209
Development 120, 209-218 (1994)
Printed in Great Britain  The Company of Biologists Limited 1994
Bone morphogenetic proteins and a signalling pathway that controls
patterning in the developing chick limb
Philippa H. Francis1,2,*, Michael K. Richardson1, Paul M. Brickell2 and Cheryll Tickle1
1Department
of Anatomy and Developmental Biology and 2Medical Molecular Biology Unit, Department of Biochemistry and
Molecular Biology, University College London Medical School, The Windeyer Building, Cleveland Street, London W1P 6DB, UK
*Author for correspondence
SUMMARY
We show here that bone morphogenetic protein 2 (BMP-2)
is involved in patterning the developing chick limb. During
early stages of limb development, mesenchymal expression
of the Bmp-2 gene is restricted to the posterior part of the
bud, in a domain that colocalizes with the polarizing region.
The polarizing region is a group of cells at the posterior
margin of the limb bud that can respecify the anteroposterior axis of the limb when grafted anteriorly and can
activate expression of genes of the HoxD complex. We
dissect possible roles of BMP-2 in the polarizing region signalling pathway by manipulating the developing wing bud.
Retinoic acid application, which mimics the effects of
polarizing region grafts, activates Bmp-2 gene expression
in anterior cells. This shows that changes in anteroposterior pattern are correlated with changes in Bmp-2
expression. When polarizing region grafts are placed at the
anterior margin of the wing bud, the grafts continue to
express the Bmp-2 gene and also activate Bmp-2 expression
in the adjacent anterior host mesenchyme. These data
suggest that BMP-2 is part of the response pathway to the
polarizing signal, rather than being the signal itself. In
support of this, BMP-2 protein does not appear to have any
detectable polarizing activity when applied to the wing bud.
The pattern of Bmp-4 gene expression in the developing
wing bud raises the possibility that BMP-2 and BMP-4
could act in concert. There is a close relationship, both
temporal and spatial, between the activation of the Bmp-2
and Hoxd-13 genes in response to retinoic acid and polarizing region grafts, suggesting that expression of the two
genes might be linked.
INTRODUCTION
polarizing region, a group of cells in the posterior mesenchyme
of the limb bud that specifies pattern across the a-p axis of the
bud. Grafts of the polarizing region to the anterior mesenchyme result in the duplication of digits, such that anterior
cells form posterior structures (Tickle et al., 1975). The effect
of the polarizing region can be mimicked by the application of
retinoic acid to the anterior margin of the limb bud (Tickle et
al., 1982). Both polarizing region grafts and locally applied
retinoic acid activate genes of the HoxD complex in anterior
cells (Izpisúa-Belmonte et al., 1991; Nohno et al., 1991).
However, this activation occurs at least 16 hours after
treatment and the intervening steps are unknown. In addition,
activation of HoxD genes requires cooperation with a signal
from the apical ridge (Izpisúa-Belmonte et al., 1992a).
Transcripts of the Bmp-2 and Bmp-4 genes, which encode
BMP-2 and BMP-4, have been found in developing mouse
limb buds (Lyons et al., 1990; Jones et al., 1991). The BMPs
were originally identified in extracts of bovine bone that could
induce ectopic cartilage formation when implanted subcutaneously in rats. Biochemical analysis of this activity led to the
discovery of seven evolutionarily conserved proteins, named
BMP-1 to BMP-7 (reviewed by Rosen and Thies, 1992). BMP2 to BMP-7 are structurally related to each other and are
A fundamental problem in embryonic development is how
patterns of differentiated cells are established. The complex
pattern of skeletal elements found in the adult limb develops
from a mass of undifferentiated mesenchymal cells in the limb
bud. Patterning in the limb involves signalling between mesenchymal cells and between mesenchyme and epithelium, and
some of the molecules involved in these signalling pathways
have been identified. For example, certain retinoids and growth
factors have been implicated as signalling molecules and a
number of homeobox-containing genes have been found to
respond to these signals (reviewed by Tabin, 1991; Tickle and
Brickell, 1991). Here we present evidence that the bone morphogenetic protein BMP-2 is involved in a signalling pathway
that controls patterning across the anteroposterior (a-p) axis of
the developing limb.
The signalling pathways in the developing limb are complex
and incompletely understood. Two regions in the limb bud are
known to be important sources of signals that control the patterning of the developing limb. One region is the apical ectodermal ridge, which is required for the outgrowth of the limb
and is involved in proximodistal specification. The other is the
Key words: bone morphogenetic protein, Bmp-2, Bmp-4, Hoxd-13,
polarizing region, retinoic acid, chick limb bud, limb development
210
P. H. Francis and others
members of the TGF-β family, while BMP-1 has a completely
different structure and may be involved in the proteolytic activation of the other BMPs. BMP-2 and BMP-4 are structurally
more closely related to each other than to other BMPs, and
have a high level of amino acid sequence identity to the product
of the Drosophila decapentaplegic (dpp) gene. There is
evidence that BMP-2, BMP-4 and DPP may be signalling
molecules that regulate pattern formation and morphogenesis
during embryonic development. For example, in Drosophila,
DPP is thought to specify dorsal-ventral polarity in the embryo
(Ferguson and Anderson, 1992) and to pattern the embryonic
midgut and the imaginal discs (reviewed by Hoffman, 1991).
In Xenopus, Bmp-4 mRNA can induce ventral mesoderm when
injected into the animal hemisphere of fertilized embryos, and
so BMP-4 is thought to be involved in mesodermal patterning
(Dale et al., 1992; Jones et al., 1992).
Recent descriptions of the distribution of Bmp gene transcripts in mouse embryos suggest that BMPs may play a role
in limb development. To investigate this possibility, we
isolated and characterized chicken Bmp-2 and Bmp-4 cDNA
clones so that we could dissect the roles of the Bmp-2 and Bmp4 genes, by carrying out experimental manipulations of developing chick wings. We show that the Bmp-2 gene is part of the
response component of the polarizing region signalling
pathway and is closely related to expression of HoxD genes.
MATERIALS AND METHODS
Isolation of chicken Bmp-2 and Bmp-4 cDNA clones
Approximately 4×105 recombinant bacteriophage from a 10-day
(stage-36) chick embryo cDNA library constructed in λgt11
(Clontech) were screened with a 1227 bp fragment prepared by PCR
from the mouse Bmp-4 cDNA clone 1321 (Jones et al., 1991) and
labelled with [α-32P]dCTP (New England Nuclear) by random
priming (Feinberg and Vogelstein, 1984). The probe corresponded
precisely to the mouse BMP-4 coding region. Plaque lifts on to
Hybond-N were performed according to the manufacturer’s instructions and duplicate filters were hybridized overnight at 65°C in 6×
SSC, 1% (w/v) SDS, 5× Denhardt’s solution, 0.1 mg ml−1 denatured
sonicated herring testis DNA and 106 cts minute−1 ml−1 of radiolabelled probe. The most stringent post-hybridization wash was for 30
minutes at 50°C in 2× SSC, 0.1% (w/v) SDS. Positive clones were
picked, purified by rescreening under the same conditions, and their
EcoRI inserts sub-cloned into plasmid vector pBluescript SK+ for
further analysis.
The longest chicken Bmp-4 cDNA clone had an insert of 772 bp
and was designated p6. To obtain the complete coding region of
chicken BMP-4, polyadenylated RNA isolated from 10-day chick
embryo limb was reverse transcribed and the cDNA product amplified
by PCR using oligonucleotide primers with the sequences 5′CCCACGTCGCTGAAATCCACAT-3′
(complementary
to
nucleotides 953-932) and 5′-ATGATTCCTGGTAACCGAATGCTG-3′. The latter sequence corresponds to the first 24 nucleotides
of the Xenopus (Dale et al., 1992), human (HSBMP2B; EMBL
Nucleotide Sequence Data Library) and mouse (Brigid Hogan,
personal communication) BMP-4 coding regions. The PCR product
(nucleotides 1-953) was cloned into pBluescript KS+ using a TA
cloning system (Invitrogen), and designated p6.1.
The longest chicken Bmp-2 cDNA clone had an insert of 1120 bp
and was designated p5.
Nucleotide sequencing
Dideoxy sequencing of double-stranded templates was performed
using Sequenase Version 2.0 (US Biochemicals) according to the
manufacturer’s instructions. Oligonucleotides were synthesized in our
laboratory. Nucleotide sequences were analyzed using the PC-GENE
software package (Intelligenetics).
Construction of probes for northern hybridization and in
situ hybridization
32P-labelled RNA probes for northern hybridization and 35S-labelled
probes for in situ hybridization to tissue sections were synthesized as
previously described (Devlin et al., 1988). Digoxigenin-labelled RNA
probes for in situ hybridization to wholemount preparations were synthesized using a kit from Boehringer, according to the manufacturer’s
instructions.
Antisense RNA probes specific for chicken Bmp-4 transcripts were
synthesized with T3 RNA polymerase, using BamHI-linearized p6.1
as a template (nucleotides 1-953). Sense RNA probes for use as
controls were synthesized with T7 RNA polymerase, using HindIIIlinearized p6.1 as a template.
To construct a template for the synthesis of Bmp-2-specific probes,
nucleotides 1-797 of p5 were amplified by PCR. The PCR product
was cloned into pBluescript SK+ using a TA cloning system (Invitrogen), and designated p5.1. Antisense RNA probes specific for
chicken Bmp-2 transcripts were synthesized with T3 RNA polymerase, using HindIII-linearized p5.1 as a template and sense RNA
control probes were synthesized with T7 RNA polymerase, using
BamHI-linearized p5.1 as a template.
The Bmp-2- and Bmp-4-specific probes share approximately 57%
nucleotide sequence identity and have never been observed to crosshybridize under the hybridization and washing conditions used.
Antisense RNA probes specific for mouse Bmp-2 transcripts were
synthesized as described by Lyons et al. (1989). Antisense RNA
probes specific for chicken Hoxd-13 transcripts were synthesized as
described previously (Izpisúa-Belmonte et al., 1991).
Chick embryos
Fertilized chicken eggs were obtained from Poyndon Farm, Waltham
Cross, Herts, and were incubated at 38±1°C. Embryos were staged
according to Hamburger and Hamilton (1951), dissected into fresh
PBS and processed for RNA isolation or in situ hybridization as
described below.
Retinoic acid treatment of chick wing buds
AG1-X2 beads soaked in a solution of all-trans retinoic acid (0.1 mg
ml−1 or 1 mg ml−1) in dimethyl sulphoxide were implanted at the
anterior margin of the wing bud of stage-19/20 embryos as previously
described (Tickle et al., 1985). At a series of times between 16 and
48 hours later, the beads were removed and the embryos pinned and
fixed in 4% (w/v) paraformaldehyde. To confirm the efficacy of the
retinoic acid, one embryo from each series of experiments was
allowed to develop for 6 days after retinoic acid treatment. These
control embryos were fixed in 5% (w/v) trichloroacetic acid and
stained with alcian green to show the skeletal pattern.
Grafting of mouse polarizing region to chick wing buds
The forelimb buds were removed from 10.5-day-old C57/BL/H-at
mouse embryos and trypsinized at 4°C for 30-60 minutes to remove
the epithelium (Szabo, 1955). The polarizing region, a cube of mesenchyme from the posterior margin of the bud, was dissected out and
grafted to the anterior margin of stage-19/20 chick embryo wing buds,
as described previously (Izpisúa-Belmonte et al., 1992b). The position
of the graft was determined by staining tissue sections with Biebrich
Scarlet.
Application of BMP-2 protein to chick wing buds
Recombinant human BMP-2 protein was supplied by Dr Elizabeth
Wang, Genetics Institute, Cambridge, Massachusetts, at a concentration of 2 mg ml−1 in aqueous solution containing 0.5 M arginine, 0.01
Bmp-2 and Bmp-4 expression in the chick limb bud
A
211
B
Fig. 1. (A) Comparison between the predicted amino acid sequences of chicken, human (Wozney et al., 1988) and mouse (Dickinson et al.,
1990) pre-pro-BMP-4. The complete mouse sequence has not been published. (B) Comparison between the predicted amino acid sequences of
chicken, human (Wozney et al., 1988) and mouse (Dickinson et al., 1990) pre-pro-BMP-2. We have not yet determined the amino-terminal
sequence of chicken pre-pro-BMP-2. Amino acids identical to those found in the chicken sequences are indicated by dots. Gaps introduced to
maintain optimal alignment are indicated by dashes. For each sequence, the predicted site of cleavage to yield mature carboxyl-terminal peptide
is underlined and shown in bold type (Wozney et al., 1988).
M histidine, pH 6.5. Where necessary, this stock was diluted in 0.02
M sodium acetate, pH 5.0, containing 0.2% (w/v) BSA. The protein
was active, as judged by its ability to enhance cartilage formation
when added to micromass cultures of chick wing bud mesenchyme
cells (P. Francis and A. Page, unpublished data). Heparin-acrylic
beads (200-250 µm, H5263, Sigma) were soaked in 2 µl of BMP-2
solution (at concentrations of 2 µg ml−1 to 2 mg ml−1) for at least 1
hour at room temperature. Control beads were soaked in aqueous
solution containing 0.5 M arginine, 0.01 M histidine, pH 6.5. Beads
were placed into slits cut in the anterior margins of stage-20 wing
buds. The embryos were allowed to develop for a further 6 days and
the wings were then stained with alcian green to determine the skeletal
pattern.
RNA preparation and northern analysis
Total RNA was extracted from fresh tissue by the acid guanidium
thiocyanate-phenol-chloroform method (Chomczynski and Sacchi,
1987) and polyadenylated mRNA was selected by two passages
through an oligo(dT) cellulose column. Polyadenylated mRNA (1 µg
per track) was size fractionated on 1% (w/v) agarose Mops-formaldehyde gels and blotted onto Genescreen Plus nylon membranes
(Dupont). Membranes were hybridized with 32P-labelled antisense
RNA probes specific for Bmp-2 or Bmp-4 transcripts, prepared as
described above. Hybridization and washing conditions were as
described previously (Rowe et al., 1991). The sizes of Bmp-2 and
Bmp-4 mRNAs were estimated by reference to RNA size markers
(Boehringer) run in adjacent tracks.
In situ hybridisation to tissue sections
Embryos were fixed in 4% (w/v) paraformaldehyde, processed and
embedded in wax as described by Davidson et al., (1988). Tissue
sections were prepared and in situ hybridization was carried out as
described by Rowe et al. (1991). The final wash after hybridization
with Bmp-2- and Bmp-4-specific probes was performed at 65°C in
0.1× SSC. Hybridization and washing conditions for the Hoxd-13specific probe were as described previously (Ispizúa-Belmonte et al.,
1991).
In situ hybridization to whole-mount preparations
Embryos were processed, hybridized, washed and developed as
described by Wilkinson (1993).
RESULTS
Isolation of chicken Bmp-2 and Bmp-4 cDNA clones
Screening of a chick embryo cDNA library at low stringency
with a mouse Bmp-4 cDNA probe resulted in the isolation of
six clones. These were sequenced and identified as chicken
Bmp-2 and Bmp-4 cDNA clones by comparing the predicted
amino acid sequences of the proteins that they would encode
with those of human BMP-2 and BMP-4 (Wozney et al., 1988).
The chicken Bmp-4 cDNA nucleotide sequence has been
Fig. 2. Northern analysis of Bmp-2 and Bmp4 mRNAs. Each track contained 1 µg of
polyadenylated mRNA isolated from 10-day
(stage-36) chick embryo limbs. Membranes
were hybridized with probes specific for
chicken Bmp-4 transcripts (track 1) or
chicken Bmp-2 transcripts (track 2). The
sizes of the transcripts are indicated in
kilobases.
212
P. H. Francis and others
deposited in the EMBL Nucleotide Sequence Data Library.
There is approximately 84.5% identity between the predicted
amino acid sequences of chicken and human pre-pro-BMP-4
(Fig. 1A). The carboxyl-terminal domain of chicken BMP-4,
which would be predicted to form mature dimers, shares
approximately 95% amino acid identity with that of both
human and mouse BMP-4 (Fig. 1A).
The chicken Bmp-2 cDNA nucleotide sequence has been
deposited in the EMBL Nucleotide Sequence Data Library.
Chicken pre-pro-BMP-2 is predicted to share approximately
80% amino acid identity with both human and mouse BMP-2
in the region of overlap (Fig.
1B). The carboxyl-terminal
domain of chicken BMP-2
shares approximately 97%
amino acid identity with that of
both human and mouse BMP-2
(Fig. 1B).
transcripts were found in two regions, one at the anterior
margin of the limb bud and one at the posterior margin (Figs
3D, 4A). Lower levels of Bmp-4 transcripts were found in the
mesenchyme directly beneath the apical ridge (Figs 3D, 4A).
At stages 25 and 26, mesenchymal expression of Bmp-4 in the
wing bud was found predominantly at the anterior margin
although a small posterior zone of low expression persisted
(Figs 3G, 4D). In the leg bud at these stages, however, Bmp-4
transcripts were still found at approximately equal levels in
both posterior and anterior mesenchyme (data not shown).
Anterior expression in the leg bud was found in two regions
Northern analysis
Chicken Bmp-2 and Bmp-4
mRNAs were analyzed by
hybridization of specific probes
to northern blots of polyadenylated mRNA isolated from 10day chick embryo limbs. The
Bmp-4-specific probe hybridised
to a single transcript of approximately 1.9 kb (Fig. 2, track 1),
while the Bmp-2-specific probe
hybridised to two transcripts, of
approximately 2.6 and 3.2 kb
(Fig. 2, track 2). Genomic
Southern blotting and the characterization of chicken Bmp-2
genomic clones demonstrated
that there is a single Bmp-2 gene
within the chicken genome
(Forbes-Robertson, Francis and
Brickell, data not shown).
Distribution of Bmp-4
transcripts in the
developing chick limb
Bmp-4
transcripts
were
detectable throughout the prewing mesenchyme of stage-16
and -16/17 embryos and in the
overlying ectoderm (Fig. 3A). At
stage 18, transcripts were found
throughout the limb bud mesenchyme and in the apical ectodermal ridge (data not shown).
In the apical ridge, Bmp-4 transcripts remained detectable until
stage 26, as the bud elongated
(Figs 3G, 4A). In the mesenchyme, however, Bmp-4
expression became progressively restricted between stages
19 and 24. High levels of Bmp-4
Fig. 3. Distribution of Bmp-4 (A,D,G,J), Bmp-2 (B,E,H,K) and Hoxd-13 (C,F,I,L) transcripts in
adjacent longitudinal sections of wing buds at stage 16/17 (A,B,C), stage 22 (D,E,F), stage 26 (G,H,I)
and stage 28 (J,K,L). The anterior margin of each bud is uppermost. Autoradiographic signal is visible
as white grains under dark-field illumination. Scale bars, 250 mm.
Bmp-2 and Bmp-4 expression in the chick limb bud
on either side of the indentation that marks the presumptive
knee. At stages 28 and 30, in both wing and leg, Bmp-4 transcripts were found in the necrotic zone between the developing digits and in the mesenchyme surrounding chondrogenic
regions corresponding to the long bones (Figs 3J, 4F). As
development of the digits proceeded, interdigital expression
became confined to mesenchyme immediately surrounding
chondrogenic regions (data not shown).
Distribution of Bmp-2 transcripts in the developing
chick limb
In contrast to Bmp-4, Bmp-2 transcripts could not be detected
in the pre-wing flank mesenchyme at stage 16. Bmp-2 transcripts were first detectable in the pre-wing flank mesenchyme
at stage 16/17 and were restricted to the posterior part of the
mesenchyme (Fig. 3B). Bmp-2 transcripts were also detectable
in the overlying ectoderm at this stage (Fig. 3B). Between
stages 17 and 26, Bmp-2 transcripts were still clearly restricted
to the posterior mesenchyme and were also detectable in the
apical ridge (Figs 3E, 4B,C). Expression of Bmp-2 in the apical
ridge continued until stage 26 (data not shown). As the bud
grew out, between stages 25 and
26, the posterior domain of
expression remained distal, so
that strongest expression was
always found near the tip of the
bud (Figs 3H, 4E). At stage 25
and 26, an additional weak
domain of expression was
detectable in the anterior
proximal part of the limb bud
(Figs 3H, 4E). Between stages
26 and 28, the anterior domain of
expression became progressively stronger and larger, so that
by stage 28, Bmp-2 transcripts
could be detected all along the
anterior margin of the limb
except in the distal hand plate
(Fig. 3K). At stage 28, Bmp-2
transcripts were also found interdigitally and around regions of
cartilage differentiation (Fig.
3K). A similar distribution was
found in the leg at stage 30 (Fig.
4G).
Comparison of the
distribution and time of
appearance of Bmp-2 and
Hoxd-13 transcripts in
wing buds
The expression domain of Bmp2 in limb bud mesenchyme is
similar to that of Hoxd-13, a
gene that responds to a signal
from the polarizing region
(Izpisúa-Belmonte et al., 1991).
Both Bmp-2 and Hoxd-13 are
expressed posteriorly, with
maximum expression towards
213
the distal tip of the limb. In order to compare the expression
domains of the two genes in detail, we examined the distribution of Bmp-2 and Hoxd-13 transcripts in adjacent sections.
Examination of sections through stage-18 wing buds showed
that the domains of Hoxd-13 and Bmp-2 expression overlapped
considerably at the posterior margin of the limb bud (Fig. 5).
The Hoxd-13 domain extended more anteriorly than the Bmp2 domain (Fig. 5A,B) and the Bmp-2 domain extended more
ventrally than the Hoxd-13 domain (Fig. 5C,D). A similar relationship between the two domains was maintained through
subsequent stages of bud outgrowth (Fig. 3E,F and H,I). The
positions of maximal Bmp-2 and Hoxd-13 expression in these
domains did not coincide. The highest levels of Hoxd-13 transcripts were found at the very tip of the limb bud, under the
apical ridge, whilst the highest levels of Bmp-2 transcripts were
found slightly more proximally, just behind the tip (Fig.
3E,F,H,I). By stage 28, both genes were expressed distally,
around developing digits (Fig. 3K,L).
The similarity between the domains of Bmp-2 and Hoxd-13
expression in posterior limb bud mesenchyme suggests that the
two genes might be regulated by the same factors. Alterna-
Fig. 4. Distribution of Bmp-4 (A,D,F) and Bmp-2 (B,C,E,G) transcripts in wholemount preparations of
wing buds at stage 20 (B), stage 21 (A), stage 22 (C) and stage 25 (D,E) and of legs at stage 30 (F,G).
Specimens are photographed from their dorsal aspect, with anterior margins uppermost. Regions to
which probes have hybridized are stained purple. Scale bars, 250 µm.
214
P. H. Francis and others
tively, BMP-2 could activate Hoxd-13 gene expression or vice
versa. To address this question, we determined the order in
which Hoxd-13 and Bmp-2 transcripts were first detectable in
the wing bud by examining adjacent sections of wing buds
from stage-16, -16/17 and -17 embryos. Bmp-2 transcripts were
not found in pre-wing mesenchyme at stage 16, but were
present in all three stage-16/17 embryos examined (Fig. 3B).
In contrast, Hoxd-13 transcripts were not detectable in any of
the three stage-16/17 embryos examined (Fig. 3C). Hoxd-13
transcripts were first detectable in a stage-17 embryo, in the
same region as Bmp-2 transcripts (data not shown).
the bud, Bmp-4 transcripts were also found around the bead
(Fig. 6C). Unlike the ectopic Bmp-2 domain, which was distal
to the bead, Bmp-4 transcripts were present both proximal and
distal to the bead (Fig. 6C). The anterior domain of Bmp-4
expression overlapped with the ectopic anterior domain of
Bmp-2 expression, with the Bmp-4 domain extending more
proximally. Since there is an anterior domain of Bmp-4
expression in normal limb buds (Figs 6C, 3D, 4A,D), it was
not clear whether the levels of Bmp-4 transcripts had changed
in response to retinoic acid treatment.
Activation of Bmp-2 and Hoxd-13 gene expression
Activation of Bmp-2 gene expression by retinoic
by polarizing region grafts
acid
The activation of Bmp-2 expression by retinoic acid shows that
The expression domain of Bmp-2 in the posterior mesenchyme
this gene is part of the polarizing region signalling pathway.
of the limb bud has a similar location to that of the polarizing
However, since it has been suggested that retinoic acid acts by
region (Maccabe et al., 1973; Honig and Summerbell, 1985).
inducing a new polarizing region, it is not clear whether BMPThis suggests that BMP-2 might be involved in the polarizing
2 is itself a polarizing molecule or whether it acts further downregion signalling pathway. Retinoic acid can mimic the action
stream. To investigate these questions we examined Bmp-2
of the polarizing region. When it is applied to the anterior
expression in wing buds following grafting of a polarizing
margin of a limb bud, expression of the HoxD gene complex is
region to the anterior margin. Such grafts induce digit dupliactivated in anterior cells and anterior cells give rise to posterior
cations (Tickle et al., 1976) and activate expression of genes
structures (Izpisúa-Belmonte et al., 1991). We examined
of the HoxD complex anteriorly (Ispizúa-Belmonte et al.,
whether application of retinoic acid to the anterior margin of a
1992). However, they do not induce polarizing activity in
limb bud could activate Bmp-2 expression anteriorly and, if so,
adjacent host mesenchymal cells (Smith, 1979). Thus, if BMPhow this activation compared with that of Hoxd-13.
2 is the polarizing signal, Bmp-2 expression should be mainTreatment with retinoic acid resulted in the appearance of
tained in the graft but not activated in adjacent host mesectopic domains of both Bmp-2 and Hoxd-13 transcripts in the
enchyme cells. We used mouse, rather than chick, polarizing
anterior mesenchyme of the bud (Fig. 6A,B,D,E; Table 1). In
region tissue so that species-specific probes could be used to
both cases, ectopic domains were
detectable 24 and 48 hours after
treatment but not at 16 or 20 hours.
The ectopic domains were located
distal to the retinoic-acid-soaked
bead, beneath the apical ridge at the
tip of the bud (Fig. 6A,B,D,E). At 24
hours, the ectopic Hoxd-13 domain
was located distally within the ectopic
Bmp-2 domain (Fig. 6A,B). By 48
hours, the ectopic Bmp-2 and Hoxd-13
domains were larger and partially
overlapping, with the Hoxd-13
domain extending more distally and
the Bmp-2 domain extending more
proximally, closer to the bead (Fig.
6D,E). The ectopic Hoxd-13 domain
also extended further towards the
posterior part of the bud than the
ectopic Bmp-2 domain (Fig. 6D,E).
The spatial relationship between the
overlapping ectopic Bmp-2 and Hoxd13 domains was similar to that
between the normal posterior Bmp-2
and Hoxd-13 domains. The relationship was maintained when retinoic
Fig. 5. Distribution of Bmp-2 (A,C) and Hoxd-13 (B,D) transcripts in transverse sections of wing
acid was applied to the posterior buds at stage 18. Sections in A and B are adjacent and are 210 µm more anterior than the sections
margin of the limb bud (Fig. 6F,G), in C and D, which are also adjacent to each other. The dorsal side of each bud is uppermost.
with the posterior Bmp-2 and Hoxd-13 Arrows indicate the apical ridge. Autoradiographic signal is visible as white grains under darkdomains both being shifted anteriorly. field illumination. Hoxd-13 transcripts, but not Bmp-2 transcripts, were detectable in the more
When a retinoic-acid-soaked bead anterior sections (A,B). In the more posterior sections (C,D), the Bmp-2 domain extended more
was placed at the anterior margin of ventrally than the Hoxd-13 domain. Scale bars, 50 µm.
Bmp-2 and Bmp-4 expression in the chick limb bud
distinguish mouse Bmp-2 transcripts in the graft from ectopic
chicken Bmp-2 transcripts in the host.
Mouse polarizing region grafts continued to express Bmp-2
after grafting to the anterior margin of stage-19/20 chick
embryo wing buds (Fig. 7C,F; Table 2). In addition, the polarizing region grafts induced anterior host mesenchyme cells to
express Bmp-2 (Fig. 7A,D; Table 2). Chicken Bmp-2 transcripts were found in cells adjacent to the graft, beneath the
apical ridge (Fig. 7A,D). We compared the timing of this
induction with that of Hoxd-13 by hybridizing adjacent
sections with probes for Bmp-2 and Hoxd-13 transcripts.
Ectopic expression of both genes was detectable by 30 hours
after grafting, at which time the ectopic domain of Hoxd-13
expression was restricted distally within that of Bmp-2
expression (Fig. 7A,B; Table 2). By 42 hours after grafting, the
Hoxd-13 and Bmp-2 domains were larger than at 30 hours and
partially overlapped (Fig. 7D,E; Table 2). The Bmp-2 domain
extended more proximally around the graft while the Hoxd-13
domain extended more distally. In addition, the Hoxd-13
domain extended further towards the posterior part of the bud
(Fig. 7D,E). This sequence of events is similar to that seen
following local application of retinoic acid to the anterior
margin of the bud (Fig. 6).
Application of BMP-2 protein to chick wing buds
The effects of polarizing region grafts on Bmp-2 expression
suggest that BMP-2 is not itself
a polarizing molecule. To test
this directly, beads were soaked
in recombinant human BMP-2
protein at concentrations of 0.2
mg ml−1 (n=5), 1 mg ml−1 (n=2)
or 2 mg ml−1 (n=4) and were
placed at the anterior margin of
chick wing buds. In all 11 cases
the pattern of digits that
developed was normal and no
additional digits formed. This
confirms that BMP-2 protein
alone does not have polarizing
activity. However, application of
BMP-2 protein to the anterior
margin of wing buds did result in
defects in the shoulder girdle
(11/11 cases) and radius (5/11
cases). Beads soaked in buffer
alone (n=4) or in recombinant
human BMP-2 protein at very
low concentrations (2 µg ml−1,
n=1; 20 µg ml−1, n=2) gave no
shoulder or digit abnormalities.
These data indicate that active
BMP-2 protein was released
from the grafted beads. Thus,
although BMP-2 did not polarize
the wing, it did affect the development of proximal structures.
DISCUSSION
We isolated and characterized
215
chicken Bmp-2 and Bmp-4 cDNA clones and determined the
distribution of Bmp-2 and Bmp-4 transcripts in the developing
chick limb. Both genes are expressed in the apical ectodermal
ridge and in discrete, and hitherto unsuspected, mesenchymal
domains. Whereas Bmp-4 is expressed both anteriorly and posteriorly in the mesenchyme with some weak expression
beneath the apical ridge, Bmp-2 expression is confined to the
posterior mesenchyme during early limb development. This
posterior domain of Bmp-2 expression colocalizes with the
polarizing region and overlaps with the domain of Hoxd-13
expression. Application of retinoic acid or grafting of a mouse
polarizing region to the anterior margin of chick wing buds
activates ectopic expression of Bmp-2 in anterior cells.
The data presented here identify Bmp-2 as a component of
the polarizing region signalling pathway. This is the first report
implicating a growth factor in a-p patterning in the limb bud.
The Bmp-2 gene is expressed by mesenchyme cells in the
posterior part of the limb bud, where the polarizing region has
been mapped (Maccabe et al., 1973; Honig and Summerbell,
1985). Grafts of polarizing region cells, which respecify cell
position, contain Bmp-2 transcripts. In addition, Bmp-2
expression is activated in anterior mesenchyme following
either local application of retinoic acid or grafting of a mouse
polarizing region to the anterior margin of the bud. Finally, the
domain of Bmp-2 expression overlaps substantially with that
of Hoxd-13.
Fig. 6. Distribution of Bmp-2
(A,D,F), Hoxd-13 (B,E,G) and
Bmp-4 (C) transcripts in
adjacent sections of retinoicacid-treated wing buds.
(A-C) A bead soaked in 1 mg
ml−1 all-trans-retinoic acid was
placed at the anterior margin of
the right-hand bud 24 hours
before fixation. The left-hand
bud was untreated. (D,E) A
bead soaked in 1 mg ml−1 alltrans-retinoic acid was placed
at the anterior margin of the
bud 48 hours before fixation.
(F,G) A bead soaked in 0.1 mg
ml−1 all-trans-retinoic acid was
placed at the posterior margin
of the bud 24 hours before
fixation. Positions of beads are
indicated with open
arrowheads. Ectopic domains
of Bmp-2 and Hoxd-13
transcripts in anterior mesenchyme of treated buds are indicated with closed arrowheads.
Autoradiographic signal is visible as white grains under dark-field illumination. Scale bars, 250 µm.
216
P. H. Francis and others
How might BMP-2 operate in the polarizing region sigHoxd-13 domain becomes slightly larger than the Bmp-2
nalling pathway? Retinoic acid is a good candidate for the
domain. In normal buds, Bmp-2 transcripts are detectable
polarizing region signal. However, an alternative view is that
before Hoxd-13 transcripts. This also suggests that BMP-2
locally applied retinoic acid induces a new polarizing region
could be involved in activating Hoxd-13 in normal limb develthat then signals by means of another, unidentified, molecule
opment.
(Wanek et al., 1991). BMP-2 could be such a molecule.
However, anterior cells exposed to retinoic acid acquire some
Table 1. Activation of ectopic Bmp-2 and Hoxd-13 gene
polarizing region activity after 15 hours (Wanek et al., 1991),
expression following application of retinoic acid to the
but activation of Bmp-2 expression in anterior mesenchyme is
anterior margin of the wing bud
not detectable until 24 hours after retinoic acid treatment. In
Ectopic domain of
addition, Bmp-2 expression is activated by polarizing region
Retinoic acid
Time after
transcripts
concentration
treatment
Number of
grafts, but Smith (1979) showed that polarizing region grafts
(mg ml−1)
(hours)*
cases
Bmp-2
Hoxd-13
do not appear to induce polarizing activity in adjacent host
0.1
16
1
−
n.d.
tissue. Moreover, Bmp-2 expression is not detectable in the
20
4
−
−
pre-wing mesenchyme of stage-16 embryos, which has polar24
2
+
+†
izing activity (Maccabe et al., 1973; Honig and Summerbell,
48
2
+
+
1985; Hornbruch and Wolpert, 1991). It therefore appears that
1
16
1
−
n.d.
BMP-2 may be a downstream component of the polarizing
20
2
−
−
24
2
+
+
region signalling pathway rather than being the polarizing
48
2
+
+
region signal itself. As would be predicted, application of
recombinant BMP-2 protein to the anterior margin of chick
n.d., not done
wing buds does not induce digit duplications. However, appli*At the 16- and 20-hour time points, every second section through the
thickness of the bud was probed to ensure that ectopic domains would not be
cation of the protein does result in shoulder girdle abnormalimissed.
ties. Interestingly, shoulder girdle abnormalities also result
†The ectopic Hoxd-13 domain was very small.
when either retinoic acid or polarizing region grafts are placed
at the anterior margin of the wing bud (Oliver et al., 1990).
Genes of the HoxD complex
are also activated in response to
either retinoic acid or polarizing
region grafts. This suggests that
the Bmp-2 and HoxD complex
genes could be connected components of the response to
signals that re-specify position
in the limb bud. Indeed, the
temporal and spatial relationship
between the activation of Bmp-2
and Hoxd-13 expression in
manipulated buds is most
striking. The two genes are
activated in anterior mesenchyme, over a similar time
period, in response to retinoic
acid or polarizing region grafts.
Following both types of manipulation, Hoxd-13 transcripts are
first detected in a small domain
located within the Bmp-2
domain, and then spread to form
a domain that overlaps with and
spreads beyond the Bmp-2
domain. This suggests that cells
might activate Hoxd-13 shortly
after they have activated Bmp-2
and raises the possibility that
BMP-2 is involved in the activation of Hoxd-13. Limited Fig. 7. Distribution of chicken Bmp-2 (A,D), chicken Hoxd-13 (B,E) and mouse Bmp-2 (C,F) transcripts
diffusion of BMP-2, such as in adjacent sections of wing buds 30 hours (A,B,C) or 42 hours (D,E,F) after a mouse polarizing region
occurs for Drosophila DPP was grafted to the anterior margin of the bud. Autoradiographic signal is visible as white grains under
(Panganiban et al., 1990), could dark-field illumination. Ectopic domains of Bmp-2 and Hoxd-13 transcripts in anterior mesenchyme of
account for the finding that the treated buds are indicated with arrows. Scale bars, 250 µm.
Bmp-2 and Bmp-4 expression in the chick limb bud
Table 2. Activation of ectopic Bmp-2 and Hoxd-13 gene
expression following grafting of mouse polarizing region
to the anterior margin of the wing bud
Time after
grafting
(hours)
30
42
Ectopic domain of
transcripts
Number
of cases
Mouse Bmp-2
transcripts
in graft
Bmp-2
Hoxd-13
2
2
+
+
+
+
+*
+†
*In one embryo, the ectopic Hoxd-13 domain was very small compared
with the ectopic Bmp-2 domain. In the other embryo, the ectopic Hoxd-13
domain was confined distally within the ectopic Bmp-2 domain.
†In both embryos, the Hoxd-13 domain extended more distally and
posteriorly than the Bmp-2 domain. In one embryo, the Bmp-2 domain
extended more proximally than the Hoxd-13 domain.
The expression of HoxD complex genes appears to be
related spatially to the maintenance of the apical ridge in the
wing bud (Izpisúa-Belmonte et al., 1992a). Therefore, another
attractive possibility is that BMP-2 is part of the mechanism
that links patterning across the a-p axis with bud outgrowth.
When anterior cells are re-specified to form posterior structures
in response to retinoic acid or a polarizing region graft, the
apical ridge is maintained anteriorly by a signal from the mesenchyme (Tickle et al., 1989; Oliver et al., 1990). If BMP-2 is
such a signal from mesenchyme to epithelium, it would
resemble DPP, which is synthesized by mesoderm and
activates expression of the homeotic gene, labial, in neighbouring endoderm (Panganiban et al., 1990). In mouse limb
buds, Fgf-4 gene expression is restricted to the posterior part
of the apical ridge (Niswander and Martin, 1992). It is possible
that BMP-2, synthesized by posterior mesenchymal cells
beneath the apical ridge, could regulate Fgf-4 gene expression.
BMP-2 could function in the polarizing region signalling
pathway in concert with BMP-4. The Bmp-2 domain overlaps
with that of Bmp-4 in the posterior mesenchyme of the normal
limb bud. In the anterior mesenchyme of manipulated buds, the
ectopic Bmp-2 domain overlaps with a Bmp-4 domain.
Members of the TGF-β family, including BMPs, have been
shown to form both homodimers and heterodimers (Sampath
et al., 1990). BMP-2 and BMP-4 might therefore act as homodimers and/or heterodimers in the limb bud. This introduces
further complexity because homodimers and heterodimers can
have opposing actions, as in the case of the inhibins, which are
also members of the TGF-β family (Hsueh et al., 1987).
However, the distribution of Bmp-2 and Bmp-4 transcripts may
not accurately reflect the distribution of active protein, since
there is the possibility of further control at the levels of translation, secretion and proteolytic activation after secretion
(reviewed by Wozney, 1989).
The distribution of Bmp-2 and Bmp-4 transcripts in the
developing limb suggests that BMP-2 and BMP-4 could have
at least two other distinct functions in limb development. Both
genes are expressed in the apical ridge. Signals from the apical
ridge are responsible for maintaining a zone of undifferentiated mesenchymal cells at the tip of the limb bud and BMP-2
and BMP-4 could be involved in this, as previously suggested
in the mouse (Lyons et al., 1990; Jones et al., 1991; Niswander
and Martin, 1993). Finally, both genes are expressed in chondrogenic regions in the later bud, suggesting a role in cartilage
differentiation and morphogenesis (Lyons et al., 1989).
217
It is very interesting that members of the TGF-β family,
including BMPs, are emerging as common components in a
range of signalling systems in developing embryos. For
example, in Drosophila, DPP appears to specify position along
the dorsoventral axis of the embryo (Ferguson and Anderson,
1992), whilst in Xenopus, the activins, BMP-4 and Vg1 are
thought to act together to specify dorsal and ventral mesoderm
(Smith et al., 1989; Dale et al., 1992; Jones et al., 1992;
Thomsen and Melton, 1993). Here we suggest that BMP-2 is
involved in the polarizing region signalling pathway that
specifies position across the a-p axis of the vetebrate limb bud.
This work was funded by the Agriculture and Food Research
Council. We are grateful to Brigid Hogan for the gift of mouse Bmp2 and Bmp-4 cDNA clones, to Denis Duboule and Juan-Carlos
Izpisúa-Belmonte for the gift of the chicken Hoxd-13 probe, to
Elizabeth Wang for the gift of recombinant BMP-2 protein, to David
Darling for the gift of chick RNA and to Anne Crawley for help with
tissue preparation and photography.
REFERENCES
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt.
Biochem. 162, 156-159.
Dale, L., Howes, G., Price, B. M. J. and Smith, J. C. (1992). Bone
morphogenetic protein 4: a ventralizing factor in early Xenopus
development. Development 115, 573-585.
Davidson, D., Graham, E., Sime, C. and Hill, R. (1988). A gene with
sequence similarity to Drosophila engrailed is expressed during the
development of the neural tube and vertebrate in the mouse. Development
104, 305-316.
Devlin, C. J., Brickell, P. M., Taylor, E. R., Hornbruch, A., Craig, R. K. and
Wolpert, L. (1988). In situ hybridisation reveals different spatial localisation
of mRNAs for type I and type II collagen in the chick limb bud. Development
103, 111-118.
Dickinson, M. E., Kobrin, M. S., Silan, C. M., Kingsley, D. M., Justice, M.
J., Miller, D. A., Ceci, J. D., Lock, L. F., Lee, A., Buchberg, A. M.,
Siracusa, L. D., Lyons, K. M., Derynck, R., Hogan, B. L. M., Copeland,
N. G. and Jenkins, N. A. (1990). Chromosomal localization of seven
members of the murine TGF-β superfamily suggests close linkage to several
morphogenetic mutant loci. Genomics 6, 505-520.
Feinberg, A. P. and Vogelstein, B. (1984). A technique for radiolabelling
DNA restriction endonuclease fragments to a high specific activity. Analyt.
Biochem. 137, 266-267.
Ferguson, E. L. and Anderson, K. V. (1992). decapentaplegic acts as a
morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell
71, 451-461.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the
development of the chick embryo. J. Morph. 88, 49-92.
Hoffman, F. M. (1991). Transforming growth factor-β-related genes in
Drosophila and vertebrate development. Current Opinion Cell Biol. 3, 947952.
Honig, L. S. and Summerbell, D. (1985). Maps of strength of positional
signalling activity in the developing chick wing bud. J. Embryol. Exp.
Morph. 87, 163-174.
Hornbruch, A. and Wolpert, L. (1991). The spatial and temporal distribution
of polarizing activity in the flank of the pre-limb-bud stages in the chick
embryo. Development 111, 725-731.
Hsueh, A. J. W., Dahl, K. D., Vaughan, J., Tucker, E., Rivier, J., Bardin, C.
W. and Vale, W. (1987). Heterodimers and homodimers of inhibin subunits
have different paracrine action in the modulation of luteinizing hormonestimulated androgen biosynthesis. Proc. natn. Acad. Sci. USA 84, 50825086.
Izpisúa-Belmonte, J.-C., Tickle, C., Dollé, P., Wolpert, L. and Duboule, D.
(1991). Expression of the homeobox Hox-4 genes and the specification of
position in chick wing development. Nature 350, 585-589.
Izpisúa-Belmonte, J.-C., Brown, J. M., Duboule, D. and Tickle, C. (1992a).
Expression of Hox-4 genes in the chick wing links pattern formation to the
218
P. H. Francis and others
epithelial-mesenchymal interactions that mediate growth. EMBO J. 11,
1451-1457.
Izpisúa-Belmonte. J.-C., Brown, J. M., Crawley, A., Duboule, D. and
Tickle, C. (1992b). Hox-4 gene expression in mouse/chicken heterospecific
grafts of signalling regions to limb buds reveals similarities in patterning
mechanisms. Development 115, 553-560.
Jones, C. M., Lyons, K. M. and Hogan, B. L. M. (1991). Involvement of Bone
Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and
neurogenesis in the mouse. Development 111, 531-542.
Jones, C. M., Lyons, K. M. Lapan, P. M., Wright, C. V. E. and Hogan, B. L.
M. (1992). DVR-4 (Bone Morphogenetic Protein-4) as a posteriorventralizing factor in Xenopus mesoderm induction. Development 115, 639647.
Lyons, K. M., Pelton, R. W. and Hogan, B. L. M. (1989). Patterns of
expression of murine Vgr-1 and BMP-2a RNA suggest that transforming
growth factor-β-like genes coordinately regulate aspects of embryonic
development. Genes Dev. 3, 1657-1668.
Lyons, K. M., Pelton, R. W. and Hogan, B. L. M. (1990). Organogenesis and
pattern formation in the mouse: RNA distribution patterns suggest a role for
Bone Morphogenetic Protein-2A (BMP2A). Development 109, 833-844.
Maccabe, A. B., Gasseling, M. T. and Saunders, J. W. (1973).
Spatiotemporal distribution of mechanisms that control outgrowth and
anteroposterior polarization of the limb bud in the chick embryo. Mech.
Ageing Develop. 2, 1-12.
Niswander, L. and Martin, G. R. (1992). Fgf-4 expression during
gastrulation, myogenesis, limb and tooth development in the mouse.
Development 114, 755-768.
Niswander, L. and Martin, G. R. (1993). FGF 4 and BMP-2 have opposite
effects on limb growth. Nature 361, 68-71.
Nohno, T., Noji, S., Koyama, E., Ohyama, K., Myokai, F., Kuroiwa, A.,
Saito, T. and Taniguchi, S. (1991). Involvement of the Chox-4 chicken
homeobox genes in determination of anteroposterior axial polarity during
limb development. Cell 64, 1197-1205.
Oliver, G., De Robertis, E. M., Wolpert, L. and Tickle, C. (1990).
Expression of a homeobox gene in the chick wing bud following application
of retinoic acid and grafts of polarizing region tissue. EMBO J. 9, 3093-3101.
Panganiban, G. E. F., Reuter, R., Scott, M. P. and Hoffman, F. M. (1990). A
Drosophila growth factor homologue, decapentaplegic, regulates homeotic
gene expression within and across germ layers during midgut
morphogenesis. Development 110, 1041-1050.
Rosen, V. and Thies, R. S. (1992). The BMP proteins in bone formation and
repair. Trends Genet. 8, 97-102.
Rowe, A., Richman, J. M. and Brickell, P. M. (1991). Retinoic acid treatment
alters the distribution of retinoic acid receptor-β transcripts in the embryonic
chick face. Development 111, 1007-1016.
Sampath, T. K., Coughlin, J. E., Whetstone, R. M., Banach, D., Corbett, C.,
Ridge, R. J., Özkaynak, E., Oppermann, H. and Rueger, D. C. (1990).
Bovine osteogenic protein is composed of OP-1 and BMP-2A, two members
of the transforming growth factor-β superfamily. J. Biol. Chem. 265, 1319813205.
Smith, J. C. (1979). Evidence for a positional memory in the development of
the chick wing bud. J. Embryol. Exp. Morph. 52, 105-113.
Smith, J. C., Cooke, J., Green, J. B. A., Howes, G. and Symes, K. (1989).
Inducing factors and the control of mesodermal pattern in Xenopus laevis.
Development 107 Supplement, 149-160.
Szabo, G. (1955). A modification of the technique of ‘skin splitting’ with
trypsin. J. Path. Bact. 70, 545.
Tabin, C.J. (1991). Retinoids, homeoboxes, and growth factors: toward
molecular models for limb development. Cell 66, 199-217.
Thomsen, G.H. and Melton, D.A. (1993). Processed Vg1 protein is an axial
mesoderm inducer in Xenopus. Cell 74, 433-441.
Tickle, C. and Brickell, P. M. (1991). Retinoic acid and limb development.
Sem. Dev. Biol. 2, 189-197.
Tickle, C. A., Summerbell, D. and Wolpert, L. (1975). Positional signalling
and specification of digits in chick limb morphogenesis. Nature 254, 199-202.
Tickle, C., Shellswell, G., Crawley, A. and Wolpert, L. (1976). Positional
signalling by mouse limb polarising region in the chick wing bud. Nature
259, 396-397.
Tickle, C., Alberts, B., Lee, J. and Wolpert, L. (1982). Local application of
retinoic acid to the limb bud mimics the action of the polarising region.
Nature 296, 564-565.
Tickle, C., Lee, J. and Eichele, G. (1985). A quantitative analysis of the effect
of all-trans-retinoic acid on the pattern of chick wing development. Dev. Biol.
197, 27-36.
Tickle, C., Crawley, A. and Farrar, J. (1989). Retinoic acid application to
chick wing buds leads to a dose-dependent reorganization of the apical
ectodermal ridge that is mediated by the mesenchyme. Development 106,
691-705.
Wanek, N., Gardiner, D. M., Muneoka, K. and Bryant, S. V. (1991).
Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing
bud. Nature 350, 81-83.
Wilkinson, D. G. (1993). Whole mount in situ hybridization of vertebrate
embryos. In In Situ Hybridization (ed. D.G. Wilkinson), pp. 75-83. Oxford:
Oxford University Press.
Wozney, J. M. (1989). Bone morphogenetic proteins. Prog. Growth Factor
Res. 1, 267-280.
Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J.,
Kriz, R. W., Hewick, R. M. and Wang, E. A. (1988). Novel regulators of
bone formation: molecular clones and activities. Science 242, 1528-1534.
(Accepted 15 October 1993)