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First published online 19 April 2006
doi: 10.1242/dev.02368
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1 Department of Cell and Developmental Biology, Oregon Health and Sciences
University, School of Medicine, 3181 SW Sam Jackson Park Road, Portland, OR
97239-3098, USA.
2 Vanderbilt University Medical Center, Division of Gastroenterology, D4108
Medical Center North, Nashville, TN 37232-2279, USA.
Author for correspondence (e-mail:
christia{at}ohsu.edu)
Accepted 16 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONREFERENCES |
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Key words: Bone morphogenetic protein, Proprotein convertase, Proteolytic activation, Embryonic patterning, Cleavage mutant mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONREFERENCES |
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;
Hogan, 1996;
Kishigami and Mishina, 2005).
Bmp4 is widely expressed throughout embryogenesis and its earliest
known function is during gastrulation. Most mice homozygous for a null allele
of Bmp4 (Bmp4-/- mice) die at embryonic day (E)
6.5, with little or no mesoderm (Winnier
et al., 1995). In the rare mutants that survive past this time,
primordial germ cells (PGCs) are not specified
(Lawson et al., 1999) and lens
induction does not occur (Furuta and
Hogan, 1998). Chimeric analysis has shown that BMP4 is required
for chorioallantoic fusion, vascularization of the allantois, migration and
survival of PGCs, and establishment of left-right asymmetry
(Fujiwara et al., 2001;
Fujiwara, 2002). Tissue-specific inactivation of Bmp4 has revealed
additional roles for BMP4 in development of the heart, limbs and craniofacial
structures (Jiao et al., 2003;
Liu et al., 2005;
Liu et al., 2004;
Selever et al., 2004).
Strict regulation of BMP4 dosage is essential for normal development, as
evidenced by patterning defects observed in mice with reduced or elevated
levels of BMP4 activity. Bmp4 null heterozygotes
(Bmp4+/- mice) on a C57BL/6J background display several
phenotypes of variable penetrance, including reduced numbers of PGCs,
polydactyly, failure to maintain spermatogenesis and defects in the kidneys,
eyes and craniofacial structures (Dunn et
al., 1997; Katagiri et al.,
1998; Lawson et al.,
1999; Miyazaki et al.,
2000). By contrast, mice that lack the BMP antagonists chordin
and/or noggin are stillborn, show loss of ventral cell fates in the spinal
cord and defects in the development of the forebrain, somites and skeleton
(Bachiller et al., 2000;
Brunet et al., 1998;
McMahon et al., 1998).
BMP4 dosage is regulated at multiple levels, including at the level of
proteolytic activation (Miyazono et al.,
2005; Nakayama et al.,
2000; Yanagita,
2005). BMP4 is synthesized as an inactive precursor that is
cleaved by FURIN and/or other members of the proprotein convertase (PC) family
(Cui et al., 1998) at two
evolutionarily conserved sites within the inactive prodomain. An initial
cleavage occurs at an optimal FURIN consensus motif adjacent to the mature
ligand domain (-RSKR-, denoted the S1 site) and this allows for subsequent
cleavage at an upstream minimal FURIN motif (-RISR-, the S2 site) within the
prodomain (Cui et al., 2001).
In Xenopus embryos, BMP4 synthesized from exogenous precursor in
which the S2 site is non-cleavable is less active, signals over a shorter
range and accumulates at lower levels than does BMP4 cleaved from native
precursor (Cui et al., 2001).
Biochemical analysis of BMP4 cleavage in Xenopus oocytes revealed
that mature BMP4 remains noncovalently attached to the prodomain following
cleavage at the S1 site (Degnin et al.,
2004). If cleavage at the S2 site does not occur, this complex is
targeted to the lysosome for degradation, either within the biosynthetic
pathway, or within the endocytic pathway following receptor activation and
internalization. As a result, mature BMP4 in complex with the prodomain
signals only at short range, to nearby cells. Cleavage at the S2 site occurs
when the mature/prodomain complex traffics to a more acidic environment, which
unmasks and facilitates cleavage of the S2 site. This leads to dissociation of
the prodomain fragments from mature BMP4, and the free ligand is stable and
able to signal over long range. Cleavage at the S2 site might therefore
determine how much BMP4 is available for signaling.
The ability of cleavages within the prodomain to regulate the signaling
range of mature BMP4 is of particular interest because BMP4 and its
Drosophila ortholog, decapentaplegic (DPP) can function as either
short- or long-range signaling molecules depending on the tissue in which they
are expressed. Xenopus BMP4, for example, acts over multiple cells
within the embryonic mesoderm (Dosch et
al., 1997) but acts only within the immediate environment of its
synthesis in ectodermal explants (Jones et
al., 1996). Similarly, DPP forms a long-range concentration
gradient that specifies cell fate in a dose-dependent manner in the wing disc
but signals only to adjacent cells between germ layers of the gut
(Neumann and Cohen, 1997).
Tissue-specific mechanisms that regulate the range of action of BMP4 have not been identified, but it is tempting to speculate that tissue-specific use of the upstream cleavage site could contribute to this process. Accordingly, failure to cleave the S2 site in some tissues would generate a short-range ligand that accumulates at low levels, whereas cleavage at both sites would generate an identical ligand that accumulates at higher levels and possesses long range signaling properties. Given that proper regulation of BMP4 activity is critical for normal embryonic patterning, mutations that disrupt ordered cleavage of proBMP4 are expected to lead to developmental defects.
To study the physiological relevance of cleavage at the S2 site, and to begin to test the hypothesis that tissue-specific cleavage of this site regulates BMP4 activity in vivo, we generated mice carrying a point mutation that prevents cleavage at the S2 site. If the S2 site is normally cleaved in vivo, this mutation should generate a hypomorphic Bmp4 allele. Furthermore, if the S2 site is cleaved in a tissue-specific fashion as proposed, mutants would be predicted to show phenotypic defects and lower levels of mature BMP4 protein in only a subset of tissues that are known to be sensitive to BMP dosage. As described below, biochemical and phenotypic analysis of these cleavage mutant mice provide strong support for this hypothesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONREFERENCES |
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) and
introduces a single amino acid change that disrupts the S2 cleavage site, a
silent point mutation that changes a PvuI site to a BglII
site (to aid in screening) and a neomycin selectable marker flanked by loxP
sites (Fig. 1C). Linearized
vector was electroporated into R1 ES cells and homologous recombinants were
selected with G418 and gancyclovir. Correctly targeted ES cell clones were
identified by Southern blotting (Fig.
1D) and mutations were verified by sequencing DNA fragments
PCR-amplified from genomic DNA. Heterozygous ES cells were injected into
C57BL/6J blastocysts, and the resulting chimeras were mated with C57BL/6J
females to obtain Bmp4S2Gneo heterozygotes.
Bmp4S2Gneo females from each line were mated to male Cre
deleter mice (Schwenk et al.,
1995) to remove the neomycin gene. Genotypes were determined by
PCR amplification of tail DNA (Fig.
1E) using the 5' primer:
5'-TCTTGAATGTCGAGATGGGGCTGTG-3' and the 3' primer:
5'-TAAGATGAGCCCGGAAGTCCCAAAC-3' under the following conditions:
94°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, 35
cycles.
Bmp4lacZ/+ mice were obtained from Dr B. Hogan (Duke
University) and genotyped as described previously
(Lawson et al., 1999).
Bmp4lacZ/+ mice were backcrossed for a minimum of six
generations to C57BL/6J prior to mating with Bmp4S2G
mice.
cDNA constructs and transient transfections
cDNAs encoding HA- and myc-epitope-tagged forms of BMP4 or BMP4(S2G)
(Degnin et al., 2004) were
transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen).
Seventy-two hours post-transfection, cells were lysed and supernatants
collected and TCA precipitated for western blot analysis.
Western blotting
Transfected cells and mouse tissues were homogenized in RIPA buffer (150 mM
NaCl, 1.0% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris 8.0, 1 mM
phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4,
1x Roche protease inhibitor cocktail) on ice for 20 minutes, followed by
centrifugation to remove cellular debris. Proteins were separated on a 12%
polyacrylamide gel and transferred to a PVDF membrane. Blots of transfected
cells were probed with anti-HA monoclonal 12ca5 antibody (1:1000) and blots of
mouse tissue lysates were probed with anti-BMP4 antibody (1:1000; R&D
Systems cat. No. MAB757). Both were probed with HRP-conjugated secondary
antibody (Zymed; 1:5000) and visualized by chemiluminescence.
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).
Immunostaining, in situ hybridization, and ß-galactosidase staining
For phosphoSmad staining, E6 deciduas from Bmp4+/+ and
Bmp4S2G/S2G inter se crosses were dissected into
phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS on ice
for 30 minutes, incubated overnight in 30% sucrose in PBS at 4°C and then
embedded in OCT (TissueTek). 8 µm cryosections were incubated overnight at
4°C with an anti-phosphoSmad1/5/8 antibody (1:1000; a gift from Dr E.
Laufer, Columbia University) in PBS with 2% horse serum and 0.1% Triton X-100.
Staining was visualized using anti-rabbit Alexa Fluor 488-conjugated secondary
antibody (1:500; Molecular Probes). Embryos were processed for in situ
hybridization with digoxigenin-labeled Tbx5 and Gata4
riboprobes as described previously
(Wilkinson and Nieto, 1993).
ß-galactosidase staining of Bmp4lacZ/+ and
Bmp4lacZ/S2G placentas was performed as described
previously (Lawson et al.,
1999) using Red gal (Research Organics, Cleveland, OH) as a
substrate. Whole-mount PECAM staining (1:500; BD Pharmingen) was performed as
described (Schlaeger et al.,
1995).
Histology and TUNEL assays
Isolated embryos and organs were fixed in 4% paraformaldehyde in PBS,
dehydrated and embedded in paraffin wax. Sections (10 µm) were stained with
Hematoxylin and Eosin. 6 µm testis sections were analyzed for apoptosis
using the Dead End fluorometric TUNEL system (Promega). Labeled sections were
mounted with Vectashield anti-fade mounting medium containing DAPI (Vector
Labs).
Skeletal preparations
Skeletons were fixed and stained with Alcian Blue and Alizarin Red as
described previously (Hogan et al.,
1994). Isolated embryonic limbs were stained with Alcian Blue and
cleared as described previously (Jegalian
and De Robertis, 1992).
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Results |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONREFERENCES |
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) or in
mammalian cells (Fig. 1B).
Thus, the same mature BMP4 ligand is generated from both precursors but
subsequent cleavage at the S2 site of proBMP4(mS2G) does not occur.
Two independent mouse lines carrying the S2G mutation and a floxed
neomycin cassette (Bmp4S2Gneo) were generated
(see Materials and methods and Fig.
1C-E) and subsequently mated to Cre-expressing mice to remove the
neomycin gene. Prior to phenotypic analysis,
Bmp4S2G individuals from each line were backcrossed to
C57BL/6J for a minimum of seven generations to remove potential modifier genes
present in the 129/Sv strain that can partially rescue Bmp4
deficiency (Dunn et al.,
1997). No phenotypic differences were noted between the two
backcrossed lines.
Based on the significantly reduced activity of an analogous cleavage mutant
when ectopically expressed in Xenopus
(Cui et al., 2001),
Bmp4S2G is predicted to be a severe hypomorphic allele.
Thus, if the S2 site is normally cleaved in all tissues in wild-type mice in
vivo, Bmp4S2G/S2G mice are expected to have defects
similar to or more severe than those observed in Bmp4 null
heterozygotes. Contrary to this prediction, whereas
Bmp4-/- mice die early in embryonic development
(Winnier et al., 1995), and
Bmp4+/- mice display a significant degree of mortality
prior to weaning (Dunn et al.,
1997), Bmp4S2G/S2G and
Bmp4S2G/+ mice appear grossly normal and survive to
adulthood in the expected Mendelian ratios
(Table 1).
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).
Previous studies have shown that BMP4 synthesized in extraembryonic ectoderm
(ExE) prior to gastrulation (E6.5) is essential for specifying PGC fate in the
epiblast (Lawson et al., 1999;
Ohinata et al., 2005), whereas
BMP4 expressed in the extraembryonic mesoderm (ExM) during headfold stages is
required for localization and survival of PGCs
(Fujiwara et al., 2001).
Bmp4S2G/S2G and Bmp4S2G/+ embryos had
dramatically reduced numbers of PGCs relative to Bmp4+/+
littermates (Fig. 2A). This
reduction was already apparent at the presomite stage, prior to PGC migration
(data not shown), indicating that the founding population of PGCs was smaller
in mutants. The initial decrease in PGC number in
Bmp4S2G/S2G and Bmp4S2G/+ embryos was
almost as severe as that reported in Bmp4 null homozygotes and
heterozygotes, respectively, suggesting that failure to cleave the S2 site
almost completely ablates BMP signaling from the ExE. The steeper slope of the
regression line on the plot of PGC number versus developmental age for
wild-type mice relative to mutant littermates
(Fig. 2A) suggests that the
rate of expansion and/or survival of PGCs may also be reduced in mutants. We
did not, however, detect defects in localization of the remaining germ cells
(data not shown). These data demonstrate that cleavage of proBMP4 at both the
S1 and the S2 sites is required in order to provide sufficient BMP4 to
generate the proper number of PGCs, but is dispensable for their appropriate
localization.
To determine the extent to which BMP4 signal transduction was reduced in
the absence of S2 cleavage, an antibody directed against the active,
phosphorylated forms of the BMP pathway-specific SMADs (SMAD1, 5 and 8) was
used to detect BMP activity in E6-6.25 embryos. It has recently been shown
that BMP4 produced in the ExE signals to the epiblast both directly, and
indirectly via the visceral endoderm (VE), to specify germ cells
(de Sousa Lopes et al., 2004).
Consistent with our data demonstrating a dramatic reduction in PGC number,
SMAD phosphorylation was significantly reduced both in the proximal region of
the epiblast (Fig. 2B, arrows)
and in the VE (arrowheads) of Bmp4S2G/S2G embryos. These
data demonstrate that failure to cleave proBMP4 at the S2 site reduces the
ability of mature BMP4 to signal from the ExE to adjacent tissues during early
gastrulation.
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). At
8-12 weeks of age, 50% of Bmp4S2G/S2G males examined
(n=7/14) had testes that were drastically smaller than those of
age-matched controls (Fig. 3A).
Histological analysis revealed extensive degeneration of a large fraction
(30-100%) of seminiferous tubules in all undersized testes
(Fig. 3B-C). This degeneration
was due to widespread apoptosis, as revealed by TUNEL staining
(Fig. 3D-G). Notably, testes
from 6-day-old (P6) wild-type and Bmp4S2G/S2G mice were
morphologically and histologically indistinguishable
(Fig. 3H-I), suggesting that
the appearance of `empty' tubules at later stages
(Fig. 3C) is not the result of
failed colonization of the embryonic testes by PGCs, but is a consequence of
defective maintenance of spermatogenesis in adults.
We have previously shown that S2 processing prevents accelerated
degradation of the cleaved ligand (Degnin
et al., 2004), and thus we would predict that if the S2 site is
normally cleaved in the testes, then levels of mature BMP4 protein would be
lower in testes of Bmp4S2G/S2G males. After 2 weeks of
age, Bmp4 is expressed throughout the seminiferous tubules
(Hu et al., 2004), and in
adults, BMP4 expression is restricted to immature germ cells
(Baleato et al., 2005). Since
the degeneration of BMP4-expressing testicular cells in adult
Bmp4S2G/S2G males would confound the interpretation of our
results, we analyzed protein levels in testes isolated at P6, when expression
of Bmp4 is restricted to Sertoli cells
(Pellegrini et al., 2003) and
testicular degeneration is not yet detectable. Less mature BMP4 was detected
in individual testes from Bmp4S2G/S2G males compared to
that of wild-type littermates (Fig.
3J), consistent with the possibility that the S2 site of proBMP4
is normally cleaved in the testes and that failure to do so targets the mature
ligand for degradation.
Cleavage of BMP4 at the S1 site alone generates sufficient BMP4 activity for normal patterning of the skeleton, eyes and kidneys
The loss of PGCs and testicular degeneration observed in
Bmp4S2G/S2G mice is more severe than that in
Bmp4+/- mice, demonstrating that BMP4 dosage is reduced by
greater than 50% in these tissues. In other tissues, however, this appears not
to be the case since Bmp4S2G/S2G mice are fully viable
(Table 1), unlike Bmp4
null heterozygotes. To examine this issue more closely, we compared
development of several other tissues (skeleton, eyes and kidneys) that are
sensitive to BMP4 dosage in Bmp4lacZ/+ mice [in which exon
3 is replaced with lacZ to generate a null allele
(Lawson et al., 1999)] with
that of Bmp4S2G/S2G mice.
Analysis of the appendicular skeleton of Bmp4lacZ/+
mice revealed extra postaxial cartilaginous elements in the forelimbs of
E15.5-P0 embryos (Fig. 4A,
n=12/16) and adults (n=20/42) with a combined frequency of
nearly 60%. A similar phenotype is seen in mice in which Bmp4 is
inactivated in the limb bud mesenchyme
(Selever et al., 2004). We
also observed right hindlimb preaxial polydactyly (data not shown,
n=1/12), as previously reported
(Dunn et al., 1997). By
contrast, none of the E15.5 to newborn Bmp4S2G/S2G mice
analyzed (n>130) displayed hindlimb or forelimb polydactyly
(Fig. 4B).
Examination of the axial skeleton of Bmp4lacZ/+ adults showed a highly penetrant (n=7/10) defect in dorsal vertebral fusion and/or formation of the spinous processes of one or more cervical (Fig. 4C) and/or thoracic vertebrae (Fig. 4E). In addition, the 13th ribs were either missing or very small in 50% (n=5/10) of Bmp4lacZ/+ mice (Fig. 4E). Defects in fusion of the dorsal vertebrae were never observed (Fig. 4D,F; n=0/11), and small or missing ribs were observed in only 1% (n=1/76) of Bmp4S2G/S2G animals (Fig. 4F).
Gross visual examination of Bmp4lacZ/+ mice revealed small or missing eyes in 13% (n=13/97) of animals, whereas eye defects were never observed in Bmp4S2G/S2G mice (n>80). Similarly, we detected polycystic or enlarged kidneys in 8% of Bmp4lacZ/+ mice (n=1/12), but in none of over 100 Bmp4S2G/S2G mice. Thus, cleavage of proBMP4 at the S1 site alone generates sufficient BMP activity for normal eye, kidney and skeletal morphogenesis.
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) (Fig. 5A),
whereas the allantois of selected Bmp4lacZ/S2G embryos was
smaller and contained a densely packed mesenchymal core, indicating that
cavitation had not occurred (Fig.
5B and data not shown). In addition, expression of the BMP4 target
gene, Gata4 (Rojas et al.,
2005), was decreased in the allantois of a subset of
Bmp4lacZ/S2G embryos relative to wild-type littermates
(Fig. 5A,B).
To test whether cleavage at the S2 site is required for normal
differentiation or morphogenesis of allantois-derived placental blood vessels,
we compared expression of Bmp4 in the placenta of
Bmp4lacZ/+ and Bmp4lacZ/S2G embryos by
staining for ß-galactosidase. Bmp4 is highly expressed
throughout the mesothelium and endothelium of the allantois both before and
after it fuses with the chorion (Downs et
al., 2004), and it continues to be expressed in the
chorioallantoic placenta in a pattern that outlines the embryonic vasculature
until at least E14 (Fig. 5G and
data not shown). Thus, ß-galactosidase staining can be used to follow the
development of these allantoic derivatives. In Bmp4lacZ/+
embryos, ß-galactosidase-positive allantoic cells spread out over the
surface of the chorion (Fig.
5C,E) and penetrated into the embryonic layer of the placenta
(Fig. 5E, inset) between
E9.5-10.5. By E11.5, Bmp4-expressing cells were present at highest
concentration in the mesothelium immediately adjacent to the developing blood
vessels, which had branched and become organized into an extensive vascular
network (Fig. 5G). By contrast,
in the most severely affected Bmp4lacZ/S2G embryos, only a
few scattered ß-galactosidase-positive cells were observed on the surface
of the placenta at E9.5-10.5 (Fig.
5D, Table 3),
possibly due to aberrant chorioallantoic fusion. With few exceptions, the
remaining placentas derived from E9.5-11.5 Bmp4lacZ/S2G
embryos had significantly fewer stained cells and/or the embryonic placental
vascular network was smaller and more poorly organized than that of age
matched Bmp4lacZ/+ embryos
(Fig. 5E-H,
Table 3). We confirmed these
changes in the superficial placental vascular network of compound mutants by
analyzing the expression of the endothelial marker PECAM in whole placentas
(Fig. 5I,J). These data
demonstrate that S2 cleavage is essential for the function of BMP4 in
promoting differentiation and assembly of allantoic vascular endothelial cells
into organized blood vessels within the placenta
(Fujiwara et al., 2001). This
defect in placental vascularization most probably contributes to embryonic
lethality.
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). We
also noted an increase in the frequency of missing or small eyes in
Bmp4lacZ/S2G mutants at E12-P0 (n=13/22;
Fig. 6C,D) that exceeds that
observed in Bmp4lacZ/+ mice (n=13/97), suggesting
that S2 site cleavage is also required for normal BMP function in eye
development. Consistent with this possibility, expression of the dorsal
retinal marker Tbx5, which is sensitive to Bmp4 dosage
(Murali et al., 2005), was
reduced or absent in the eyes, but not in the heart or the limbs, of
Bmp4lacZ/S2G embryos relative to wild-type littermates
(Fig. 6E,F).
Bmp4lacZ/S2G embryos showed a more severe and frequent
loss of retinal expression of Tbx5 than did
Bmp4lacZ/+ littermates
(Table 4).
|
),
ventricle, and/or outflow tract (Jiao et
al., 2003; Liu et al.,
2004) raising the possibility that heart anomalies also contribute
to the late embryonic or perinatal lethality observed in
Bmp4lacZ/S2G and Bmp4lacZ/+embryos.
Histological analysis of mice recovered at E15-E17.5 revealed that
approximately 10% of Bmp4lacZ/+embryos examined (1/11) had
defects in closure of the membranous portion of the ventricular septum
(Fig. 7E, arrow) whereas these
defects were more frequent in Bmp4lacZ/S2G embryos
(Fig. 7F; n=4/8).
Membranous ventriculoseptal defects were never observed in
Bmp4S2G/S2G embryos
(Fig. 7D; n=0/11) or
wild-type controls (data not shown). Defects in septation of the outflow tract
(Fig. 7A-C) or the atrium
(Fig. 7G-I) were not detected
in any mice that were examined. These data demonstrate that cleavage at the S2
site is essential for normal BMP function in closure of the membranous
ventricular septum. Furthermore, they suggest that septation of the ventricle
is more sensitive to BMP dosage than is septation of the atrium or outflow
tract.
A single copy of Bmp4S2G generates sufficient BMP4 activity for patterning of the skeleton and kidneys
Although analysis of BMP4 compound mutants carrying null and S2G alleles
revealed additional tissues in which the Bmp4S2G allele is
hypomorphic, we did not detect any evidence for this in the skeleton or
kidney. Bmp4lacZ/S2G mutant embryos did not show an
increase in the frequency of polycystic kidneys (n=0/9) relative to
that found in age matched Bmp4lacZ/+ mice
(n=1/12). Likewise, the presence of the Bmp4S2G
allele did not enhance the severity or frequency of hindlimb preaxial
polydactyly (n=1/12) or forelimb postaxial duplications
(n=8/12) compared to that seen in Bmp4lacZ/+
controls (n=1/12 and 13/16, respectively). Thus, even when endogenous
BMP4 levels are halved, the S2G mutation is silent in the kidney and limbs,
suggesting that S2 site processing does not occur, or is not required for
normal BMP4 activity in these tissues.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONREFERENCES |
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) such
that it is less active and signals at shorter range
(Cui et al., 2001). These
studies involved overexpression of exogenous protein, however, and did not
address whether cleavage of the S2 site was physiologically relevant. In the
current studies, we show that mice expressing a single allele of Bmp4
containing a point mutation that prevents S2 processing have defects in
multiple organ systems and die during embryogenesis and early postnatal life.
Thus, cleavage of the S2 site of endogenous proBMP4 is essential for normal
development in vivo. An intriguing possibility is that BMP4 activity and/or signaling range is regulated in a tissue-specific fashion by cleavage of the S2 site. Consistent with this idea, inability to cleave the S2 site of proBMP4 in all tissues leads to phenotypic defects in only a subset of tissues where full Bmp4 dosage is required. The lack of defects in some tissues could potentially be explained if these tissues have a lower threshold requirement for Bmp4, or express functionally redundant Bmp family ligands. This is inconsistent, however, with the spectrum of phenotypes observed in Bmp4+/- mice relative to those in Bmp4S2G mutants. Specifically, we show that Bmp4S2G functions as a silent mutation in some tissues that are sensitive to a 50% reduction in Bmp4 dosage, such as the limb, dorsal vertebrae and kidney, yet leads to a much greater than 50% reduction in Bmp4 dosage in other tissues, such as the testes and PGCs. Furthermore, levels of mature BMP4 are reduced in some tissues (e.g. testes), but are equivalent in other tissues (e.g. kidneys and limbs, D.C.G., S.S. and J.L.C., data not shown) of Bmp4S2G/S2G mice relative to wild-type littermates. The simplest interpretation of our data is that the S2 site is cleaved in a tissue-specific manner and that reduced levels of mature BMP4 protein and/or phenotypic defects are observed only in tissues where the S2 site is normally cleaved. A less likely possibility is that the S2 site is ubiquitously cleaved and generates a long range gradient of BMP4 activity in all tissues, but that S2 cleavage is irrelevant in some tissues because a short range signal is sufficient for patterning. At present, it is not possible to distinguish between these possibilities in mammals since the range of action of BMP4 cannot be tracked in vivo and we have been unable to detect endogenous cleaved prodomain on western blots using any of four antibodies generated against epitopes in this region. These studies may not be feasible if the prodomain is rapidly degraded in vivo, or may require the development of more sensitive reagents.
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;
He et al., 2004).
Bmp4 mRNA and protein levels are significantly reduced in the
intestine of Bmp4lacZ/S2G mice, yet we do not detect any
reduction in phosphoSmad staining, nor do we see histological abnormalities in
intestinal architecture (D.C.G., R.H. and J.L.C., unpublished). It is probable
that the related family members Bmp2 and/or Bmp7 can
compensate for reduced levels of Bmp4 in the intestine, given that
their expression domains overlap extensively in this tissue
(Lyons et al., 1995).
Consistent with this possibility, expression of Bmp4 from the
Bmp7 locus can rescue kidney defects in Bmp7 mutants
suggesting that these ligands can function interchangeably in at least some
tissues (Oxburgh et al.,
2005). Furthermore, S1 and S2 FURIN consensus motifs are conserved
in all BMP2 and BMP4 precursor proteins
(Cui et al., 2001), suggesting
that cleavage of proBMP2 is most probably regulated in a fashion identical to
that of proBMP4. Thus, in tissues of Bmp4S2G/S2G mice
where cleavage of the S2 site is required, and where Bmp2 is
co-expressed, the S2 site of proBMP2 is also likely to be cleaved and this may
generate sufficient levels of mature protein to compensate for reduced levels
of BMP4.
Analysis of cleavage mutant mice reveals novel roles for BMP4
In addition to confirming and/or extending previous studies showing that
Bmp4 is required for specification of PGCs, maintenance of
spermatogenesis, and development of other organ systems such as the allantois,
heart and eye, our studies provide the first genetic evidence that
Bmp4 function is required for dorsal fusion of the vertebrae and for
ventral body wall closure.
The dorsal portion of the vertebral body, which gives rise to the spinous
process, is derived from mesenchymal cells of somitic origin. These cells
migrate and begin to express the Bmp4 target genes, Msx1 and
Msx2, after taking a position between the surface ectoderm and the
roof plate of the neural tube, both of which express Bmp4 (reviewed
by Christ et al., 2004).
Classical embryological experiments in chick showed that grafts of BMP2- or
BMP4-producing cells dorsal to the neural tube led to hypertrophy of the
spinous process (Monsoro-Burq et al.,
1996; Watanabe et al.,
1998; Watanabe and Le Douarin,
1996), whereas grafts of a ventral structure, the notochord, or of
recombinant sonic hedgehog (SHH), which is produced by the notochord,
inhibited expression of Bmp4 and prevented differentiation of the
spinous process (Watanabe et al.,
1998). These studies led to a model in which dorsal patterning of
the vertebrae is accomplished by BMP4, which is opposed by ventrally derived
SHH. Our data showing that formation of the spinous process is defective in
Bmp4 null heterozygotes provide strong genetic evidence in support of
this model. Interestingly, Bmp2 is co-expressed with Bmp4 in
the surface ectoderm and dorsal neural tube
(Dudley and Robertson, 1997),
and we observe dorsal vertebral defects at a similar frequency in
Bmp2+/- mice (D.C.G. and J.L.C., unpublished data),
underscoring the importance of achieving full BMP dosage for development of
the axial skeleton. This exquisite dosage sensitivity, together with the lack
of vertebral defects in Bmp4S2G/S2G mice, suggests that
BMP signals at short range from the dorsal ectoderm or roof plate to the cells
that will form the spinous process, and that S2 site processing is not
essential to generate these signals.
Defects in closure of the ventral body wall are fairly common in humans and
yet the underlying genetic and environmental causes are poorly understood
(Brewer and Williams, 2004).
Members of the TGFß family, including BMPs, have been implicated in this
process since deletion of Tgfß2 plus
Tgfß3, or of Bmp1 (a metalloproteinase that
boosts BMP activity by inactivating a BMP antagonist) leads to defects in
ventral body wall fusion (Dunker and
Krieglstein, 2002; Suzuki et
al., 1996). In Drosophila, Dpp is required for dorsal
closure, a process that has been proposed to be analogous to ventral closure
in mammals (Brewer and Williams,
2004). DPP is responsible for the induction of cell shape changes
that draw together and `zipper' close the two epithelial sheets during dorsal
closure (Ricos et al., 1999).
It plays an analogous role in regulating cytoskeletal organization during
pupal thorax closure (Martin-Blanco et
al., 2000) and morphogenesis of the wing
(Gibson and Perrimon, 2005;
Shen and Dahmann, 2005). These
observations have led to the suggestion that DPP plays a broadly conserved
role in driving cell shape changes that are required for patterned
morphogenesis of developing epithelia. Our demonstration that Bmp4 is
required for ventral closure in the mouse supports the assertion that this
role is conserved in vertebrates as well.
Regulation of proBMP4 cleavage
This analysis of Bmp4S2G mutant mice illustrates the
importance of proteolytic processing in regulating BMP activity, yet the
identity of the proprotein convertase (PC) that cleaves the S1 and/or S2 sites
of proBMP4 remains ambiguous. In vertebrates, seven PCs have been identified
and, among these, FURIN, PACE4 (PCSK6 - Mouse Genome Informatics), PC6 and PC7
are eligible to cleave proBMP4 since they are all broadly expressed and
function within the constitutive secretory pathway. Earlier studies involving
the use of a selective PC inhibitor in Xenopus embryos suggested that
FURIN and/or PC6 are the best candidates for endogenous proBMP4 convertases
(Cui et al., 1998). Consistent
with this possibility, mouse embryos lacking Furin die by E11.5 and
show an early defect in chorioallantoic fusion similar to that observed in
Bmp4lacZ/S2G mutant mice
(Roebroek et al., 1998). Loss
of Furin has less severe consequences than loss of Bmp4,
however, suggesting that other PCs function redundantly to cleave proBMP4.
Pc6 (Pcsk5 - Mouse Genome Informatics) mutant mice die prior
to E7.5 (Essalmani et al.,
2006), precluding analysis of its requirement in later
BMP4-dependent patterning events. By contrast, proteolytic maturation of
proBMP4 is reported to be intact in mice lacking both Furin and
Pace4, at least during pregastrula stages of development
(Beck et al., 2002),
eliminating PACE4 as an essential BMP4 convertase in the early embryo.
Pc7 (Pcsk7 - Mouse Genome Informatics) mutants develop
normally (Taylor et al.,
2003), ruling out the possibility that this protease plays a
dominant role in the maturation of proBMP4. Furthermore, we have previously
shown that recombinant PC7 can cleave the S1, but not the S2 site of proBMP4
in vitro (Cui et al., 1998),
consistent with other studies showing that PC7 has a strict requirement for a
basic residue at the P2 position (van de
Loo et al., 1997). It is possible, however, that PC7 functions
redundantly with FURIN in cleaving the S1 site of proBMP4. The generation of
mice carrying tissue-specific mutations in Pc6, Furin and/or
Pace4 will facilitate analysis of the potential role of these
proteases in cleaving proBMP4.
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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