(Received for publication, March 13, 1995; and in revised form, July 7, 1995)
From the
Bone morphogenetic protein-4 (BMP-4) is one of a member of related polypeptides that are important in bone formation and other developmental processes. We isolated the BMP-4 gene from a mouse genomic library and characterized the exon-intron structure and one of the candidate promoters. Two alternative 5`-noncoding exons, 1A and 1B, were identified by reverse transcription polymerase chain reaction assays. Quantitative competitive polymerase chain reaction using Exon 1A, Exon 1B, and Exon 3 primers indicate the 1A-containing transcript is the primary BMP-4 mRNA expressed in bone cell cultures. Primer extension analysis supports that 1A is the major promoter utilized in bone cell cultures as well as in 9.5-day mouse embryos. 1A promoter activity indicate selective DNA regions functional in bone cells. We found potential regulatory response regions in the 1A 5`-flanking region of the BMP-4 gene for the chicken ovalbumin upstream-transcription factor I (COUP-TFI). Specific binding to the COUP-TFI response regions in the BMP-4 1A promoter was demonstrated. By co-transfection of a COUP-TFI expression plasmid with the BMP-4 1A promoter in fetal rat calvarial osteoblasts, we demonstrated that COUP-TFI inhibits the BMP-4 promoter activity. This suggests that COUP-TFI could act as a silencer for BMP-4 transcription in vivo.
The bone morphogenetic protein (BMP) family
comprises a group of closely related polypeptides in the transforming
growth factor
superfamily, which were identified initially by
their capacity to stimulate ectopic bone formation in vivo (Wozney et al., 1988; Celeste et al., 1990).
Recently, we have found that mRNAs for BMP-2 and BMP-4 are expressed in
FRC osteoblasts as they differentiate in primary culture prior to
forming mineralized bone nodules (Harris et al., 1994a).
Expression of BMP-2 and BMP-4 mRNA coincides with expression of several
osteoblast differentiation markers by these cells, including
osteocalcin, osteopontin, and alkaline phosphatase (Harris et
al., 1994a). BMP-4 is synthesized as a large precursor, as are
other transforming growth factor
members, and is processed to a
mature dimer form, which is then secreted into the extracellular matrix
(Suzuki et al., 1993). Recombinant BMP-4 stimulates new
cartilage and bone formation when implanted near long bones (Shimizu et al., 1994). Expression patterns of BMP-4 mRNA in the apical
ectodermal ridge and limb suggest important roles during limb
development (Wozney, 1992). BMP-4 has also been shown to be an
important factor in dorsal region specification in mouse embryos and
high levels of BMP-4 expression are seen at the gastrula stage of Xenopus embryos (Nishimatsu et al., 1993; Jones et al., 1992).
The physiological factors regulating BMP-4 gene expression and function are still largely unknown. Recently, we and others have found that there are at least two different BMP-4 noncoding first exons (Exon 1A and 1B) (Chen et al., 1993; Kurihara et al., 1993). This suggests the BMP-4 gene may have at least two promoters. Analysis of the BMP-4 gene structure and functional studies in bone and other cells should provide a foundation for understanding the environmental and cellular factors which regulate BMP-4 gene transcription and alternate promoter usage.
In this
study, we have isolated the mouse BMP-4 gene and determined its primary
DNA sequence. We have demonstrated, with the limits of the constructs
tested, that the promoter 1A is active in bone cells by transfection
studies. With Exon 1A and 1B probes and poly(A) RNA
from primary FRC cell cultures, Northern analysis supports this
conclusion. These data were confirmed by quantitative competitive
RT-PCR using Exon 1A or 1B primers and an Exon 3 primer with a common
competitor containing Exon 1A, Exon 1B, and Exon 3 priming sites.
We have also demonstrated that COUP-TFI, a member of the steroid/thyroid hormone receptor superfamily, negatively modulates BMP-4 gene transcription in transient transfection assays using primary fetal rat calvariae cells. We have shown that two elements in the 1A promoter bind to the COUP-TFI protein and that COUP-TFI mRNA is expressed in these cultures during bone cell differentiation.
Figure 1:
A, partial
restriction enzyme map of mouse genomic BMP-4 and diagram of two
transcripts. The mouse BMP-4 gene transcription unit is 7 kb and
contains 2 coding exons (filled boxes) and 3 noncoding exons
labeled Exon 1A, Exon 1B, and Exon 2. The original 19-kb clone has
6-kb 5`-flanking region and an
7-kb 3`-flanking region. The
diagram shows approximately 2.4 kb of 5`-flanking region, and a small
region (
200 bp) of 3`-flanking area. The lower panel shows two alternative transcripts of BMP-4 gene. Both have the
same Exons 2, 3, and 4 but a different Exon 1. Transcript A has Exon
1A, and transcript B has Exon 1B. B, DNA sequence of mouse
genomic BMP-4 DNA and predicted amino acid sequences within the coding
exons. The sequence of the exons is presented. The numbers on
the right show the position of nucleotide sequence, and the bold numbers indicate the location of amino acid sequence of
the coding region. The end of the transcription unit was estimated
based on a 1.8-kb mRNA transcript size. Primer 1 in Exon 1A, primer 2
in Exon 1B, and primer 3 in Exon 3 were used in RT-PCR analysis. Primer
B1 and B2 were used in primer extension reactions. C, BMP-4 1A
5`-flanking region and potential response elements in mouse BMP-4 1A
promoter. The sequence of 2678-bp mouse BMP-4 gene are shown.
Nucleotides are numbered on the left with +1
corresponding to the major transcription start site of the 1A promoter.
The response elements of DR-1A Proximal and DR-1A Distal sequences are
indicated. The other potential response DNA elements in the boxes are
p53, retinoblastoma (RB), SP-1, AP-1, and AP-2. The primer A,
indicated as the dotted line above the DNA sequence at
+114/+96, was used for primer extension analysis for Exon
1A-containing transcripts.
Figure 4:
Quantitation of BMP-4 A and B transcript
levels in FRC cells. A, generation of a BMP-4 competitor to
quantitate relative BMP-4 A and B transcript levels in FRC bone cells.
The BMP-4 competitor is used to compete for PCR of Exon
1A-2-3-containing transcript when primer 1 (Exon 1A primer) is
utilized with primer 3 (Exon 3 primer). The same BMP-4 competitor is
used to compete for PCR of Exon 1B-2-3-containing transcripts
when primer 2 (Exon 1B primer) is utilized with primer 3 (Exon 3
primer). B, quantitative competitive RT-PCR results for BMP-4
transcript A. C, quantitative competitive RT-PCR results of
BMP-4 transcripts B and B`. The upper panel of B and C shows electrophoretic pattern of the BMP-4A and 4B products.
The primer 1 (Exon 1A), primer 2 (Exon 1B), and primer 3 (Exon 3) were
utilized. The ethidium bromide-stained PCR products were separated on a
2% agarose gel. The 952-bp BMP-4 competitor template has both Exon 1A
and 1B priming sites on the 5` end and Exon 3 priming site on the 3`
end. The first lane shows the PCR products without BMP-4 competitor in
the PCR reaction. The remaining lanes show the PCR products with
increasing amounts of BMP-4 competitor (0.025-250 ng).
[-
P]dCTP was added to the PCR reaction for
quantitation of the PCR products. The relative amounts of BMP-4A and 4B
products to the competitor products were calculated after size
correction. The logarithm of the ratios of BMP-4A products (455 bp) and
the sum of the two different size BMP-4B products (495 and 245
bp) to the BMP-4 competitor products (952 and 935 bp) was graphed as a
function of the logarithm of the amounts of the BMP-4 competitor added
to the PCR reaction. These calculations are shown in the lower
panel of B and C. The calculated amounts of the
A and B transcripts are marked by the arrows, where the ratios
of BMP-4A or 4B transcripts to corresponding competitor products are
equal 1 (log 1 = 0).
The PCR
reaction conditions used for the quantitative PCR are as described
above. 1 µCi of [-
P]dCTP was added into
each PCR reaction. Reverse-transcribed FRC cell cDNA, 2 mM MgCl
, 10-fold serial dilutions of the BMP-4 competitor
(0.025-250 pg), 0.25 mM dNTP, Taq polymerase,
primer 1 or primer 2, and primer 3 (thus, the same lower primer for
transcripts A and B) were placed into a master mixture. Primer 1 was
used for BMP-4 A transcript quantification, and primer 2 was used for
BMP-4 B transcript quantification. After amplification, a 10-µl
sample from each 50-µl reaction was resolved by agarose gel
electrophoresis. Gels were stained with ethidium bromide (50
µg/ml), photographed, dried, and scanned with the AMBIS Image
Acquisition and Analysis System (AMBIS, San Diego, CA). The logarithm
of the ratio of rat BMP-4 products (containing Exon 1A or Exon 1B) to
BMP-4 competitor PCR products was graphed as a function of the
logarithm of the amount of BMP-4 competitor added after correction for
the size difference between the rat BMP-4 PCR products and BMP-4
competitor PCR products.
The isolated FRC
cells, enriched for the osteoblast phenotype, were used as recipient
cells for transient transfection assays. The technique of
electroporation was used for DNA transfection (Potter, 1988; van den
Hoff et al., 1992). After electroporation, the cells were
divided into aliquots, replated in 100-mm diameter culture dishes and
cultured for 48 h in -minimal essential medium (Life Technologies,
Inc.) with 10% fetal calf serum. The cell lysates were assayed for CAT
activity according to the method described by Gorman (1988), and CAT
activity was normalized by
-galactosidase assay from a
co-transfected Rous sarcoma virus-
-galactosidase at one-tenth the
concentration of the CAT plasmids, according to the method of Rouet et al.(1992).
Double-stranded oligodeoxynucleotides corresponding to consensus
COUP RE (AAGTCA A AAGTCA) (Cooney et al., 1992) was prepared
and used in competition assays. BMP-4 DR-1A Proximal (-865 to
-878) (GGGCCA A AGGGCA) or BMP-4 DR-1A Distal (-2095 to
-2108) (GGGCCA A AGGTCA) binding sites were labeled with
[-
P]dATP to specific activities of 2 to 8
10
cpm/µg. 0.1-0.2 ng of labeled DNA
probe was incubated with 1-4 µl of in vitro translated COUP-TFI for 15 min at room temperature under the
following conditions: 60 mM KCl, 10% glycerol, 3 mM
MgCl
, 1 mM dithiothreitol, 20 mM HEPES
(pH 7.9), 0.2-2 µg/ml poly(dI-dC) (Pharmacia Biotech Inc.).
The reactions were resolved by electrophoresis in a 5% (w/v)
polyacrylamide gel in 0.5
Tris borate-EDTA running buffer. The
gels were dried and then autoradiographed at -70 °C with a
DuPont Cronex Lightning Plus intensifying screens with Kodak X-Omat
x-ray film. For antibody supershift analysis, the COUP-TFI antiserum
was preincubated with the in vitro translation products at
room temperature for 5 min prior to the addition of the labeled DNA
probes.
Figure 2: Primer extension assay. The total RNAs prepared from FRC cells (on the left) and 9.5-day mouse embryo (on the right) were used with primer A (Fig. 1C) or primer B1 (Fig. 1B). Two major extended fragments, 67 and 115 bp in lanes marked A are obtained from primer A. No major fragments are detected using the B1 primer. Primer B2 (Fig. 1B) also gave negative results with both FRC and mouse embryo total RNA as templates (data not shown).
To test whether the BMP-4 1B exon is expressed in FRC
cells, we designed oligonucleotide primers to ascertain whether spliced
1B-2-3 exon products and 1A-2-3 exon products (control)
could be obtained by RT-PCR from FRC poly(A) RNA. The
3` primer was in Exon 3 (primer 3) and the 5` primers were either in
Exon 1A (primer 1) or 1B (primer 2) (see Fig. 1B).
The RT-PCR products were cloned and sequenced. A photograph and diagram of the products obtained are presented in Fig. 3. Both 1A-2-3 and 1B-2-3 products were obtained. The results indicate that FRC osteoblasts produce transcripts with either a 1A exon or a 1B exon, but not both. This suggests that the intron region between 1A and 1B exon may contain regulatory response elements under certain cellular contexts, resulting in BMP-4 B transcripts. Of the 1B-2-3 RT-PCR products obtained from FRC osteoblasts, we obtained two products with different 3` splice sites for Exon 1B. By comparison with the genomic DNA, both 3` ends of the two Exon 1Bs have reasonable 5` splice consensus sequences, consistent with an alternate splicing pattern obtained for the 1B-2-3 RT-PCR products. Most importantly, no 1A-1B-2-3 RT-PCR-spliced products of the BMP-4 gene were obtained. Thus, 1B does not appear to be an alternatively spliced 5`-noncoding exon and suggests the BMP-4 gene may have at least two promoters.
Figure 3: Gel electrophoresis of Exon 1A-2-3 (lane 2) and Exon 1B-2-3 RT-PCR products (lane 1). RT-PCR was performed with two pairs of primers using reverse transcribed FRC cDNA as the template. The products were verified by the DNA sequence analysis. Schematic diagram of spliced BMP-4 RT-PCR products with 1A and 1B exons are shown for FRC cells (right). The primers and their locations are noted in Fig. 1B. RT-PCR product 1A-2-3 which contains Exon 1A, Exon 2, and the 5` region of Exon 3, is produced with primer 1 and primer 3. Primer 2 (Exon 1B) and primer 3 (Exon 3) generate two RT-PCR products with Exon 1B`-2-3 pattern (245 bp) and the 1B-2-3 pattern (495 bp).
After 48 h of transfection with various BMP-4-CAT reporter gene constructs, the cells were harvested and the CAT activity was determined. As indicated in Fig. 5(A and B), pCAT-0.24 plasmid (-25/+212) has measurable CAT activity. The CAT activity of this pCAT-0.24 plasmid was 3-fold lower than that the background parent pBL3CAT plasmid. This suggests the -25 to +212 has transcriptional or translational inhibitory activity. The pCAT-0.5 (-260/+212), pCAT-1.3 (-1144/+212), and pCAT-2.6 (-2372/+258) showed progressive increasing promoter activity when transfected into primary FRC cells. These data are shown in Fig. 5B. With pCAT-0.5 (-260/+212) there is a 10-fold increase in CAT activity relative to pCAT-0.24 (-25/+212). pCAT-1.3 (-1144/+212) shows an additional 6-fold increase in CAT activity over pCAT-0.5, and pCAT-2.6 (-2372/+258) shows an additional 2-fold increase in CAT activity over pCAT-1.3 (-1144/+212). Thus the net increase in CAT activity between the pCAT-0.24 (-25/+212) and the pCAT-2.6 (-2372/+258) in FRC cells is approximately 100-fold.
Figure 5: Map of the BMP-4 1A 5`-flanking-CAT plasmid and promoter activity in FRC cells. A, the 2.6-kb EcoRI and XbaI fragment, 1.3-kb PstI fragment, 0.5-kb SphI and PstI fragment, and 0.24-kb PCR fragment were inserted into pBLCAT3 as described under ``Materials and Methods.'' The closed box indicates the noncoding Exon 1A. The CAT box represents the CAT reporter gene. The values are percentages of CAT activity relative to that of pCAT-2.6 (100%) and represent the average of four independent assays. Similar results were obtained with independent FRC cultures. B, autoradiogram of the CAT assays using the FRC cells transfected with the BMP-4 1A 5`-flanking CAT plasmids in A.
Since COUP-TFI has been shown to be an important modulator of transcription of a variety of genes (Ladias and Karathanasis, 1991; Lehmann et al., 1991; Tran et al., 1992; Kliewer et al., 1992a, 1992b), and BMP-4 mRNA is transiently expressed during bone cell differentiation, we examined these potential DR-1 COUP-TFI response region in the 1A promoter.
A
consensus COUP-TFI binding oligonucleotide (AGGTCA A AGGTCA), referred
to as COUP RE, was used as positive control. The result in Fig. 6A shows that in vitro transcribed/translated COUP-TFI binds both the proximal and distal
BMP-4 DR-1A elements (lanes 6 and 11).
Non-radioactive COUP-TFI RE oligonucleotide competes the binding of
labeled BMP-4 DR-1A oligonucleotides to the COUP-TFI produced in
vitro (lanes 7 and 12). In a supershift assay,
COUP-TFI antibodies further retarded the
COUP-TFI/-
P-labeled BMP-4 distal and proximal BMP-4
DR-1A elements (lanes 9 and 14). The COUP-TFI thus
forms a high affinity stable complex with the distal and proximal DR-1A
BMP-4 elements. An oligonucleotide containing a progesterone response
element (PRE) at 25-fold excess over the labeled BMP-4 DR-1A elements
could not compete the binding to the COUP-TFI protein (Fig. 6A, lanes 8 and 13). As shown
in Fig. 6B, the proximal DR-1A-COUP-TF interaction
required more non-radioactive consensus COUP-TF oligonucleotide to
compete the COUP-TFI binding. This suggests the proximal BMP-4 DR-1A
binds to COUP-TFI with higher affinity than the distal BMP-4 DR-1A
element (Fig. 6B).
Figure 6: COUP-TFI specifically binds to a Distal and Proximal DR-1A elements in the BMP-4 1A promoter. A, COUP-TFI obtained from in vitro translation was incubated with the labeled ``wild type'' COUP RE or the BMP-4 DR-1A Proximal or Distal REs. COUP is the non-radioactive wild type COUP RE. PRE is the progesterone response element oligonucleotide (Cooney et al., 1992). COUP-TFI Ab is a polyclonal antibody to COUP-TFI. All reaction products were electrophoresed on nondenaturing 6% polyacrylamide gels. B, the labeled double-stranded oligonucleotide probes of the three COUP RE (wild type, DR-1A proximal, and DR-1A distal) were incubated in the presence of increasing concentrations of cold nonlabeled double-stranded oligonucleotides of wild type COUP RE, AGGTCAAAGGTCA. The BMP-4 DR-1A Proximal RE (middle, GGGCCAAAGGCA) binds COUP-TFI specifically. The wild type COUP RE competes with this DR-1A proximal RE. The BMP-4 DR-1A Distal RE also specifically binds to COUP-TFI (right). The DR-1A Proximal element requires higher concentration of non-radioactive wild type COUP-TF RE to compete the BMP-4 DR-1A binding.
Figure 7:
BMP-4 1A promoter activity is inhibited by
COUP-TFI. Left top panel shows that COUP-TFI has little effect
on the pBL2CAT (ptk-CAT) activity. The tk promoter contains no COUP-TF
response elements. The right top panel shows that equivalent
CMV-driven COUP-TF expression plasmid inhibits BMP4 1A promoter
activity. CAT activity is normalized to -galactosidase activity.
10 µg of pCAT 2.6 was used per transfection with 1-5 µg
of pCMV-COUP-TFI. The normalized data are quantitated and shown in the bottom left (pBL2-CAT) and bottom right (pCAT
2.6).
Figure 8:
COUP-TFI mRNA expression in FRC cells
during in vitro bone cell differentiation and nodule
formation. The fold change of COUP-TFI mRNA has been normalized to
glyceraldehyde-3-phosphate dehydrogenase mRNA levels as described under
``Materials and Methods'' and then calculated based on the
level at day 0 set at 1.0. The 1.8-kb BMP-4A mRNA levels are shown for
comparison. Poly(A) RNA (5 µg) was electrophoresed
on denaturing gels, transferred, and hybridized with the appropriate
probes as described (Harris et al., 1994a).
, BMP-4
mRNA;
, COUP-TFI mRNA.
In this study, we have isolated the murine BMP-4 gene transcription unit and flanking regions and examined its DNA sequence in order to lay the foundation for understanding factors and signaling pathways that may regulate transcription of the BMP 4 gene in the context of primary cultures of rat bone cells and other tissues. Over 6 kb of 5`-flanking region have been isolated, and the exon and intron structure has been defined by DNA sequence analysis. The transcription unit comprises 7030 bp with three 5`-noncoding exons. These results are similar and extend those reported by Kurihara et al.(1993). The first noncoding exon, Exon 1A, defines a transcription start site utilized in bone cells and the 9.5-day mouse embryo. The candidate 1B promoter has not been extensively investigated and the transcription start site is not known. However, by quantitative RT-PCR we demonstrate that 1B transcript are present in bone cells but at a 10-fold lower concentration than 1A transcripts.
A 2360-bp 5`-flanking region of Exon 1A has been sequenced, and several potential regulatory elements have been identified by their sequence homology to known transcription factor binding sites. Four BMP-4 1A 5`-flanking CAT reporter gene plasmid constructs with different size deletions were transfected into primary FRC osteoblasts to establish regions of the BMP-4 1A promoter that are important for transcriptional regulation in bone cells. A region between -23 and -260 shows over a 10-fold stimulation of transcription. Between -23 and -1144 there is over a 60-fold stimulation of transcription.
Two direct repeat
elements (DR-1 sites) similar to RXR-COUP-TFI binding sites were found
to bind the transcription factor COUP-TFI, and by transfection studies
in primary osteoblast cultures, we demonstrated that COUP-TFI is a
negative regulator of the BMP-4 1A promoter in vitro. We have
also demonstrated that COUP-TFI mRNA is expressed in primary cultures
of FRC osteoblasts and undergoes marginal (2-fold) changes in
level during differentiation, in parallel with BMP 4 expression.
Mammalian BMP 4 gene structure is very similar to the fruit fly homologous gene, decapentaplegic or dpp (St. Johnston et al., 1990). The dpp gene has two coding exons and at least three 5`-noncoding exons, which define three alternate promoters. In the dpp gene there are other promoters further upstream and regulatory DNA region in the 3`-flanking area (St. Johnston et al., 1990). The dpp mature coding regions share over 75% identity to BMP 4 mature coding region. The position of the splice site between the two coding exons is also conserved (St. Johnston et al., 1990). The dpp gene is critical for dorsal-ventral specification during embryogenesis and imaginal disc differentiation during pupation (Shimell et al., 1991). What is of interest for this study is that different promoters for the dpp gene are used at different developmental stages and in different tissues, presumable with unique sets or combinations of transcription factors for each promoter. This supports the hypothesis that the mammalian BMP 4 gene will utilize different promoters in different developmental context. We believe we have identified at least two BMP 4 promoters and have shown that both are utilized in heterogeneous primary bone cell cultures, which contain isolated areas of rapid bone formation (mineralizing nodules) between areas that do not differentiate (Harris et al., 1994a, 1994b). We are now in a position to identify differential utilization of the 1B and 1A exons using in situ RT-PCR during different stages of our in vitro bone cultures. This analysis is being extended, using mouse embryo sections at different developmental stages. Deer antler tissue and prostate tissue express much higher 1B transcripts and should allow us to develop more specific probes and map the transcription start sites for the 1B promoter (Chen et al., 1993; Feng et al., 1995).
In situ hybridization analysis of BMP 4 expression has shown a wide but selective expression pattern during embryogenesis (Jones et al., 1991). BMP 4 mRNA is detectable in Xenopus oocytes (Nishimatsu et al., 1992). The BMP 4 mRNA expression greatly increases between blastula and gastrulae and subsequently declines in Xenopus during later embryogenesis (Nishimatsu et al., 1992). BMP 4 mRNA expression domains in chick and mammalian embryos are highly focal and specific (compared to BMP 2) during limb bud/limb formation (Francis et al., 1994; Jones et al., 1991; Jones et al., 1992;Wozney et al., 1992). BMP 4 is expressed at high levels in select regions of the developing nervous system, in the developing branchial arches and later, during tooth development (Jones et al., 1991; Vainio et al., 1993). BMP 4 is selectively expressed in rhombomere 3 and 5 at stage 5 in the chick embryo (Graham et al., 1994). In two of the above studies, the biologically active BMP 4 protein was shown to 1) stimulate differentiation of dental mesenchyme and autoregulate BMP 4 mRNA expression, and 2) selectively control apoptosis of neural crest cells in rhombomere 3 and 5 of the developing hindbrain (Vainio et al., 1993; Graham et al., 1994). Thus we propose, that with the large variety of developmental contexts in which BMP 4 is expressed, the use of alternate BMP 4 promoters will allow selected tissue and developmental specific gene networks to operate within a given cell on the BMP 4 gene. The consequence of having BMP 4 transcripts in a cell with alternate 5`-noncoding exons could result in controlling where the BMP 4 mRNA is localized in the cell and selective rates of translation of BMP 4 1B mRNA versus BMP 4 1A mRNA transcripts in these different cellular contexts (for review, see Curtis et al.(1995)). Our analysis of 1B and 1A expression patterns in embryos will help in developing more extensive models.
Analysis of predicted secondary structures of Exon
1A and 1B indicates dramatic differences using the M FOLD program in
GCG. The G levels for all structures were between -92 and
-95 kcal/mol. These predicted secondary structures in the
5`-noncoding region could play important roles in BMP 4 cellular
localization and translation by binding select sets of RNA binding
proteins in different cells or tissues (Curtis et al., 1995).
COUP-TFI negatively modulate RXR-dependent pathways (Cooney et al., 1992; Kliewer et al., 1992a, 1992b). RXR heterodimerizes with thyroid, retinoid, and vitamin D receptors, and the early response gene, NUR77 or NGF 1B (Cooney et al., 1992; Perlmann and Jansson, 1995). Thus COUP-TFI levels to RXR levels in a given cell may allow the balancing of diverse hormonal and growth factor signals during embryogenesis and homeostasis (Lu et al., 1994).
The potential in vivo significance of COUP-TFI levels and expression on the BMP 4 gene can be addressed by first analyzing areas of overlapping expression of COUP-TFI and BMP 4 expression. Several areas in mouse embryos indicate a strong correlation between BMP 4 expression and COUP-TFI expression in both spatial and temporal aspects. First, in 10.5 post coitus embryos, there are high levels of both COUP-TFI and BMP-4 mRNAs in nasal pits and facial processes, and in the otic vesicles (Jones et al., 1991; Jonk et al., 1994; Lu et al., 1994). Several areas of the diencephalon and rhombomeres overlap in expression domains for both genes that may be relevant to the control of brain development and selected apoptosis of neural crest cells by BMP 4 in rhombomere 3 and 5 (Lu et al., 1994; Graham et al., 1994).
There are high levels of both COUP-TFI and BMP 4 mRNA in the mesenchyme surrounding whisker follicles (Jones et al., 1991; Jonk et al., 1994). Targeted overexpression of BMP 4 using a cytokeratin promoter to whisker follicles and other tissues results in some of the surviving mice with a large number of developmental defects, including craniofacial malformations, fusion of dentary and maxillary bones, growth retardation, cleft palate, absence of whiskers, and defects in hair regeneration (Blessing et al., 1993). This indicates that controlling the level and spatial and temporal aspects of BMP 4 expression are extremely important for proper differentiation in a variety of tissues. COUP-TFI could play a role in regulating the level of BMP 4 expression from the 1A promoter.
Finally, both BMP 4 and COUP-TFI expression patterns overlap in an interesting ways in the developing limb bud. In 10.5-day post coitus embryos, there is a distal-to-proximal gradient of BMP 4 expression. COUP-TFI expression shows a proximal-to-distal gradient (Jones et al., 1991; Lu et al., 1994). This type of reciprocal expression pattern would be ideal for controlling the level of BMP 4 expression. In the limbs of later 14.5-day post coitus embryos, there is an overlap in BMP 4 expression and COUP-TFI expression in the mesenchyme surrounding the chondrogenic region of the long bones as well as in the apoptotic region between the developing digits (Jones et al., 1991; Lu et al., 1994). Thus there are now two direct associations between BMP 4 expression and COUP-TFI expression in cell death associated with limb/digit development and in neural crest apoptosis in rhombomeres 3 and 5. Further analysis of BMP 4 expression pattern in which the COUP-TFI gene has been inactivated may help unravel the relationship between COUP-TFI and the BMP 4 gene.
In summary, we have characterized the BMP-4 gene and one of the promoters in the context of primary cultures of fetal rat osteoblasts. Bone cells utilize primarily the upstream 1A promoter. Mouse embryos (9.5 days) also appear to utilize predominantly the 1A promoter. Analysis of the 1A promoter indicates the transcription factor COUP-TFI could modulate BMP-4 gene transcription in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L47480[GenBank].