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DEVELOPMENTAL DYNAMICS 207:439-449 (1996)
Transcripts for Two Members of the Transforming Growth
Factor-p Superfamily BMP-3 and BMP-7 are Expressed in
Developing Rat Embryos
HIROSHI TAKAHASHI AND TOHRU IKEDA
Department of Pathology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173 (H.T.), and Department
Oral Pathology, School of Dentistry, Showa University, Shinagawa-ku, Tokyo 142 ( T J J , Japan
ABSTRACT
Bone morphogenetic proteind
(BMP-3) and BMP-7 are members of the transforming growth factor p superfamily that have
been implicated in the formation of cartilage and
bone. Using in situ hybridization, we localized
mRNAs for BMP-3 and BMP-7 during organogenesis in rats. Both mRNAs were expressed in a variety of cells, in particular, in the developing hair
follicle, tooth, kidney, and lung tissues, in which
reciprocal epithelial-mesenchymal interactions
are essential. In some tissues, the distribution of
BMP-3 and BMP-7 mRNAs overlapped. In other
tissues, the patterns of expression were quite different. Moreover, the site of expression of the
transcripts changed from one cell type to another
during organogenesis. These results suggest that
BMP-3 and BMP-7 play important roles in organogenesis and that the differential patterns of
their expression might reflect their distinct roles
in embryogenesis. o 1996 Wiley-Liss, Inc.
Key words: Bone morphogenetic protein-3, Bone
morphogenetic protein-7, Epithelialmesenchymal interaction, In situ hybridization
INTRODUCTION
The members of the transforming growth factor beta
(TGF-P) superfamily are polypeptide signaling molecules that play important roles in growth and development (for review, see Massague, 1990). Based on sequence homology, the members of the TGF-P
superfamily have been subdivided into several subfamilies: the TGF-p subfamily; the activin subfamily; the
DVR group and others. The DVR group has been further subdivided into the dpp subfamily (bone morphogenetic protein-2 (BMP8), BMP-4, dpp), the 6 0 8 subfamily (BMP-5, BMP-6IVgr-1, BMP-7/0P-1, BMP-8/
OP-2, and 60A) and other molecules (BMPS, Vg-1,
GDF-1, Vgr-2IGDF-3, GDF-9, nodal, ~ O F S U (for
~ ) review, see Kingsley, 1994).
BMPs were originally identified in extracts of bone
on the basis of their ability to induce ectopic bone formation (Wozney et al., 1988). In addition to their presence in developing bone and cartilage, BMP-2, BMP-4
and BMP-6 are expressed in a variety of cells in various
0 1996 WILEY-LISS, INC.
of
tissues, in particular, in the developing organs in
which reciprocal epithelial-mesenchymal interactions
are essential (Lyons et al., 1989a,b, 1990; Jones et al.,
1991).
Recent studies indicate that BMPs play important
roles in various stages of embryogenesis (for review,
see Lyons et al., 1991). BMP-4 is involved in the mesodermal induction as a posterior ventralizing signal
(Jones et al., 19921, and it also mediates epithelialmesenchymal interactions during early tooth development (Vainio et al., 1993). BMP-2 may be an epithelial
signal in early limb development (Niswander and Martin, 1993). Mutant short-eared mice have a broad range
of skeletal defects which include alterations in the size,
shape, and number of many different skeletal elements, as well as marked reduction in the size of the
external ear. A genetic study shows that the mouse
short ear locus is associated with defects in BMP-5
(Kingsley et al., 1992). Therefore, each BMP molecule
seems to have distinct roles in development.
Previous studies have shown that mRNAs for
BMP-2, BMP-4, and BMP-6 are expressed during embryogenesis in spatially and temporally specialized
patterns, suggesting that coordinated expression of
BMPs is needed for the regulation of organogenesis
(Lyons et al., 1989a,b, 1990; Jones et al., 1991). However, little is known about the role of BMP-3 and
BMP-7 in embryonic development.
In order better to understand the precise role of each
members of the TGFP superfamily in cell-cell interactions and embryonic organogenesis in vivo, it is necessary to define the tissues and cell types that express the
different proteins.
In the present study, we have examined the patterns
of expression of BMP-3 and BMP-7 mRNA in the developing rat tissues by in situ hybridization. We discuss these patterns of expression in a comparison with
those of BMP-2, -4, and -6 mRNAs.
Received August 22, 1995; accepted July 22, 1996.
Address reprint requestdcorrespondence to Hiroshi Takahashi’s
current address: Laboratory of Histochemistry, Mitsubishi Kasei Institute of Life Sciences, 11Minamiooya, Machida-shi, Tokyo-194, Japan.
440
TAKAHASHI AND IKEDA
RESULTS
Expression of mRNAs for BMP-3 and BMP-7 in
the Embryos
Figure 1 showed that two BMP-3 transcripts of approximately 6.4 kb and 2.9 kb, and two BMP-7 transcripts of approximately 4.6 kb and 2.7 kb, were detected in Northern blot analysis using embryonic day
(El15rat embryos, The existence of two BMP-7 transcripts was consistent with the mouse data reported
previously (Ozkaynak et al., 1991). These data also
showed that each probe for BMPs specifically recognized each molecule. To examine the localization of
BMP-3 and BMP-7 mRNA in detail, in situ hybridization was performed. Before presenting the pattern of
expression of these molecules in each organ, representative pictures indicating differences of expression pattern of these BMPs were shown in Figure 2. In E l 8
embryos, strong expression of BMP-3 mRNA was detected in bone and cartilage, developing tooth, nasal
epithelium, lung, kidney and small intestine (Fig. 2A).
In contrast, the strong signals of BMP-7 transcripts
were observed in the skin, nasal epithelium, hair follicle, developing tooth, heart, adrenal gland, kidney,
esophagus, meningeal tissue, inner ear, and weaker in
the developing intestine, lung and bone and cartilage
(Fig. 2C).Although the distribution of these two BMPs
overlapped in some tissues such as kidney, lung, and
developing tooth, cell types expressing each BMPs
were different. For instance, expression of BMP-3
showed tubular pattern as BMP-3 was expressed in the
developing tubules in the kidney (Fig. 2A, arrowhead).
On the other hand, expression of BMP-7 showed spotty
pattern as BMP-7 was expressed in the developing
glomeruli (Fig. 2C, arrowhead). No hybridization signal was seen in the sections hybridized with sense
probes for BMP-3 or BMP-7 (Fig. 2B and D).
Expression of mRNAs for BMP-3 and BMP-7 in
Skin and Hair Follicles
In rats from embryonic day 11( E l l ) to postnatal day
5 (P5),BMP-3 mRNA was not detected in developing
skin. During development of hair follicle, low-level of
expression of the mRNA was detected in the mesenchyma1 condensation adjacent to the epidermis and in the
outer and inner root sheath at E l 5 (data not shown). At
E18, BMP-3 mRNA was localized in the dermal sheath
(Fig. 3A). This pattern of expression of BMP-3 mRNA
continued a t least until P5, although the intensity of
the signal declined (Fig. 3B). Expression of BMP-3
mRNA was also detected in the interdigital mesoderm
in early limb buds (data not shown).
At E l l , BMP-7 mRNA was detected in the somites
and epithelium adjacent to the somites. At E13, it was
weakly expressed in the oral epithelium (data not
shown). At E l 5 and El$,it was strongly expressed in
basal cell layers (Fig. 3C). No or little signal was detected in the suprabasal cells in the epidermis. Expression of BMP-7 mRNA declined postnatally (Fig. 3D).
Fig. 1. Northern blot analysis of rat BMP-3 and BMP-7 RNA blots
were prepared using 1 pg of poly(A)+RNA isolated from E l 5 rat embryos. Two transcripts of 6.4 kb and 2.9 kb in rat BMP-3 and two transcripts of 4.6 k and 2.7 kb in rat BMP-7 are observed. Smaller transcripts
in each lane are more abundant than larger transcripts.
During development of hair follicle, BMP-7 mRNA was
not detected prior to E15. At El$, higher-level expression of BMP-7 mRNA was detected in the inner and
outer root sheaths and lower level of expression was
detected in the dermal sheath (Fig. 3C). Later in hair
follicle development, BMP-7 mRNA was detected in
the dermal papilla and in the outer root sheath, with
the highest levels being found in the lower half of each
follicle (Fig. 3D).
Expression of BMP-3 and BMP-7 mRNAs in the
Developing Tooth
No expression of BMP-3 or BMP-7 mRNA was detected at the tooth bud stage. At the cap stage of tooth
development, high level of expression of BMP-3 mRNA
was detected in the dental follicle, in particular, adjacent to the outer enamel epithelium (Fig. 3E). This
pattern of expression of BMP-3 mRNA persisted
through the early and late bell stage of tooth development (Fig. 3F).
Strong signals due to BMP-7 mRNAs were seen in
the inner enamel epithelium with relatively weak signals in the dental papilla below the enamel epithelium
during the cap stage and the bell stage of tooth development (Fig. 3G). At the bell stage, expression of
mRNA was detected in the inner enamel epithelium,
stratum intermedium, and odontoblasts (Fig. 3H).
Expression of BMP-3 and BMP-7 mRNAs in the
Nasal Epithelium
At E18, expression of BMP-3 mRNA was detected in
the mesenchyme adjacent to the folded nasal epithe-
BMP-3 AND BMP-7 IN RAT EMBRYOS
Fig. 2. Expression of BMP-3 and BMP-7 mRNAs in €18 rat embryos.
Parasagittal embryonic sections hybridized with BMP-3 antisense probe
(A) or sense probe (B). BMP-3 transcripts are localized in the developing
bone and cartilage, tooth, nasal epithelium, skin and hair follicle, lung,
kidney, and small intestine (A). Parasagittal embryonic sections hybridized with BMP-7 antisense probe (C) or sense probe (D). Strong signals
441
of BMP-7 mRNA can be detected in the skin, nasal epithelium, hair fol-
licle, developing tooth, heart, adrenal gland, kidney, esophagus,
meningeal tissue, inner ear, and weaker ones in the developing intestine,
lung, and bone and cartilage (C). No obvious signal is recognized in B
and D. Expressions of BMP-3 and BMP-7 in the kidney are indicated
(A$, arrowheads).
442
TAKAHASHI AND IKEDA
Fig. 3. Expression of BMP-3 and BMP-7 rnRNAs in the developing
skin and hair follicle, and tooth. Skin and hair follicle hybridized with
BMP-3 probe at E l 8 (A) and P5 (B). A: BMP-3 transcripts are localized
in the dermal sheath (arrow) and in the mesenchyme adjacent to the
epithelium (arrowhead). No hybridization signal can be seen in the epidermis. B: BMP-3 mRNA is located in the dermal sheath (arrow). Skin
and hair follicle hybridized with BMP-7 probe at E l 8 (C) and P5 (0). C:
BMP-7 transcripts are detected in the innerjouter root sheath and the
dermal sheath at E18. Strong signal can be seen in the basal cell layer of
the epidermis. D: BMP-7 RNAs are seen in the outer root sheath, espe-
cially in the lower half of the follicle (arrow). Developing tooth hybridized
with BMP-3 probe at E l 8 (E) and PO (F). Strong signal of BMP-3 is seen
in the dental follicle adjacent to the outer enamel epithelium at the cap
stage (E) and bell stage (F). No signal can be detected in the enamel
epithelium nor dental papilla. Developing tooth hybridized with BMP-7
probe at E l 8 (0)and P5 (H).BMP-7 RNA is expressed in the inner
enamel epithelium (arrow) and dental papilla (arrowheads) at cap stage
(G). BMP-7 expression is located in the inner enamel epithelium (arrow),
and odontoblasts (arrowheads) at bell stage (H). Bars = 100 pm (AE,G,H); 500 pm (F).
BMP-3 AND BMP-7 IN RAT EMBRYOS
lium (Fig. 4A). BMP-7 mRNA was detected in some
parts of the mesenchyme below the nasal epithelium a t
E l 8 (Fig. 4B). Expression of BMP-7 mRNA declined
postnatally.
Expression of BMP-3 and BMP-7 mRNAs in the
Developing Lung
BMP-3 mRNA was first detected in the epithelium
and mesenchyme a t E l 5 (data not shown). Subsequently, it was detected in the bronchial epithelium
(Fig. 4C), with little or none in the alveolar epithelium
(EM). BMP-7 mRNA was not expressed in the lung
prior to E15. At E15, low level of expression of BMP-7
mRNA was detected in the bronchial epithelium and
the mesenchyme around the bronchus (data not
shown). During development, expression of BMP-7
mRNA declined gradually.
Expression of BMP-3 and BMP-7 mRNAs in the
Digestive Organs
At E15, low-level expression of BMP-3 mRNA was
found in the mesenchymal tissues around the epithelium (data not shown). At E18, BMP-3 mRNA was
strongly expressed in the mesenchyme at the top of the
villi of the small intestine (Fig. 4D). Weaker expression
of BMP-3 mRNA was found in the surrounding mesenchyme (Fig. 4D). At E18, BMP-3 mRNA was expressed
in the mesenchyme adjacent to the epithelium of the
esophagus (data not shown).
At E13, low level of expSession of BMP-7 mRNA was
first detected in the epithelium of the intestine (data
not shown), and this pattern of expression continued
until E15. At E18, BMP-7 mRNA was located in the
mesenchyme, with especially strong signals at the base
of the villi (Fig. 4F). A strong signal was detected in the
epithelium (in particular, in the basal cell layer) of the
esophagus (Fig. 4E).
Expression of BMP-3 and BMP-7 mRNAs in the
Kidney and Adrenal Gland
Prior to E15, expression of BMP-3 mRNA was first
detected in the mesonephric tubules (data not shown).
At E18, BMP-3 mRNA accumulated specifically in
some parts of the developing tubules (Fig. 4G). At E13,
BMP-7 mRNA was first detected in the tubular portion
(data not shown). Strong signals of BMP-7 were detected in the cap portion (glomerular epithelium),
which surrounds the developing glomerulus, and
weaker signals were found in the tubular portion of the
developing kidney (Fig. 4H,I). Expression of BMP-7
mRNA was also detected in the developing adrenal
gland (Fig. 4H).
Expression of BMP-3 and BMP-7 mRNAs in
Developing Bone and Cartilage
BMP-3 mRNA was expressed in the perichondrium
(Fig. 5A,B). It was expressed in the osteoblasts and
osteocytes of developing endochondral and intramembranous bones (Fig. 5E,F and I,J), but it was absent
443
from the hypertrophic chondrocytes (Fig. 5A,B and
E,F).
By contrast, BMP-7 mRNA was found in resting
chondrocytes, in proliferating chondrocytes and hypertrophic chondrocytes in cartilage and in endochondral
bone (Fig. 5C,D and G,H). In the intramembranous
bone, BMP-7 was detected in osteoblasts and osteocytes
(Fig. 5K,L).
Expression of BMP-3 and BMP-7 mRNAs in
Other Organs
BMP-7 mRNA was expressed in the ducts of the sublingual gland and the ducts of the minor salivary gland
in the tongue, but not in acini (Fig. 6A,B). No BMP-3
mRNA was detected in the heart. From Ell, BMP-7
mRNA was expressed in both the atria and the ventricles of the heart (Fig. 6C). This pattern continued postnatally.
During the early postnatal period, BMP-3 mRNA
was specifically expressed in the postomitotic neurons
in the entorhinal cortex (Fig. 6D). BMP-7 mRNA, but
not BMP-3 mRNA, was expressed in the meningeal
tissues (Fig. 6E). This pattern of expression continued
from E l 5 to adulthood. In the brain on E13,expression
of BMP-7 mRNA was detected in some neuronal stem
cells in the diencephalon (data not shown).
DISCUSSION
It is important to compare the patterns of expression
of BMPs because these patterns should reflect the distinct roles of various BMPs in embryogenesis. Previous
studies have shown that BMP-2, -4, and -6 mRNAs are
expressed during embryogenesis in spatially and temporally restricted patterns (Lyons et al., 1989a,b, 1990;
Jones et al., 1991). However, little information has
been available on the localization of BMP-3 and BMP-7
mRNAs in the developing organs. In this study, we
used in situ hybridization to localize BMP-3 and
BMP-7 mRNAs in developing rat embryos. Each BMP
was expressed in a variety of cells in various tissues, in
particular, in the organs in which reciprocal mesenchymal-epithelial interactions are essential. Our results
suggest that BMP-3 and BMP-7 play important roles in
organogenesis. Distribution of human OP-l(BMP-7)
protein was examined with an antibody against this
protein (Vukicevic et al., 1994). The organs in which
BMP-7 was detected are almost identical to the organs
in which BMP-7 mRNA was detected in our study, although precise localizations in the various organs differ between the protein and the mRNA. The OP-1
(BMP-7) protein was localized in the basement membrane (rich in type IV collagen) of bronchi in the lung,
and in the basement membrane between the dermis
and epidermis of the skin. By contrast, BMP-7 mRNA
was detected in the bronchial epithelium and the surrounding mesenchyme, as well as in the basal cells of
the epidermis in our data. It is possible that the distribution of the protein is different from that of the
mRNA. OP-l(BMP-7) has high affinity for type IV col-
444
TAKAHASHI AND IKEDA
Fig. 4. Expression of BMP-3 and BMP-7 mRNAs in the nasal epithelium, lung, esophagus, small intestine, kidney and adrenal gland. Nasal
epithelium hybridized with BMP-3 (A) and BMP-7 (B) probes at Ei8. Both
BMP-3 and BMP-7 transcripts are located in the mesenchyme under
nasal epithelium. Signal of BMP-3 mRNA is strong in the folded portion.
Developing lung hybridized with BMP-3 probe at El8 (C). BMP-3 signal
can be seen in the bronchial epithelium (arrow), not in the alveolar epithelium. D: Small intestine hybridized with BMP-3 probe at Ei8. Strong
signal is detected at the top of the villi (arrows) and weak signal in the
surrounding mesenchyme. E: Esophagus hybridized with BMP-7 probe
at El 8. Strong signal is detected in the basal cell layer of the esophageal
epithelium (arrow). F: Small intestine hybridized with BMP-7 probe at
E18. BMP-7 transcripts are located in the mesenchyme at the base of the
villi (arrows). 0: Developing kidney hybridized with BMP-3 probe. BMP-3
is expressed in some portion of the developing tubules (arrows). H,I:
Developing kidney and adrenal gland hybridized with BMP-7 probe.
Strong signal can be seen in the cap portion (glomerular epithelium;
arrows in I) and weak one in the tubular portion (arrowheads in I).BMP-7
is also expressed in the adrenal gland (arrow in H). Bars = 100 pm
(A-F,H,I); 50 pm (G).
BMP-3 AND BMP-7 IN RAT EMBRYOS
Fig. 5. Expression of BMP-3 and BMP-7 mRNAs in developing bone
and cartilage A, C, E,G, I, K are brightfield figures. 6, D, F, H,J, L are
darkfield figures corresponding to A, C, E, G, I, K, respectively. A and B:
Developing cartilage hybridized with BMP-3 probe. Strong signal of
BMP-3 transcripts is seen in the perichondrium (arrow). C and D: Developing cartilage hybridized with BMP-7 probe. BMP-7 transcripts are seen
in the perichondrium and hypertrophic chondrocytes. E and F: Developing endochondral bone hybridized with BMP-3 probe. Signal of EMP-3
mRNA is detected in perichondrium (arrowheads) and osteoblasts (arrow). Note that no signal can be detected in hypertrophic chondrocyle.
445
G and H: Developing endochondral bone hybridized with BMP-7 probe.
Signal is detected not only in the proliferating chondrocytes and resting
chondrocytes, but also in hypertrophic chondrocytes. BMP7 mRNA transcripts are also seen in the osteoblasts. I and J: Developing intramembranous bone hybridized with BMP9 probe. Strong signal is detected in
the osteoblasts and osteocytes (arrows). K and L: Developing intramembranous bone hybridized with BMP-7 probe. Strong signal is detected in
the osteoblasts and osteocytes (arrows). Bars = 100 pm (A, C,E, G, I,
K).
446
TAKAHASHI AND IKEDA
Fig. 6. Expression of BMP-3 and BMP-7 mRNA in various organs.
A,B: BMP-7 transcripts are detected in the duct of sublingual gland (arrows in A) and the duct of minor salivary gland (B) in PO. Note that there
is no signal in the acinus. C: BMP-7 expression in the heart. BMP-7
lagen (Vukicevic et al., 1994). Thus, BMP-7 might be
secreted into the surrounding mesenchyme or the secreted protein might enter into the blood stream and be
trapped at sites with high affhity for it. Another possibility is that BMP-7 mRNA is not translated into
protein in some tissues. It is known that localization of
the DVR-6 (BMP-6)protein is different from that of the
DVR-6 (BMP-6) mRNA. Moreover, Wall et al. (1993)
failed to detect DVR-6 (BMP-6) protein in a cell line
that expressed DVR-6 RNA. It seems likely that posttranscriptional regulation or modification of BMP-6 oc-
mRNA is detected in both atrium and ventricle at E15. D: BMP-3 is
expressed in the neurons (arrow) of the entorhinalcortex at PO. E: Strong
signal of BMP-7 is detected in the meningeal tissues. Bars
(A,B,E); 500 pm (C,D).
=
100 pm
before expression of BMP-2 in the ectodermal placode.
Expression of BMP-4 in the mesenchyme is transient;
no expression has been detected at the stage of which
the dermal papilla is formed (Jones et al., 1991; Ikeda
and Takahashi, unpublished data). In the present study,
BMP-3 mRNA was also expressed in the mesenchymal
condensation below the ectodermal placode. Thus, both
BMP-3 and BMP-4 might play an important role in the
initial events in hair development. Furthermore,
BMPS mRNA was expressed in the dermal sheath in
the immature and mature follicle. By contrast, BMP-7
CWS.
mRNA was expressed in the inner and outer root
From our data and the data reported previously (Ly- sheaths and the dermal sheath of the immature follicle.
ons et al., 1989a,b, 1990; Jones et al., 19911, it appears In the mature follicle, expression of BMP-7 mRNA was
that BMP-2, -3, -4, -6 and -7 are expressed in organs detected in the dermal papilla and the outer root sheath.
that develop through mesenchymal-epithelial interac- No expression of BMP-4 or BMPS mRNA was observed
tions (e.g., hair follicles, tooth buds, kidneys and in the mature hair follicle (Jones et al., 1991;Ikeda and
lungs). Therefore, we shall discuss the patterns of their Takahashi, unpublished data). The other members of
expression and their possible roles in organogenesis.
the TGF-f3 superfamily and fibroblast growth factors
The development of hair and whisker follicle involves (FGFs) are expressed in immature and mature hair
a sequence of reciprocal interactions between the epi- follicle (Pelton et al., 1990; du Cros et al., 1993). It has
dermis and underlying dermis (see review, Hardy, been reported that activin, BMP-4 and FGF are re1992). A previous report showed that expression of quired for mesoderm induction (for review, see Sive,
BMP-4 is the earliest event in the hair development. 1993). Also, it is known that FGF-4 and BMP-2 have
BMP-4 is expressed in the mesenchymal condensation opposite effects on limb growth: FGF-4 stimulates the
BMP-3 AND BMP-7 IN RAT EMBRYOS
proliferation of mesenchyme in the early development
of mouse limbs, while BMP-2 inhibits limb growth
(Niswander and Martin, 1993). Not only interactions
among members of the TGF-P superfamily but also interactions between the TGF-P superfamily and the FGF
family (or other molecule) must be required for the
appropriate development of hair.
Only BMP-6 is known to be expressed in the thickened epidermis in prenatal skin and expression of
BMPS protein continues in the supra-basal cell layers
of the skin until birth (Lyons et al., 1989b; Wall et al.,
1993). In the present study, BMP-7 mRNA was localized in the basal cell layer of the epidermis in the developing skin, and the level of expression declined postnatally. The presence of BMP-7 mRNA in the basal
cells of the epidermis and its absence from the suprabasal layers suggest that BMP-7 might act as an autocrine mitogen for skin keratinocytes. By contrast,
BMP-6 is expressed in the differentiated, postmitotic
cells. Thus, BMPS might act as a differentiation factor.
Tooth development is also controlled by reciprocal
epithelial-mesenchymal interactions. At the cap stage,
BMP-4 mRNA is detectable in mesenchymal cells of
the dental papilla below the enamel epithelium. At the
late bell stage, BMP-4 mRNA is expressed in both differentiating (mesenchymal) odontoblasts and differentiating (epithelial) ameloblasts. On the other hand,
BMP-2 is expressed in the central cells of dental papilla
at the cap stage and in ameloblasts and preodontoblasts transiently a t the bell stage (Vainio et al., 1993;
Ikeda and Takahashi, unpublished data). Patterns of
expression of BMP-3 and BMP-7 are very different
from those of BMP-4 and BMP-2. No expression of
BMP-3 or BMP-’7 mRNA was detected before the cap
stage. Expression of BMP-3 mRNA was detected in the
dental follicles, in particular, adjacent to the outer
enamel epithelium and this pattern of expression persisted to the bell stage. BMP-7 mRNA was strongly
expressed in the inner enamel epithelium and relatively more weakly in the dental papilla below the
enamel epithelium during the cap stage and the early
bell stage of tooth development. In late bell stage, expression of BMP-7 mRNA was detected in the inner
enamel epithelium and odontoblasts. Coordinate expression of BMP-7, -4, -2 might regulate the differentiation of ameloblasts and odontoblasts in an autocrine
or paracrine manner. By contrast, BMP-3 might be involved in follicular formation during the tooth development andlor in the differentiation of the outer
enamel epithelium.
The morphogenesis of the kidney is a prime example
of the importance of the mesenchymal-epithelial interactions. During glomerular development, BMP-7
mRNA was strongly expressed in the cap portion; the
glomerular epithelium. BMP-4 mRNA was also located
in the cap portion, although expression of BMP-4
mRNA was also observed in mesenchymal tissues
around the tubules (Ikeda and Takahashi, unpublished
447
data). BMP-3 and BMP-6 are not expressed in the developing glomerulus. BMP-7 and BMP-4 might play
important roles in the formation of the glomerulus. Expression of BMP-3 mRNA was localized within a restricted portion of the developing tubule. Weak expression of BMP-7 mRNA was seen in the tubular portion.
BMP-2 mRNA was expressed in the mesenchyme of the
medulla (Ikeda and Takahashi, unpublished data). Experiments using organotypic culture and antisense oligonucleotides or antibodies should help to clarify the
role of each BMP in renal development.
Patterns of expression of BMPs were also distinct in
the digestive organs. BMP-3 mRNA was localized in
the mesenchyme at the top of villi in the small intestine. BMP-7 mRNA was strongly expressed in the mesenchyme at the base of the villi. Expression of BMP-4
mRNA was seen diffusely in the surrounding mesenchyme. No expression of BMP-2 has been detected in
developing digestive organs (Ikeda and Takahashi, unpublished data). The mesenchyme of the developing
gut is not homogeneous. For example, the level of expression of the extracellular matrix differs between the
top of the villi, the base of the villi and the surrounding
mesenchyme of the gut. Before formation of villi, laminin, type IV collagen and nidogen are present a t the
epithelial/mesenchymal interface, whereas fibronectin
and type I11 procollagen are found throughout the mesenchyme. During the formation of the villi, fibronectin
and type I11 procollagen disappear transiently from the
mesenchyme a t the top of the villi. (Simon-Assmann et
al., 1986). Moreover, each BMP has a different ability
to induce cell adhesion molecules in mesenchymal and
neural cells (Perides et al., 1992, 1993, 1994). This observation suggests that BMPs might differentially affect the extracellular matrix and differential expression of BMPs might contribute to formation of the
specific shape of the organ.
Previous studies and our data show that patterns of
expression of BMPs overlap, but there are some differences (Lyons et al., 198913,1990;Ikeda and Takahashi,
unpublished data). BMP-2, BMPS, and BMP-7 mRNAs
‘areexpressed in hypertrophic chondrocytes, but BMP-3
or BMP-4 mRNAs are not. BMP-3, BMP-7 and BMP-2
mRNAs are expressed in osteoblasts, where expression
of BMP-4 and BMPS mRNA cannot be detected. Furthermore, human OP-l (BMP-7) protein is distributed
in hypertrophic chondrocytes, osteoblasts, and the periosteum (Vukicevic et al., 1994). These distributions
suggest different physiological roles for the various
BMPs in skeletogenesis. To clarify the physiological
functions of BMPs more precisely, their interactions
should be studied.
Concluding Remarks
The data presented in this study provide information
relevant to identification of the functions of the TGF-P
superfamily in morphogenesis. BMP-3 and BMP-7
mRNAs were detected in various tissues, in particular,
in organs in which reciprocal mesenchymal-epithelial
448
TAKAHASHI AND IKEDA
exposed to the imaging Plate for 24 hours and the result
was analysed using a Biolmage Analyzer (BAS 2000,
Fuji Photo Film Co., Japan). [3SSlUTP-labelledsinglestranded RNA probes for in situ hybridization were
synthesized by in vitro transcription. Treatment of the
slides and hybridization conditions were the same as
described previously (Ikeda et al., 1992)and the protocol
was essentially that of Nomura et al. (1988) In brief,
sections were pretreated with proteinase K (BoehringEXPERIMENTAL PROCEDURES
er-Mannheim, Germany) and acetylated with acetic anPreparation of Tissues
hydride/triethanolamine-HC1.Tissue sections were alEmbryos were obtained from female rats of Wistar lowed to hybridize with probes overnight in a moist
strain. Embryonic age was determined on the basis of chamber a t 55°C. After hybridization, the sections were
detection of the vaginal plug (day 0).Embryonic tissues treated with 12.5 pgh1 of RNAse A in 10 mM Tris-HC1,
were fixed with 4% paraformaldehyde in phosphate pH 7.6,l mM EDTA and 500 mM NaCl at 37°C for 30
buffer a t 4°C for 24 hr, dehydrated and embedded in min, and washed with 2 x SSC and 0.2x SSC at 55°C.
paraffin. Tissues from postnatal rats were also fixed in Dried slides were dipped in NTB-3 emulsion (Eastman
4% paraformaldehyde solution by cardiac perfusion Kodak: Rochester, NY) that had been diluted (1:l) with
and embedded in paraffin. Four-pm-thick serial sec- 2% glycerol, dried and stored for 2 weeks at 4°C in
tions of tissue were cut and transferred to siliconized decicated slide boxes. After development and fixation,
the sections were stained with hematoxylin and eosin.
slides.
interactions are essential, and the patterns of expression of these mRNAs are temporally and spatially regulated. Identification and study of receptors for BMPs
will help us to elucidate the functions of BMPs.
Analysis of gene-targetted mice and studies of organotypic cultures should also help to identify the precise
functions of these molecules during vertebrate organogenesis.
Preparation of Probes, Northern Blotting and In
Situ Hybridization
Poly(A)+RNAwas isolated from whole bodies of rats
on E l 5 with a Fast Track mRNA isolation kit (InVitrogen, San Diego, CAI. Single-stranded cDNA was synthesized with a cDNA Synthesis Kit plus (Amersham,
U.K.). To generate the cDNA sequences for rat BMP-3
and BMP-7, polymerase chain reactions (PCRs) were
performed with the single-stranded cDNA from E l 5
rats as the template. The degenerate oligonucleotide
pairs that were used to amplify the sequences corresponded to nucleotides 1,136-1,530 of the human gene
for BMP-3 (Wozney et al., 1988) and 924-1,349 of the
human gene for BMP-7 (Celeste et al., 19901, respectively. Products of PCR were ligated into the pGEM-T
vector (Promega, Madison, WI). They were sequenced
and identified as fragments of cDNAs for rat BMP-3
or BMP-7. (GenBank accession numbers: D29768,
D29769.1 Data base analyses showed that the fragment
of rat BMP-3 had 87% identity with human BMP-3 and
only 10-15% homology with other members of BMPs in
nucleotide level. The fragment of rat BMP-7 had 91%
identity with human BMP-7 and 63% identity with
mouse BMP-6 (which had the highest homology among
BMPs except for human BMP-7). Thus, each BMP
cDNA in the plasmid vectors is suitable as the probe for
Northern blot analysis and in situ hybridization.
Northern blot filters containing 1pg of poly(A)+RNA
from whole bodies of rats on E l 5 were prepared. DNA
probes for BMP-3 and BMP-7 were isolating from the
plasmid vectors described above and were radiolabelled
with 32P-dCTP by random-prime labelling kit (Boehringer-Mannheim, Germany). The Northern blot filters
were hybridized with each probe at 65°C for 15 hours in
hybridization buffers and washed a t room temperature
in l x SSC/O.l% SDS for 40 minutes and 65°C in
0.1 x SSC/O.l% SDS for 40 minutes. The filters were
ACKNOWLEDGMENTS
The authors thank Ms. Eiko Moriizumi for excellent
technical assistance, Dr. Shintaro Nomura for expert
advice, and Professor Shusaku Yoshiki and Dr. Akira
Yamaguchi for support during this study. The authors
also thank Dr. Chikako Nor0 and Dr. Nobuhiro Nor0
for helpful comments on the manuscript.
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