
OE neurogenesis, all raise the possibility that Foxg1 acts via inhibition of
Gdf11 activity. To assess this directly, we compared OE development in wild
type, Foxg1-/-, Gdf11-/- and
Foxg1-/-;Gdf11-/- double mutants. Morphology
and neuronal lineage markers were first examined at birth (the latest age to
which both strains survive) in sagittal sections at equivalent mediolateral
levels (Fig. 4A).
Figure 4B-D shows ISH analysis
of littermate wild-type, Foxg1-/- and
Foxg1-/-;Gdf11-/- animals, performed using a
probe to Ngn1 to highlight the neuronal progenitor layer of the OE
(Beites et al., 2005;
Wu et al., 2003). As shown in
Fig. 4B, the nasal cavity and
turbinate cartilage underlying the nasal mucosa are easily visualized in
sections from wild-type mice, with Ngn1+ progenitor cells forming a
distinct layer in the basal region of the OE proper. In sections from
Foxg1-/- mice, however, no olfactory turbinate structures
were observed: Only a small cavity filled with serous gland tubules was seen,
and no expression of Ngn1 could be detected
(Fig. 4C). By contrast, both
the gross morphology of nasal structures and the microscopic structure of the
OE are significantly rescued in Foxg1;Gdf11 double mutants
(Fig. 4D). The double mutants
have recognizable olfactory turbinates that are lined with OE, and this OE
contains Ngn1+ cells in their normal, basal, location.
This OE surrounds a substantial nasal cavity that is located dorsal and
posterior to the major maxillary incisor, as is the case in wild-type animals
(compare Fig. 4B and
4D). Thus, the absence of
Gdf11 results in substantial rescue of the defects in nasal cavity
morphogenesis and OE neuroepithelial development observed in
Foxg1-/- mice.
|
). In contrast
to wild-type and Gdf11-/- embryos,
Foxg1-/- embryos at E13.5 have essentially no OE
(Fig. 5C). When viewed at a
dorsoventral level equivalent to the wild-type example (note presence of
retina in all sections), the Foxg1-/- embryo shown in
Fig. 5C shows reduction of the
entire frontonasal region, an absence of nasal cavities, and an aberrant
(apparently enlarged) optic recess of the third ventricle. When
Foxg1-/- embryos were also made null for Gdf11,
however, both the OE and the frontonasal region were significantly rescued at
E13.5 (Fig. 5D). The OE of
Foxg1-/-;Gdf11-/- embryos was of normal
thickness, and contained cells expressing all major neuronal lineage markers
(Fig. 5D). Moreover, a
developing nasal cavity could be seen, as well as lateral folds of mesenchyme
covered by OE, evidence that basic turbinate structures are also rescued in
the double mutant (Fig. 5D).
Thus, the failure of both OE neurogenesis and nasal cavity morphogenesis
observed in Foxg1-/- animals is significantly rescued by
E13.5 when Gdf11 is absent.
Gdf11 dosage regulates the ability of Foxg1 to maintain OE neurogenesis
Genes of the TGFβ superfamily often show dose dependence in their
effects on development (Dunn et al.,
1997; Eldar et al.,
2002; Lawson et al.,
1999; Sutherland et al.,
2003). We tested whether Gdf11 might show such activity
in its effects on OE development. Mice null for Gdf11, Foxg1, as well
as Foxg1-/-;Gdf11+/- and
Foxg1-/-;Gdf11-/- mice, were examined at E16.5
(Fig. 6). At this stage in
normal development, olfactory turbinates are well developed and all cell types
in the OE can be recognized easily by their laminar position and molecular
marker expression; at this stage, the OE also possesses an apical layer of
sustentacular cells, the intrinsic glial cells of the OE
(Beites et al., 2005;
Cuschieri and Bannister, 1975a;
Cuschieri and Bannister,
1975b; Murray et al.,
2003; Smart,
1971). As demonstrated previously
(Wu et al., 2003),
Gdf11 nulls at this age have greater numbers of
Ngn1-expressing INPs and Ncam-expressing ORNs than wild
types, but show no obvious changes in the number of Mash1-expressing
early neuronal progenitors (Fig.
6A,B). We have found that Otx2, an orthodenticle homolog
that is expressed in the developing olfactory placode
(Mallamaci et al., 1996), is a
marker for sustentacular cells at E16.5 and beyond in the mouse OE.
Figure 6B shows that the layer
of Otx2-expressing sustentacular cells appears to be complete in
Gdf11 mutants, as expected.
In contrast to what was seen in wild-type and Gdf11-/- embryos at E16.5, the OE neuroepithelium itself, the nasal cavity, and molecular markers of OE neuronal and sustentacular cells were largely absent from Foxg1-/- embryos at this age, despite the fact that a septal structure could often be observed (Fig. 6C) (the presence of neural retina in all sections indicates that the horizontal sections shown have been taken at approximately the same dorsoventral level). Notably, when just one allele of Gdf11 was inactivated in Foxg1 nulls (Foxg1-/-;Gdf11+/- embryos), bilateral nasal cavities formed and were easily recognizable in the compound mutants at E16.5 (Fig. 6D). Although the surfaces of the nasal cavities were not as elaborately folded as in wild-type OE, they were lined by an OE of normal thickness. Moreover, the OE in these compound mutants contained cells expressing all neuronal and sustentacular markers tested, and these cells were present in their appropriate apical-basal positions within the OE (compare Fig. 6D to 6A). Indeed, even the layer of Ncam-expressing ORNs appeared to be as thick as that seen in wild types (Fig. 6D, inset).
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Loss of follistatin expression in Foxg1-/- nasal mucosa and its rescue by loss of Gdf11
The secreted protein follistatin (Fst) is an antagonist of activins and
Gdf11 that competes for binding of these factors to their receptors
(Gamer et al., 2001;
Nakamura et al., 1990;
Schneyer et al., 1994;
Schneyer et al., 2008;
Wu et al., 2003). Fst is
expressed in the nasal mucosa, both in the OE proper and in its underlying
mesenchymal stroma (Lander et al.,
2009; Wu et al.,
2003); during embryonic development, stromal expression of
Fst is particularly strong (Fig.
7A). The importance of Fst as an endogenous inhibitor of GDF11 is
evidenced by the phenotype of Fst-/- mice, which display
an OE at birth that is very thin and that is markedly depleted of neurons
(Wu et al., 2003).
Intriguingly, we found that Foxg1-/- embryos lack Fst expression in and around the OE from the earliest developmental stages (Fig. 7A). Even when OE remnants could be detected at late stages in Foxg1-/- embryos (e.g. example in Fig. 7A at E16.5), no Fst expression was detected. These results suggest an additional mechanism by which Foxg1 could antagonize Gdf11 activity: by promoting expression of Fst, a Gdf11 antagonist, Foxg1 could lower the effective concentration of Gdf11 in the extracellular milieu. Although this latter activity might contribute to the Foxg1-/- phenotype in the OE, however, it cannot explain it entirely, as that phenotype is both qualitatively and quantitatively different from the OE phenotype observed in Fst-/- mice (see Fig. S1 in the supplementary material; see also Discussion). It seems more likely that the Foxg1-/- phenotype arises as the result of a combination of intracellular (cell-autonomous) and extracellular (non-cell-autonomous) effects. This may help to explain why the phenotype is so severe.
Interestingly, both stromal and intraepithelial expression of Fst were completely rescued not only in Foxg1-/-;Gdf11-/- embryos, but also in compound Foxg1-/-;Gdf11+/- mutants in which only one allele of Gdf11 was inactivated (Fig. 7B). This demonstrates that Foxg1 is not itself required for Fst expression, i.e. Foxg1 does not itself induce Fst. The most parsimonious explanation is that it is the OE itself that induces Fst in surrounding tissue, with Foxg1 being required to generate an OE that is competent to do so. Exactly what signal from the OE induces Fst is unknown, but from the data in Fig. 7B, we can rule out Gdf11.
Does Foxg1 regulate Gdf11 expression?
In view of the fact that Foxg1 is a transcriptional regulator, we also
considered the possibility that a third mechanism - a repressive effect of
Foxg1 on Gdf11 expression - might also be at play in the OE. To
investigate this, we measured the expression of Gdf11 in the
different mutants discussed above. The results of these experiments, shown in
Fig. 8, revealed several
interesting findings. First, we found no evidence that Gdf11
autoregulates its own expression, as is the case for some TGFβ
superfamily members (Fig. 8A)
(Chen and Schier, 2002;
Forbes et al., 2006). Thus, in
a normal (wild-type) genetic background, inactivation of one allele of
Gdf11 led to a reduction in Gdf11 transcript levels to
35-49% of wild type; loss of both alleles led to essentially undetectable
expression of Gdf11 (Fig.
8A). Second, in the frontonasal process of E12
Foxg1-/- embryos, where loss of OE tissue was already
significant, Gdf11 expression was still detectable in the remaining
OE by ISH (Fig. 8B).
Third, Q-RT-PCR experiments, performed to determine transcript levels of Gdf11 in E11.5 frontonasal tissue (this age was chosen because there is still a reasonable amount of OE remaining in Foxg1-null animals), showed that Gdf11 transcript levels in Foxg1-/- and Foxg1-/-;Gdf11+/- mutants were significantly lower than in wild type (Fig. 8C). This was not surprising, given that Gdf11 is mainly expressed in the OE, and there is substantially less OE tissue in such mutants compared with wild type. Indeed, Q-RT-PCR showed that levels of Sox2, a marker for neuroepithelium (Fig. 1A), were also markedly decreased in Foxg1-/- and Foxg1-/-;Gdf11+/- mutants (Fig. 8D). By normalizing Gdf11 transcript levels to Sox2 transcript levels in the same samples, we could attempt to correct for the differing amounts of OE in different samples. The results (Fig. 8E) indicated that Gdf11 levels were 2- to 3-fold higher, per unit of OE, in Foxg1 nulls than in wild-type animals.
|
), we
considered the possibility that absence of Gdf11 might also rescue
some of the defects in telencephalic neurogenesis that are observed in
Foxg1 mutants (Hanashima et al.,
2007; Hanashima et al.,
2004; Hanashima et al.,
2002; Hebert and McConnell,
2000; Xuan et al.,
1995). When we examined
Foxg1-/-;Gdf11-/- embryos, however, we
found no such rescue. This is illustrated in
Fig. 9A, which shows
Hematoxylin/Eosin-stained horizontal sections through the brains of E13.5 mice
of the three different genotypes. As noted previously
(Xuan et al., 1995), the
developing cerebral hemispheres were much smaller in
Foxg1-/- embryos than in wild types and, instead of a
smooth cortical neuroepithelium, the cortical surface was buckled and uneven,
and cortical thickness differed in different areas. In
Foxg1-/-;Gdf11-/- double mutants,
there was no rescue of this phenotype: the cerebral hemispheres were still
much smaller than in wild-type embryos, and the cortex showed the same
structural aberrations that are apparent in Foxg1-/-
embryos. Thus, absence of Gdf11 fails to rescue the defects in
cortical development seen in the Foxg1-null mouse. To understand why an absence of Gdf11 should fail to rescue cortical development, while OE development is rescued so dramatically, we performed ISH to examine the expression of Gdf11 and Foxg1 in the developing forebrain from E11.5 through E15.5. As shown in Fig. 9B, Foxg1 was initially expressed throughout the telencephalon, but gradually became restricted to more dorsal structures, including the developing cerebral cortex. By contrast, Gdf11 expression at E11.5 was primarily found in a subset of the most ventral cells, but gradually disappeared by E15.5. Thus, cells that express Gdf11 appear not to be located in the vicinity of the developing cortical neuroepithelium.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Massague and Gomis, 2006;
Rodriguez et al., 2001;
Seoane et al., 2004). Although
that interaction was discovered through the study of p21Cip1
induction by TGFβ1 in cultured cells and cell lines, Gdf11 acts through
the same Smads as TGFβ1 (Nomura et
al., 2008; Tsuchida et al.,
2007), and, we have shown that Gdf11 induces p21Cip1 in
cells of the OE (Fig. 3).
Recent studies in the zebrafish, in which morpholino oligonucleotides were
used to reduce Foxg1 levels in a mosaic fashion
(Duggan et al., 2008), also
showed that Foxg1 influences OE neurogenesis in a cell-autonomous fashion, a
result opposite to what would have been expected if Foxg1 acts by controlling
the expression of a secreted growth factor, such as an FGF. The fact that we observe a higher level of Gdf11 transcripts, relative to Sox2 transcripts, in Foxg1 mutant OE (Fig. 8E) raises the additional possibility that Foxg1 regulates the expression of Gdf11 at the transcriptional level, although we cannot rule out alternative interpretations of the data. For example, loss of Foxg1 in Foxg1-/- OE might increase the number of Gdf11-expressing cells (relative to Sox2-expressing cells), rather than the level of Gdf11 transcripts per cell. Conversely, loss of Foxg1 might decrease the expression of Sox2. Even if Foxg1 does regulate Gdf11 transcription, the mechanism need not be direct (i.e. it could be mediated through other Foxg1 target genes). Likewise, loss of expression of the extracellular Gdf11 antagonist Fst in and around the OE of Foxg1-/- mice almost certainly reflects an indirect effect, as Foxg1 is not expressed in the stroma (where most Fst expression occurs), and Fst expression can be achieved in the complete absence of Foxg1 OE, by removing copies of Gdf11.
Regardless of the underlying mechanism(s), however, the finding that loss of Foxg1 is capable of leading to increased Gdf11 signaling, increased Gdf11 expression, and decreased expression of a Gdf11 antagonist collectively suggest that the relationship between Gdf11 activity and Foxg1 activity is a highly sensitive one.
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Threshold responses to signaling molecules usually imply cooperativity (or some other form of ultrasensitivity), such that a doubling in gene dosage produces more than a doubling in signaling. Because Gdf11 does not appear to regulate its own expression (Fig. 8A), we believe the likely source of ultrasensitivity lies elsewhere. If, as we suggest, the OE induces expression of Fst in its underlying stroma, then a positive-feedback loop emerges: an increase in Gdf11 activity would lead to a decrease in OE size, which would cause a decrease in Fst expression, which would in turn cause an increase in Gdf11 activity. A decrease in Gdf11 activity would be similarly self-enhancing. According to this view, Gdf11 in the embryonic OE is less of a graded regulator of neuronal production than a switch-like controller of a self-sustaining program of neurogenesis, with Foxg1 regulating when and where the switch is thrown.
Foxg1 and olfactory epithelium morphogenesis
During embryonic development of the OE, the process of neurogenesis can be
viewed as serving two distinct ends: histogenesis and morphogenesis. By
histogenesis we mean the generation of an appropriate complement and number of
OE cells at each location along the epithelium. By morphogenesis we mean the
planar growth and invagination of the epithelium to produce a deep,
characteristically folded nasal cavity. In Foxg1-/-
embryos, both processes fail from early stages. Yet when Foxg1
mutants are rescued through loss of Gdf11, the two processes are
restored to very different degrees. Histogenesis is nearly normal in both
Foxg1-/-;Gdf11-/- and
Foxg1-/-;Gdf11+/- mutants, but
morphogenesis is still impaired in
Foxg1-/-;Gdf11-/- mutants, and even
more so in Foxg1-/-;Gdf11+/- animals
(Figs 5 and
6). These results stand in
marked contrast to the phenotype of Fst-/- mice, which
exhibit severely defective histogenesis (an extremely thin OE), but relatively
normal morphogenesis (see Fig. S1 in the supplementary material). How could
excessive Gdf11 activity disrupt morphogenesis in one situation but
not the other?
We believe that the answer lies in the expression pattern of Foxg1
in the developing OE. As shown in Fig.
2, Foxg1 is initially found throughout the OE, but soon
becomes localized primarily to those areas in which planar expansion of the
epithelium is occurring. This implies that Gdf11 levels in most of the OE are
normally low enough to permit a constant, steady accumulation of ORNs, driving
normal histogenesis. At locations where Foxg1 is strongly expressed,
however, potent inhibition of Gdf11 signaling might allow the tissue to switch
into a mode of more dramatic expansion. Recently, we used mathematical
modeling to show that the only change needed to convert a tissue that adds
cells at a constant rate to one that adds cells at an exponentially increasing
rate is adjustment of the `replication probability' of a stem or
transit-amplifying cell to a level above 50%
(Lander et al., 2009). As
Gdf11 demonstrably lowers INP replication probabilities
(Lander et al., 2009;
Wu et al., 2003), the idea
that a sufficient reduction in Gdf11 activity could switch the OE
into an exponential growth mode is very plausible. In the
Fst-/- mutant, excessive levels of free extracellular
Gdf11 everywhere could account for a reduction in steady-state neurogenesis
throughout the OE (and thereby a very thin epithelium), but in regions of
Foxg1 expression, even this higher level of Gdf11 signaling might
still be effectively blocked (through the cell autonomous action of Foxg1 on
Gdf11 signaling). The result would be that planar expansion, and thus
morphogenesis, could proceed normally in the Fst-/-
mutant. By contrast, in the Foxg1-/- OE, unopposed Gdf11
activity would occur everywhere, leading to a failure of both histogenesis and
morphogenesis.
In summary, we propose that a major role of Gdf11 is to set a balance between the proliferation and differentiation of progenitor cells, whereas the primary role of Foxg1 is to tip that balance in favor of tissue expansion. A schematic depiction of the balancing act between Gdf11 and Foxg1 activities is presented in Fig. 10. It will be interesting to see whether the model of Foxg1 action through regulation of TGFβ family signaling applies to the OE only, or to other developing neural structures as well. Although Foxg1 clearly does not act through Gdf11 in the cerebral cortex (Fig. 9), a potential role for other TGFβ family ligands cannot be ruled out. Alternatively, it may be that, in some neural structures, the positive regulation of pro-neurogenic signals, such as FGFs, is of greater importance than the negative regulation of anti-neurogenic ones.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/dev.034967/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work 
Present address: Department of Neurosciences, University of California, San
Diego, La Jolla, CA 92093, USA
Present address: Vanderbilt Center for Stem Cell Biology, Vanderbilt
University, Nashville, TN 37232, USA
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DISCUSSION
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  C. L. Beites, P. L. W. Hollenbeck, J. Kim, R. Lovell-Badge, A. D. Lander, and A. L. Calof Follistatin modulates a BMP autoregulatory loop to control the size and patterning of sensory domains in the developing tongue Development, July 1, 2009; 136(13): 2187 - 2197. [Abstract] [Full Text] [PDF]
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