1 Department of Cell Biology, Duke University Medical Center, Durham, NC
27710-3709, USA
2 Victor Goodhill Ear Center, Head and Neck Surgery Division, University of
California, Los Angeles, CA 90095-1794, USA
* Author for correspondence (e-mail: kling{at}cellbio.duke.edu)
Accepted 23 July 2002
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SUMMARY |
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Key words: Chordin, Noggin, BMP, Holoprosencephaly, Prechordal plate, Forebrain, Mouse
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INTRODUCTION |
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BMPs also play important roles in development of the brain. Several BMPs
are expressed in the mouse dorsal forebrain and facial primordia
(Barlow and Francis-West, 1997;
Furuta et al., 1997
). Ectopic
application of BMP4 to the ventral forebrain leads to holoprosencephaly in
chick (Golden et al., 1999
)
and reduces expression of both Shh and Fgf8
(Ohkubo et al., 2002
). BMP4
applied to mouse forebrain explants represses anterior neural gene expression
and may promote apoptosis in forebrain, hindbrain and craniofacial neural
crest (Furuta et al., 1997
;
Graham et al., 1994
;
Graham et al., 1996
). These
observations suggest that rostral tissues require proper control of BMP
activity for normal development.
The distribution and activity of BMPs in the extracellular space is
regulated by the secreted factors chordin (CHRD) and Noggin (NOG), which
specifically bind BMP proteins and prevent ligation of their receptors
(Sasai and De Robertis, 1997).
The effects of both Chrd and Nog are mediated entirely by
antagonism of BMP signaling, acting upstream of signal transduction
(Hammerschmidt et al., 1996
;
Holley et al., 1995
;
Holley et al., 1996
). Although
similar biochemical activities suggest functional redundancy, CHRD and NOG
display different binding affinities for various BMPs
(Piccolo et al., 1996
;
Zimmerman et al., 1996
) and
interactions with extracellular modifiers
(Ashe and Levine, 1999
;
Piccolo et al., 1997
;
Scott et al., 2001
). Thus,
CHRD and NOG may have both shared and specific roles.
Both Chrd and Nog are expressed in the node and its
derivatives (Klingensmith et al.,
1999; McMahon et al.,
1998
). Later in embryogenesis, they are expressed in diverse
structures, some uniquely and others in common
(Brunet et al., 1998
;
Scott et al., 2000
). Null
mutations for mouse Chrd and Nog have been generated to
assess the role of these genes in mammalian development. Chrd null
homozygotes are fully viable in outbred backgrounds with partially penetrant
mild defects of the chondrocranium and cervical vertebrae (D. B. and J. K.
unpublished). Chrd homozygosity, however, is lethal in inbred and
defined hybrid backgrounds, resulting in severe chondrocranial and pharyngeal
arch defects (Bachiller et al.,
2000
) (R. M. A., D. B., and J. K., unpublished). Nog null
homozygotes die perinatally, with defects in dorsoventral patterning and
skeletal development (McMahon et al.,
1998
; Brunet et al.,
1998
). Analysis of double null homozygotes reveals functional
redundancy of Chrd and Nog. Regardless of genetic
background, these embryos have severe forebrain truncations in addition to
defects in dorsoventral and left-right patterning
(Bachiller et al., 2000
) (R. M.
A. and J. K., unpublished).
In this study, we have examined a second class of Chrd;Nog double mutants, Chrd-/-;Nog+/-, to further elucidate the functions of these genes in head development. Our results indicate that Chrd and Nog promote growth and patterning signals from the PrCP and the ANR, two organizing centers of rostral development.
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MATERIALS AND METHODS |
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Gene expression assays
Whole-mount in situ hybridization was performed as already described
(Belo et al., 1997). When
necessary, staining buffer was supplemented with 5% polyvinyl alcohol (Aldrich
#36, 313-8) to enhance weak signals. Standard techniques were used for
ß-galactosidase staining (Hogan et
al., 1994
). For RT-PCR, five-somite stage embryos were dissected
into three pools of tissue using glass knives: trunk, ANR and anterior
midline. The ANR region excluded rostromedial tissue to avoid potential
ambiguity caused by expression in the midline mesendoderm. Preparation of cDNA
samples and PCR for Chrd and Hprt were as described
(Stottmann et al., 2001
).
Nog was detected using the following primers:
5'-GCATGGAGCG-CTGCCCCAGC and 5'-GAGCAGCGAGCGCAGCAGCG.
Skeletal preparation and histological sectioning
Double staining of neonatal skeletons was performed as described
(McLeod, 1980). For
histological sectioning, embryos were fixed overnight in Bouin's fixative
(histology) or 4% paraformaldehyde (TUNEL/immunohistochemistry), and washed in
PBS before embedding in paraffin wax, sectioning (8 µm) and counterstaining
according to standard protocols (Hogan et
al., 1994
).
Cell death and proliferation
Nile Blue Sulfate (NBS) staining was performed as described
(Trumpp et al., 1999), except
embryos were incubated in Lactated Ringers containing 0.002% NBS (Sigma,
N-5632) for 30 minutes at 37°C. Whole-mount TUNEL was performed as
described (Conlon et al.,
1995
), except fragmented DNA was labeled with fluorescein-dUTP and
detected with anti-fluorescein IgG (Roche). For apoptosis/proliferation
assays, TUNEL was performed according to manufacturer's instructions using
Fluorescein TUNEL labeling mix and TdT (Roche). Sections were then blocked in
PBS containing 10% sheep serum, 1% blocking reagent (Roche) and 0.1% Tween-20
for 1 hour. Cells in metaphase were detected with anti-phosphorylated histone
H3 IgG (Upstate Biotechnology) and an Alexa Red secondary antibody (Molecular
Probes). Coverslips were mounted in 2.5% DABCO/90% glycerol.
At E8.5, proliferative and apoptotic indices were calculated by dividing number of metaphase-stage or TUNEL-positive cells by total number of DAPI-stained nuclei, respectively. At E9.5, indices were determined by dividing the number of labeled cells by total area of neural ectoderm. Area measurements were made using NIH image software. At least three adjacent sections were counted in each assay. Statistical significance assessed using Student's t-test.
Bead preparation and explant culture
Purified recombinant BMP2 (Genetics Institute) and BSA (Sigma) were applied
to beads as described (Furuta et al.,
1997). BMP2 was used at a concentration of 10 µg/ml unless
otherwise indicated and BSA concentration was 1 mg/ml. Cephalic explants were
isolated from five- to eight-somite embryos and contained all tissues rostral
to the mid-hindbrain region. These were cultured on Nucleopore filters
(Whatman) using media and culture conditions as described
(Furuta et al., 1997
).
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RESULTS |
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Chrd-/-;Nog+/- mice display three
types of craniofacial skeletal defects
Cranial development in Chrd-/-;Nog+/-
animals ranges from outwardly normal to nearly headless. Three classes of
defects may be distinguished among affected mutants, defined here as midline,
truncation and jaw classes. The midline class shows absence of rostroventral
midline structures often resulting in cyclopia
(Fig. 1B,B', see
Fig. 4P). The truncation class
shows an absence of rostral-most tissues
(Fig. 1C,C'). The jaw
class lacks jaw elements derived from the first branchial arch and frontonasal
mass (Fig. 1D-F). Although
these defects are not mutually exclusive, one type usually predominates
(Table 1).
|
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In this study, we present our analysis of the initial neural defects in
these mutants. Some mandibular phenotypes result from independent roles of
Chrd and Nog in the first branchial arch
(Stottmann et al., 2001). A
study of the nature and causes of other defects in the craniofacial skeleton
will be presented elsewhere (R. M. A., R. W. S. and J. K., unpublished).
Anterior neural defects
Holoprosencephaly (HPE) is a deficit of ventral midline development in the
forebrain. In its most severe form, a holosphere is generated: a thin-walled,
singly lobed cerebral structure with a common ventricle. Less severe forms and
microforms also exist with less dramatic midline deletions. HPE is frequently
associated with midline craniofacial defects in humans
(Ming and Muenke, 1998).
Affected Chrd-/-;Nog+/- mice exhibit a spectrum
of HPE phenotypes observed throughout gestation
(Fig. 2B,E,L). Significantly,
loss of medial forebrain tissue is evident before evagination of the
telencephalic vesicles, the precursors to the cerebral hemispheres
(Fig. 2G). In the most severe
cases, HPE is evident prior to neurulation (see
Fig. 4M). Truncation class
mutants that lack most of the facial skeleton also entirely lack rostral brain
structures. Some severely affected animals lack forebrain and midbrain
(Fig. 2C,N), while more
modestly affected truncation mutants lack only the forebrain
(Fig. 2M). In the mildest
examples, mutants have fused or absent olfactory bulbs
(Fig. 2I,J). Generally, mutant
neural phenotypes correlate closely with external craniofacial defects.
|
Penetrance of rostral defects in
Chrd-/-;Nog+/- animals
To reveal gestational lethality and penetrance of phenotypes in
Chrd;Nog mutants, we assessed embryo development during early and
late gestation. Prenatal lethality is apparent only in the double homozygote
(Table 2), and presence of
rostral-restricted phenotypes was perfectly correlated with the genotype
Chrd-/-;Nog+/-. Overall, HPE phenotypes were
observed in 9.0% of embryos examined from E8.5-17.5 and were first detectable
at the five-somite stage (E8.0). Furthermore,
Chrd-/-;Nog+/- animals were under-represented
by nearly 12% at weaning. This number reflects that in addition to the 9% of
animals with overt HPE phenotypes, a further 3% of this mutant class die soon
after birth with few, if any, detectable external defects. For example, most
juvenile Chrd-/-;Nog+/- mice were externally
normal, but some had mild midline defects such as an absence of upper incisors
(n=4; data not shown).
|
Haploinsufficiency of Nog for forebrain development occurs only in the absence of Chrd (Table 3). However, the penetrance of these defects is low in a random-outbred genetic background. One theoretical explanation for this is that other potential genetic modifiers influence Chrd and Nog function in the midline. Indeed, the mutant phenotypes observed in Chrd-/- mice differ significantly with genetic background (see Introduction). Because of this sensitivity, we tested whether Chrd-/-;Nog+/- animals might have full penetrance of rostral phenotypes in defined genetic backgrounds. However, we found that most Chrd-/-;Nog+/- pups of both inbred (129Sv/J) and hybrid (B6SJLF1) strains exhibited no discernible holoprosencephalic phenotypes and were fully viable (Table 3, data not shown). Given the low fecundity of inbred mice and no evidence for increased phenotypic penetrance, we conducted all subsequent studies in an outbred genetic background.
|
Expression of Chordin, Noggin and BMPs during early neural
patterning
To understand more fully the functions of Chrd and Nog in
head formation, we characterized their expression from neural plate through
early organogenesis stages (E7.5-8.5). The prechordal plate (PrCP) is
identifiable by the co-expression of Gsc, Shh and Foxa2
(Fig. 3B-D)
(Belo et al., 1998
;
Camus et al., 2000
). In situ
hybridization at headfold and early somite stages reveals axial expression of
Chrd and Nog through the caudal PrCP; neither is expressed
in the rostral PrCP at these stages (Fig.
3A,E,I,J). Thus, the initial limit of Chrd and
Nog expression defines rostral and caudal subregions within the PrCP.
By the five-somite stage, however, Chrd and Nog are
expressed throughout the axial mesendoderm, including the PrCP
(Fig. 3L,M). Chrd and
Nog are also clearly expressed in cephalic mesenchyme
(Fig. 3L,M), and Nog
is expressed near the anterior neural ridge (ANR), the boundary between
surface and neural ectoderm (Fig.
3E). We did not detect Chrd in the ANR by in situ
analysis; however, RT-PCR amplification demonstrates a low level of
Chrd expression in the non-midline ANR, at early somite stages
(Fig. 3N). In summary,
Chrd and Nog are both expressed in the axial mesendoderm
including the PrCP, although expression in the rostral-most PrCP is delayed
until the five-somite stage. They are also expressed in or around the ANR.
Thus, both genes are expressed in two of the known organizing centers of
rostral head development.
|
Because CHRD and NOG presumably influence BMP signaling only where target
BMPs are present (Piccolo et al.,
1996; Zimmerman et al.,
1996
), we examined expression of BMP2, BMP4 and BMP7 at headfold
through early somite stages (Fig.
3F-H). Each is expressed in epidermis adjacent to the neural
plate. Furthermore, Bmp2 and Bmp7 are expressed in paraxial
mesoderm adjacent to axial mesendoderm. Bmp7 is co-expressed with
Chrd and Nog in the node, notochord and caudal PrCP.
Collectively, several relevant BMPs are expressed such that they may interact
with CHRD and NOG, and influence PrCP and ANR activities.
Rostral defects involve loss of SHH signaling
Defects in Chrd-/-;Nog+/- pups resemble
rostral holoprosencephaly phenotypes seen in Shh-/- mice
(Chiang et al., 1996).
Furthermore, humans heterozygous for SHH display a range of
phenotypes almost identical to that seen in
Chrd-/-;Nog+/- mice
(Roessler et al., 1996
). We
therefore examined Shh expression and signaling in
Chrd-/-;Nog+/- mutants. In affected embryos,
Shh expression is absent from the rostral mesendoderm and the rostral
ventral neural midline (RVNM; Fig.
4A,B,E,F), while more caudal domains are unaffected. Patched 1
(Ptch1; Ptch Mouse Genome Informatics) and
Gli1 are positively regulated transcriptional targets of SHH
(Lee et al., 1997
;
Marigo and Tabin, 1996
).
Therefore, expression levels reflect SHH signal transduction. Ptch1
and Gli1 expression is diminished or lost in the RVNM of affected
Chrd-/-;Nog+/- embryos
(Fig. 4C,G; data not shown).
SHH also induces the expression of Nkx2.1 (Titf1
Mouse Genome Informatics) in the presumptive ventral diencephalon
(Dale et al., 1997
;
Shimamura and Rubenstein,
1997
), and this expression is critical for pituitary development
(Kimura et al., 1996
;
Takuma et al., 1998
).
Nkx2.1 expression is lost in affected
Chrd-/-;Nog+/- mutants
(Fig. 4D,H), resulting in
aberrant infundibulum development (Fig.
4L,P). Together, these results indicate that the most rostral
expression of Shh is lost in affected
Chrd-/-;Nog+/- embryos, with a consequential
diminishment of SHH signaling and PrCP function.
Loss of anterior FGF8 expression and signaling
A total lack of SHH results in holoprosencephaly but not truncation of
rostral structures (Chiang et al.,
1996). We therefore suspected that an additional defect in
Chrd-/-;Nog+/- mice caused the rostral
truncations. The ANR has an important role in patterning and growth of the
rostral neural tube, which is mediated at least in part by FGF8 expression
(Rubenstein and Beachy, 1998
;
Meyers et al., 1998
;
Shimamura and Rubenstein,
1997
). We therefore examined expression of Fgf8 and its
downstream target Foxg1
(Shimamura and Rubenstein,
1997
) in affected Chrd-/-;Nog+/-
embryos. At five somites (
E8.0), Fgf8 is expressed in bilateral
domains of the ANR (Fig. 4I).
However, affected Chrd-/-;Nog+/- embryos have
reduced expression of Fgf8 in a single domain, fused at the midline
(Fig. 4M). At E9.5,
Fgf8 is expressed in the commissural plate and lateral epithelial
domains of developing nasal placodes
(Crossley and Martin, 1995
)
(Fig. 4J). Moderately affected
embryos retain only a medial domain of rostral Fgf8 expression at
dramatically reduced levels (Fig.
4N). Expression of Foxg1 is correspondingly reduced in
the prosencephalon (Fig. 4K,O).
These data indicate that FGF8 expression and signaling are reduced in affected
Chrd-/-;Nog+/- embryos.
The reduction of Shh and Fgf8 expression and signaling that we have observed could be due to prior loss of cells expressing these genes. However, we have not detected morphological changes prior to the five-somite stage. Moreover, no Chrd-/-;Nog+/- embryos at late headfold stages showed evidence of increased apoptosis as measured by whole-mount TUNEL assay (n=41). Therefore we conclude that rostral neural ectoderm domains affected in Chrd-/-;Nog+/- embryos are initially present at the time Shh and Fgf8 are normally first expressed. Moreover, the overall frequency of Chrd-/-;Nog+/- embryos displaying diminished Shh or Fgf8 was similar to the penetrance of morphological phenotypes (not shown).
Cell death and proliferation
Many brain and craniofacial structures are clearly absent in affected
Chrd-/-;Nog+/- embryos by organogenesis stages.
We therefore used sectioned embryos to analyze levels of apoptosis via TUNEL
reaction and proliferation via anti-phosphorylated histone H3 antibody
staining. At eight to 10 somites (E8.5), cell proliferation is greatly
decreased in the forebrain of affected
Chrd-/-;Nog+/- embryos, although apoptosis
appeared comparable with wild type (Fig.
5A,B). By contrast, affected 20-25 somite (
E9.5)
Chrd-/-;Nog+/- embryos displayed expansion of
death in the dorsal midline, lateral head mesenchyme and the trigeminal
ganglion, but normal levels of neural proliferation
(Fig. 5C,D). As another measure
of apoptosis, we stained whole embryos with Nile Blue sulfate (NBS), which
marks non-necrotic cell death. NBS staining at eight to 10 somites reveals
mildly increased dorsal staining not detected in sections
(Fig. 5E,F). Unexpectedly,
normal ventromedial domains of apoptosis were absent, suggesting that tissues
in this region have been deleted or mis-specified. At 20-25 somites, the
dorsomedial stripe of NBS-positive cells was broadened in affected mutants
(Fig. 5G,H). Again, the ventral
midline domain of cell death was absent in affected embryos. Based on the
timing of changes in gene expression relative to cell death increases
elsewhere, we suspect that this loss of a normal domain of cell death is due
to mis-specification of this tissue, rather than an early total deletion of
the entire tissue. Collectively, these results indicate that cell
proliferation is reduced in the forebrain at eight to 10 somites, following
the first changes in rostral gene expression. Apoptosis levels may be mildly
elevated at this stage. By 20-25 somites, proliferation levels have normalized
in the forebrain, but cell death is dramatically increased in forebrain and
surrounding tissues.
|
Apoptosis via BMP activity may be mediated by Msx transcription factors
(Marazzi et al., 1997), and
Msx transcription is positively regulated by BMP2 and BMP4 in the forebrain
(Furuta et al., 1997
).
Moreover, Msx1 may repress expression of anterior neural genes
(Feledy et al., 1999
). We have
observed increased expression of Msx1 in
Chrd-/-;Nog-/- double homozygous embryos (R. M.
A. and J. K., unpublished). In addition, more severely affected
Chrd-/-;Nog+/- embryos may display robust
increases in Msx1 expression (Fig.
5I,J). However, there is no clear change in most moderately
affected Chrd-/-;Nog+/- embryos
(Fig. 5K,L), and spatial
differences reflect only the absence of midline tissue. Thus, although we
observe changes in apoptosis and proliferation in all affected
Chrd;Nog mutants, only the more severe phenotypes also display a
clear increase in Msx1 expression. Therefore it is possible that
Msx1 induction is a transient response to BMP signaling in some
contexts (see more below).
An explant assay for addressing the consequences of increased BMP
activity
Increased BMP signaling is anticipated with the loss of Chrd and
Nog; thus, unantagonized BMP signaling probably causes the defects
seen in Chrd-/-;Nog+/- embryos. To test the
effects of locally increased BMP activity, we cultured cephalic explants from
five- to eight-somite embryos in combination with beads soaked in recombinant
BMP2. Explants were prepared by placing a single bead against the ANR between
the bilateral anterior neural folds (designated type 1). To confirm the
presence of increased BMP signaling, we examined the expression of the direct
BMP target Msx1 (Table
4, part A). Msx1 was strongly induced in regions adjacent
to BMP2 beads (Fig. 6A).
Strikingly, Msx1 was induced laterally at large distances (hundreds
of µm) from the bead, but did not extend into the midline of the explant.
This suggests that BMPs may act at long range in the ANR, and that some
regions of the head are refractory to Msx1 induction by BMP
signaling. We tested this by placing a BMP bead directly on the midline
(designated type 2 explants). In these, the midline responded with
Msx1 induction, but not in the most rostral part
(Fig. 6D). Ectopic BMP2 was
probably present in this region, as Msx1 induction was seen beyond
this zone, in medial regions of the ANR.
|
|
Because we observed no clear increase in Msx1 expression in some affected Chrd-/-;Nog+/- embryos, we suspected that increased Msx1 expression may be a transient response to modestly increased BMP activity in these tissues. We used our explant assay to address the duration of Msx1 expression as a response to exogenous BMP2. Msx1 was strongly expressed in the ANR after 6 hours of culture with BMP beads (Fig. 6A). After 9 hours, expression was markedly diminished, and more so at 12 hours (Fig. 6B,C).
BMP2 inhibits Shh and Fgf8 expression in cephalic
explants
Because we observed a loss of Fgf8 and Shh expression in
affected Chrd-/-;Nog+/- embryos, we tested
whether increased BMP could elicit the same responses in wild-type explants
(Table 4, part B). In type 1
explants, we observed suppression of Fgf8 expression in nearly all
cases (93%) after 6 hours of culture using BMP2 at a concentration of 10
µg/ml (Fig. 6E).
Additionally, rostral domains of Shh expression were reduced in
43% of explants. To determine whether this was due to greater sensitivity
of the ANR or to bead placement, we used type 2 explants. In these, rostral
Shh was repressed 96% of the time. Fgf8 expression was
significantly reduced but not completely repressed in 42% of explants
(Fig. 6F). This demonstrates
that Shh may be repressed by BMP2 in some domains without coincident
Msx1 induction. Together, these data demonstrate that effects of BMP2
on cultured cephalic explants reproduces changes in gene expression observed
in affected Chrd-/-;Nog+/- embryos. Thus,
antagonism of BMP activity is crucial both in the ANR and rostral ventral
neural midline for Fgf8 and Shh expression,
respectively.
Cephalic explants deficient in Chordin and Noggin
are hypersensitive to BMP
If the primary cause of the defects in
Chrd-/-;Nog+/- embryos is increased BMP
activity resulting from reduced BMP antagonism, explants prepared from these
embryos should be more sensitive to exogenous BMP than are wild-type explants.
To test this, we first compared the response of wild-type and
Chrd-/-;Nog+/- explants to BMP2-soaked beads
(Table 4, part C). Here, two
beads were used: one between the rostral neural folds and one against the
lateral neural folds of the midbrain (type 1+). Using a BMP concentration of
10 µg/ml, Msx1 induction was consistently stronger in
Chrd-/-;Nog+/- explants than in wild type
(Fig. 6G). Next, we tested
whether mutant explants would show Msx1 induction at a lower BMP2
concentration than did wild-type explants. Whereas beads soaked in 10 µg/ml
BMP2 strongly induced Msx1 in wild-type explants, beads prepared in
0.1 µg/ml resulted in little or no induction
(Fig. 6H). However,
Chrd-/-;Nog+/- explants responded to 0.1
µg/ml BMP2-soaked beads with robust Msx1 expression
(Fig. 6H). Induction of
Msx1 in Chrd-/- explants was occasionally similar
to that of Chrd-/-;Nog+/- explants, although a
strong response in Chrd-/- explants occurred less
frequently [the relative sensitivity of different Chrd;Nog genotypes
to BMPs will be presented elsewhere (R. M. A. and J. K., unpublished)].
Together, these data demonstrate that
Chrd-/-;Nog+/- rostral tissues are sensitized
to BMP signaling, strongly supporting the premise that they are deficient in
BMP antagonism.
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DISCUSSION |
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Reduced BMP antagonism in Chrd;Nog double mutants
Molecular and genetic assays used in frog, fish and fly embryos indicate
that the function of both Chordin and Noggin is to antagonize BMP signaling
(Furthauer et al., 1999;
Holley et al., 1995
;
Holley et al., 1996
;
Piccolo et al., 1996
;
Schulte-Merker et al., 1997
;
Zimmerman et al., 1996
). Thus,
genetic ablation of Chrd and Nog in mice is expected to
reduce BMP antagonism. We confirmed this using two methods. First, expression
of Msx1, a positive transcriptional target of BMP signaling, is
upregulated and expressed ectopically in all
Chrd-/-;Nog-/- mice. This suggests increased
BMP signaling in the absence of Chrd and Nog. However,
ectopic Msx1 expression was observed only in the more severely
affected Chrd-/-;Nog+/- embryos, demonstrating
that one wild-type allele of Nog is largely sufficient to control BMP
signaling in the rostral head. Importantly, although Chrd is
transcribed at low levels after gastrulation, its expression has great
functional significance. Haploinsufficiency of Nog occurs only in the
absence of functional Chrd alleles.
We also assessed the response of Chrd-/-;Nog+/- explants to exogenous BMP. We found that cephalic explants prepared from Chrd-/-;Nog+/- embryos respond to BMP2 with greater induction of Msx1 than did wild-type explants. Moreover, Chrd-/-;Nog+/- explants exhibit robust response to BMP2 at concentrations that elicit little if any effect in wild-type explants. These results provide strong functional evidence that Chrd-/-;Nog+/- embryos possess reduced BMP antagonist activity. Complementary to these data, biochemical assays reveal that BMP signal transduction is increased in Chrd;Nog mutant embryos (R. M. A. and J. K., unpublished).
Prechordal plate function is promoted by BMP antagonists
Midline neural deletions result from compromised function of the prechordal
plate. Surgical ablation of the PrCP in mouse explants prior to four somites
results in a loss of medial neural markers
(Shimamura and Rubenstein,
1997), as does ablation of the PrCP in chicken
(Pera and Kessel, 1997
). In
addition, loss of signaling molecules expressed in the PrCP, such as
Shh in mouse (Chiang et al.,
1996
), results in similar neural midline defects. Affected
Chrd-/-;Nog+/- mutants lack Shh
expression in the prechordal mesendoderm, and consequently lose expression of
downstream SHH targets in surrounding tissues. Thus, the resulting midline
defects in Chrd-/-;Nog+/- mutants are probably
due to reduction of PrCP function.
At headfold stages, Chrd and Nog are expressed in the
notochord and the caudal PrCP, but not in the most rostral regions; however,
both genes are expressed throughout the PrCP by the five-somite stage. This
early distribution of Chrd and Nog expression reveals an
initial subdivision of the PrCP into two compartments. As we have observed a
clear reduction of rostral-most ventral midline tissue as early as the
five-somite stage, this early compartmentalization may have functional
significance. In support of this hypothesis, others have demonstrated that
caudal regions of rostral midline mesendoderm are required for the maintenance
of identity and function of the rostral PrCP at early somite stages
(Camus et al., 2000). Thus,
production of CHRD and NOG protein in the caudal compartment may be essential
for the activity of the rostral PrCP.
At least three BMPs bound by CHRD and NOG are expressed adjacent to the
ventral midline of the rostral neural plate in mouse. In chicken, others have
shown a role for BMPs in prechordal mesoderm specification
(Vesque et al., 2000). Thus,
the delay in Chrd and Nog expression in rostral PrCP may
provide a temporal window for this to occur. Additionally, BMP7 secretion from
prechordal mesoderm may be an essential co-factor with SHH in specification of
rostral ventral neural midline fates; temporal regulation of Chrd
expression may limit this induction along the rostrocaudal axis
(Dale et al., 1999
). Although
this suggests that loss of CHRD and NOG should promote rostral-ventral midline
fates by increasing BMP7 signaling, the associated loss of rostral SHH may
prevent induction of ectopic ventral fates in affected
Chrd-/-;Nog+/- embryos. Alternatively, given
significant differences in PrCP morphology and histology between mouse and
chicken (Pera and Kessel,
1997
; Sulik et al.,
1994
), it is possible that the PrCP may function differently in
mammals and birds. Taken together, our results suggest that CHRD and NOG
coordinate the location, duration and composition of BMP signaling during
rostral midline development.
Our study has demonstrated that application of BMP-soaked beads mimics the
abnormal gene expression of Chrd-/-;Nog+/-
mutants by repressing rostral expression of Shh in the CNS,
consistent with results from chicken
(Ohkubo et al., 2002). In
addition to transcriptional repression of Shh, BMPs can both
antagonize SHH signaling and alter the response to SHH in receiving cells
(Liem et al., 1995
;
Murtaugh et al., 1999
;
Watanabe et al., 1998
). Thus,
in Chrd-/-;Nog+/- animals, ectopic BMP
signaling may affect both the expression of SHH and its signal
transduction.
Chordin and noggin in the anterior neural ridge
Rostral truncation phenotypes cannot be due to impairment of SHH signaling
alone, as this type of defect never occurs in Shh-/- mice
(Chiang et al., 1996) (R. M.
A. and J. K., unpublished). However,
Chrd-/-;Nog+/- mutants recapitulate rostral
defects seen in some Fgf8 mutants
(Meyers et al., 1998
).
Furthermore, Chrd-/-;Nog+/- mutants have
reduced FGF8 expression and activity in the ANR. Taken together, these data
suggest that rostral neural deletions result from impairment of ANR function.
Expression of Nog in the ANR precedes Fgf8 expression, and
by the five-somite stage, both Chrd and Nog are co-expressed
with Fgf8 in the ANR. We have shown that several BMPs are expressed
in surface ectoderm adjacent to the ANR, and that BMP2 can repress
Fgf8 in the ANR in vitro. Indeed, similar transcriptional repression
of Fgf8 by BMPs has been observed in the chick ANR
(Ohkubo et al., 2002
), and in
the first branchial arch of mouse
(Stottmann et al., 2001
). We
therefore speculate that in addition to direct repression of the FGF8 target
Foxg1 by BMPs (Furuta et al.,
1997
), Foxg1 may be reduced indirectly by repression of
Fgf8. Our results suggest that CHRD and NOG may preserve endogenous
expression of FGF8 in the ANR.
Multiple roles for chordin and noggin in patterning the rostral
neural plate
Our data provide evidence for two distinct roles for Chrd and
Nog in patterning the rostral neural plate
(Fig. 7A,B). First, CHRD and
NOG secreted from the notochord and caudal PrCP antagonize the activity of
BMPs expressed in the midline mesendoderm and adjacent mesodermal domains.
This protects and promotes the functions of SHH and the rostral PrCP in
defining the rostral neural midline. Second, CHRD and NOG secreted from the
ANR antagonize BMPs that are secreted from adjacent non-neural ectoderm, and
thus prevent the transcriptional repression of Fgf8. In the affected
mutant embryos, reduced CHRD and NOG lead to lower BMP antagonism and thus
increased BMP signaling; this in turn results in reduced SHH signaling from
the rostral PrCP and FGF8 signaling from the ANR
(Fig. 7C).
|
BMPs are reported to have proliferative, anti-proliferative and
pro-apoptotic effects upon neural tissue
(Furuta et al., 1997;
Mabie et al., 1999
;
Mehler et al., 1997
;
Trousse et al., 2001
). Our
data support a role for CHRD and NOG in permitting proliferation during
neurulation, and later in preventing apoptosis in the neural tube. At the
eight- to 10-somite stage, cell proliferation in rostral neural ectoderm is
decreased, while apoptosis is slightly increased. By 20-25 somites,
proliferation in this tissue has essentially normalized, while apoptosis is
substantially increased. We therefore suggest that the dramatic malformations
in the forebrain of Chrd-/-;Nog+/-
mutants are due to a combination of apoptosis-promoting and anti-proliferative
activities of BMPs, together with the loss of trophic factors such as FGF8 and
SHH.
Variability and penetrance of craniofacial defects
The mutant phenotypes of
Chrd-/-;Nog+/- animals showed low
penetrance and wide variability. We suggest two explanations that may account
for these phenomena. First, variability could result from unlinked modifier
alleles that confer sensitivity to increased BMP signaling. Potential
modifiers include other BMP antagonists, such as cerberus, gremlin, Dan
(Nb11; Parn Mouse Genome Informatics) and twisted
gastrulation (Hsu et al.,
1998; Pearce et al.,
1999
; Piccolo et al.,
1999
; Scott et al.,
2001
). If this were the case, the penetrance and expressivity of
phenotypes in Chrd-/-;Nog+/- should
depend strongly on genetic background. However, we observed that penetrance
was low with wide phenotypic variability, even in a defined genetic
background.
Alternatively, there may be natural stochastic variation in levels of BMP
or BMP antagonist levels. We suggest that this variation does not approach
upper or lower critical thresholds unless the balance between BMPs and
antagonists is disrupted, as in Chrd;Nog mutants. Our studies with
Chrd-/-;Nog+/- cephalic explants would
seem to support this idea. At a given dose of BMP2 we saw variable increases
in Msx1 expression in explants of identical genotype. Consistent with
this idea, mutants of several BMP signaling components have variable defects,
including BMP2, BMP4, BMP7, and SMAD5 and SMAD6
(Chang et al., 1999;
Dudley et al., 1995
;
Dunn et al., 1997
;
Galvin et al., 2000
;
Winnier et al., 1995
;
Zhang and Bradley, 1996
). This
intrinsic variability in BMP signaling levels could normally be controlled by
the multiple regulatory feedback loops that control BMP signaling. These
include autoregulation of BMP expression levels
(Ghosh-Choudhury et al., 2001
;
Vainio et al., 1993
), as well
as positive regulation by BMPs of Chrd and Nog transcription
(Stottmann et al., 2001
) and
inhibitory SMAD expression (Afrakhte et
al., 1998
; Imamura et al.,
1997
; Nakao et al.,
1997
; Takase et al.,
1998
). Thus, regulatory mechanisms in Chrd;Nog mutants
might include reduction of BMP expression and compensatory antagonism by other
BMP antagonists and inhibitory SMADs.
BMP signaling and human holoprosencephaly
The spectrum of phenotypes in
Chrd-/-;Nog+/- mutants mirrors the
range of defects observed in human holoprosencephaly. Furthermore, human HPE
is frequently associated with micrognathia, agnathia and pituitary dysgenesis,
as in affected Chrd-/-;Nog+/- mutants.
HPE is common in man, occurring in as many as one out of 250 conceptuses
(reviewed by Ming and Muenke,
1998). The frequency of sporadic HPE in wild-type mouse
conceptuses is apparently much lower, in that we have never seen HPE in
thousands of outbred embryos and fetuses examined in our laboratory (R. M. A.,
A. R. L., R. W. S. and J. K., unpublished). This may imply that humans are
more sensitive to environmental or genetic factors that promote HPE.
Consistent with this possibility, human SHH heterozygotes can display
a partially penetrant range of HPE phenotypes, while mouse Shh
heterozygotes do not display HPE (Chiang
et al., 1996
; Ming and Muenke,
1998
). Overall, the variability, penetrance, and restricted
location of phenotypes suggest that Chrd;Nog mutant mice reproduce
many aspects of the human holoprosencephaly syndrome.
Although no BMP antagonists or BMP pathway components correspond to known
HPE loci, many genes involved in human holoprosencephaly remain unidentified
(Roessler and Muenke, 2001).
Furthermore, it is possible that mutations in CHRD or NOG
modify one or more of the known loci. Consistent with our data, other evidence
suggests that BMP signaling pathways may be involved in the pathogenesis of
HPE. Addition of BMP proteins to the chick forebrain leads to reduced SHH and
FGF8 expression as well as midline and anterior deletions
(Golden et al., 1999
;
Ohkubo et al., 2002
). Our
genetic and embryological manipulations of mouse
Chrd-/-;Nog+/- mutants lead us to
propose that insufficient BMP antagonism may underlie some human HPE by
decreasing the activity of rostral organizing centers.
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ACKNOWLEDGMENTS |
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