Isolation of Vgr-2, a Novel Member of the Transforming Growth Factor-PRelated Gene Family
C. Michael Jones, Dominique and Brigid L. M. Hogan Department of Cell Biology Vanderbilt University Medical Nashville, Tennessee 37232
Simon-Chazottes,
Jean-Louis
Guenet,
School
lnstitut Pasteur (D.S.-C., J.-L.G.) Departement d’lmmunologie 75724 Paris Cedex 15, France
A cDNA clone, Vgr-2, with homology to certain members of the transforming growth factor-p superfamily has been isolated from a mouse embryo cDNA library. The encoded protein shows significant similarity to members of the Vg-l/decapentaplegic/bone morphogenetic protein subgroup of the transforming growth factor-p family. Within this group, Vgr-2 is more similar to Xenopus Vg-1 than to any other member so far isolated. The gene is expressed at highest levels during midgestation mouse development, and transcripts are localized by in situ hybridization to the osteogenic zone of developing bone. Vgr-2 is expressed in F9 teratocarcinoma cells, and its RNA levels are down-regulated within 24 h after differentiation with retinoic acid. The genomic organization of Vgr-2 and its location on mouse chromosome 6 are reported. (Molecular Endocrinology 6: 1961-1968, 1992)
and comprises the active TGF-@-related mature molecule. Of the TGF-P superfamily, activin has received considerable attention recently, as it has been shown to be a morphogen, instructing naive amphibian tissue to differentiate into a wide variety of mesodermal derivatives (9, 10). Another member, the product of the Drosophila Dpp gene, acts initially in establishing the dorsalventral pattern of developing larvae and later in imaginal disc (11) and midgut differentiation (12-14). Transcripts of the Xenopus Vg-1 gene are localized to the vegetal hemisphere of the frog oocyte and embryo (15) and their localization makes the protein a candidate mesoderm inducing factor. However, no function has yet been shown for the gene product. The BMPs induce de nova bone formation when injected SC into rats (16), and their expression patterns during mouse embryogenesis suggest their involvement in a variety of developmental processes (17-I 9). These include early mesodermal patterning (BMP-4) and a variety of epithelial-mesenchymal interactions, including those involved in limb, heart, and whisker follicle development (BMP-2 and -4). Additionally, BMP6 (also known as Vgr-I) is expressed in the epithelial layer of both developing skin (suprabasal layer) and forestomach (regions of stratified squamous epithelium), as well as within the developing nervous system. These expression patterns suggest that the BMPs could be key intercellular signaling molecules in many developmental pathways. In an attempt to isolate additional members of the TGF-/3 family related to dpp, Vg-1 , and the BMPs. we screened, at low stringency, a mouse embryo cDNA library with a probe encoding the C-terminal region of the Xenopus Vg-1 cDNA. We report here the isolation of a novel member of this gene family, which we have named Vgr-2 (Vg-related-2). The predicted protein is more similar to that encoded by Xenopus Vg-1 than to any other member of the TGF-P family. Expression during mouse development and differentiation of embryonal carcinoma cells is discussed. In addition, the
INTRODUCTION In recent years, proteins encoded by the transforming growth factor-p (TGF-/I) gene family have been shown to be involved in a variety of developmental processes, in both vertebrates and invertebrates. The family includes members closely related to TGF-PI, including TGF-/?2 and $3 (1) as well as more divergent members such as the activins and inhibins (2-5) and Mullerian inhibiting substance (6). A third group of the superfamily includes Drosophila decapentaplegic (DPP), Xenopus Vg-1, growth/differentiation factor-l (GDF-1) (7) and the bone morphogenetic proteins (BMPs) 2-7 (reviewed in Ref. 8). This group is characterized by the conservation of seven cysteine residues located near the carboxy-terminal end of the molecule. This C-terminal region is proteolytically released from a larger precursor 0888-8809/92/1961-1968$03 00/o Molecular Endocmology Copyright 0 1992 by The Endocme Scmety
1961
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MOL 1962
ENDO.
1992
Vo16No.11
genomic organization of Vgr-2 localization are reported.
RESULTS Cloning
AND
opus Vg-1. Several of the hybridizing clones encoded mouse homologs of BMP-2, -4, and -6 (Vgr-I), while one cDNA proved to encode a novel member of the TGF-P gene family. We have designated this new gene Vgr-2. The 1181 -base pair (bp) nucleotide sequence (Fig. 1) encodes one long open reading frame with a probable translation initiation codon in the context of a consensus translation start site (20) at position 62. Upstream of this first ATG in the cDNA is an in frame stop codon, and both of the other reading frames contain multiple stop codons throughout. No polyadenylation signal (AATAAA) or poly(A)’ tail is contained within this cDNA.
and its chromosomal
DISCUSSION
and Sequence
of Vgr-2
One million recombinants of a mouse 6.5 days post coitum (dpc) cDNA library were screened at low stringency (see Materials and Methods) using a probe encoding the C-terminal TGF-/I-conserved region of Xen-
gaattccctagaccgctgagctgcgcaccccagaggctgctctaccctggctcagacgac
60
CATGCAGCCTTATCMCGGCTTCTGGCGCTTGGCTTCCTTCTGTTMCCCTGCCCTGGGG MQPYQR
120
LLALGBLLLTLPWG
CCAGACATCCGAGTTTCMGACTCTGACCTTTTGCAGTTTCTGGGATTAGAG~GCGCC
180
QTBEFQDSDLLQPLGLEXAP TTCACCTCACAGGTTCCMCCTGTGCCTCGCGTCTT~GG~TCATCCGGGCTCGAGA 8
240
PHRFQPVPRVLRKI
I
R
A
R
E
AGCCGCTGCAGCCAGTGGGGCCTCGCAGGACTTATGCTACGTGMGGAGCTGGGTGTTCG A
A
A
A
8
300
GASQDLCYVKELGVR
TGGGAACCTGCTTCAGCTTCTCCCAGACCAG'GGTTTTTTCCTT~TACACAG~CCTTT GNLLQLLPDQGF
360
PLNTQKPF
CCMGATGGCTCCTGTCTCCAGMGGTCCTCTAAC
420
QDGSCLQKVLYPN^LSAIKEK 480
GGCAAAGTTGACCATGGCCCAGCTGACTCTAGACTTGGGGCCCAGGTCCTACTATMCCT AILTMAQL
T
L
D
L
G
P
R
S
Y
Y
N
L 340
GCGACCAGAGCTGGTGGTTGCTCTGTCTGTGGTTCAGGACCGGGGCGTGTGGGGGCGATC RPELVVAL
8
V
V
Q
DRGVWGRS
CCACCCTMGGTGCGGAGATTCGTTTTTCTGCGGTCTGTCCCTGGGCCTCMGGTCAGCT H
P
RVRRFVF
600
LRSVPGPQGQL 660
CCAGTTCAACCTGCAGGGTGCGCTTMGGATTGGAGCAGC~CCGACTGMGMTTTGGA QFNLQGALKDWSSNRLRNLD CTTACACTTAGAGATTTTGGTCAAAOAOGAGGACAGATACTCCAGGGTMCTGTCCAGCCCGA L
H
L
E
I
720
LVREDRYSRVTVQPE 780
GMCCCCTGTGACCCGCTGCTCCGCTCTCTACATGCCTCGCTGCTGGTGGTMCCCTCM N
P
CDPLLRSLHAS
LLVVTLN 840
TCCTAAACACTGTCATCCTTCTTCCAG~GGAGGGCGGCCATCTCTGTCCCC~GGG P
K
w
c
I1
P
8
SRKRRAAI
8
VP
K
G 900
TTTCTGTAGGAACTTCTGCCACCGTCATCAGCTGTTCATCMCTTCCAGGACCTGGGTTG F
CRNFCHRHQLFINFODLGW 960
GCACAAGTGGGTCATCGCCCCTMGGGGTTCATGGC~TTACTGTCATGGAGAGTGCCC H
K
w
v
IAPKGFMANYCHGECP
1020
CTTCTCMTGACCACGTATTTAAATAGTTCCMTTATCGTTTCATGCAGGCTCTGATGCA F
SNYRFMOALMR
SMTTYLN-8
TATGGCTGACCCCAAGGTCCCCAAGGCTGTCTGTGTCCCCACC~GCTCTCGCCCATCTC .M
A
D
P
K
1080
TKLSPIS
VPKAVCVP
CATGCTCTATCAGGATAGTGATAAGAACGTCATTCTCCGACATTATG~GACATGGTAGT D
MLYODS
K
NV
CGATGAGTGTGGGTGTGGGTAGtCtCgggaCtaggCtagga D
Em-C
G
C
G
I
L
R
H
Y
1140 E
D
M
V
V
1181
l
Fig. 1. Nucleotide and Deduced Amino Acid Sequence of the Vgr-2 cDNA Numbers on the right represent nucleotide positions relative to the 5’-end of the cDNA. 5’- and 3’-Untranslated represented by lower case letters. The C-terminal TGF-fl conserved region is underlined. The position of the intron the arrowhead. Two potential N-linked glycosylation sites are represented by carats. The asterisk denotes the stop
regions is depicted codon.
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are by
Isolation
and Characterization
of Vgr-2
1963
A Vgr-2
266
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. /3, TGF+l
CHRHQLFINF
-QDLGWHKWV
IAPKGFMANY
CHGECPFSMT
TYLNSS----
CKKRHLYVEF CRTRRLHVSF CRRHSLYVDF CKRHPLYVDF CARRYLKVDF CRRHSLYVDF CKKHELYVSF CKKHELYVSF CKKHELYVSF CKK-QFFVSF CVR-QLYIDF
-KDVGWQNWV -REVGWHRWV -SDVGWDDWI -SDVGWNDWI -ADIGWSEWI -SDVGWNDWI -RDLGWQDWI -QDLGWQDWI -RDLGWQDWI -KDIGWNDWI RKDLGWK-WI
IAPQGYMANY IAPRGFLANF VAPLGYDAYY VAPPGYHAPY ISPKSFDAYY VAPPGYQAFY IAPEGYAAFY IAPKGYAANY IAPEGYAAYY IAPSGYHANY HEPKGYHANF
CYGECPYPLT CQGTCALPET CHGKCPFPLA CHGECPFPLA CSGACQFPMP CHGDCPFPLA CDGECSFPLN CDGECSFPLN CEGECAFPLN CEGECPSHIA CLGPCP-YIW
EILNGS---LRGPGGPPAL DHFNST---DHLNST---KSLKPS---DHLNST---AHMNAT---AHMNAT---SYMNAT---GTSGSSL--8 ----SLD--T
l
Vgr-2
312
*
l
NYRFMQALMH
MADP-K--VP
KAECVPTKLS
PISMLYQDSD
KNVILRHYED
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. on TGF-@l
NHAILQTLVH NNAVLRALMH NHAWQTLVN NHAIVQTLVN NHATIQSIVR NHAIVQTLVN NHAIVQTLVH NHAIVQTLVH NNAIVQTLVH FHSTVINHYR QYSKVLALYN
SIEPED--1P AAAPTPG-AG NMNPGK--VP SVNS-K--1P AVGWPG-IP SVNSS---IP LMFPDH--VP LMNPEY--VP FINPET--VP MRGHSPFANL --QHNPGASA
LPCCVPTKMS SPCCVPERLS KACCVPTQLD KACCVPTELS EPCCVPEKMS KACCVPTELS KPCCAPTKLN KPCCAPTKLN KPCCAPTQLN KSCCVPTKLR APCCVPQALE
PISMLFYDNN PISVLFFDNS SVAMLYLNDQ AISMLYLDEN SLSILFFDEN AISMLYLDEY AISVLYFDDS AISVLYFDDN AISVLYFDDS PMSMLYYDDG PLPIVYYVGR
DNVVLRHYEN DNVVLRHYED STWLKNYQE EKWLKNYQD KNVVLKVYPN DKWLKNYQE SNVILKKYRN SNVILKKYRN SNVILKKYRN QNIIKKDIQN KPKV-EQLSN
Vgr-2
MVVDECGCG
360
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. p,, TGF-Bl
MAVDECGCR MVVDECGCR MTWGCGCR MVVEGCGCR MTVESCACR MWEGCGCR MVVRSCGCH MVVRACGCH MVVRACGCH MIVEECGCS MIVRSCKCS l
"gr-2 "g-1 GOF-1
DPP BNP-2
*
Vgr-2
Vg-1
GDP-1
DpP
100
79 100
71 75
68 77 63 100
100
BMP-2 BHP-3 15
a3
64 71
68
66
a7 100
IMP-3
BMP-4 BMP-5 BW-6 BMP-7 Inh B,
TGF-Bl
B"P-4 74 62
67 68 100
69
88
97 69 100
w-5 67 77
68 lb lb 66 lb 100
w-6
w-7
Inhg,TGF-,?l
72 16
69
61
50
78
69
50 47
lb
78
67 62
78 64
57
46
93 91 100
67 62 59 62
49 50 50
67 77 66 lb
93 100
78
it
100
48
4a
48
48 100
Fig. 2. Comparison of Vgr-2 Amino Acid Sequence with Other Members of the TGF-P Gene Family A, Alignment of the C-terminal region of Vgr-2 with the corresponding region of all other characterized members of the Vg-1 /Dpp group. TGF-pl and inhibin PA sequences are included for comparison. Alignment begins at the first conserved cysteine of each molecule and extends to the C-terminus. Numbers represent amino acid positions encoded by the Vgr-2 cDNA. The positions of the seven conserved cysteines characteristic of the family are marked by asterisks, and the residues are shaded. Double underlining marks the valine contained in the Vgr-2 cDNA, where all other TGF-P-like molecules contain a cysteine (see text). Dashes represent gaps introduced to optimize alignments. B, Percent amino acid similarity between members of the TGF-0 family of molecules. Numbers represent percent similarity considering conservative amino acid changes using the Gap program from the sequence comparison software of Genetics Computer Group (University of Wisconsin, Madison, WI): gap wt = 5; length wt = 0.3.
The predicted protein sequence of Vgr-2 encodes a polypeptide of 366 amino acids. The amino terminus contains a core stretch of 13 hydrophobic amino acids characteristic of a leader sequence, suggesting that Vgr-2 is a secreted molecule. Two potential N-linked glycosylation sites are present at amino acid positions
113 and 308. Comparison of the C-terminal 118 amino acids of Vgr-2 with other TGF+like proteins reveals significant homology to members of the Vg-@p-related group of molecules (Fig. 2A). The predicted protein encodes a potential dibasic cleavage site preceding the presumed mature region of the molecule which contains
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Vo16No.11
MOL ENDO. 1992 1964
is located between kidney androgen-regulated protein (KAP) (23) and glyceraldehyde 3-phosphate dehydrogenase (GAPD/) (24), with calculated centimorgan (CM) distances as follows: Centromere. CM-KAP. . Fig. 3. Genomic Organization and Restriction Map of the Vgr2 Gene The protein coding sequence is contained on two exons (boxed regions). The stippled area represents the TGF-8 mature region of the encoded protein. The dashed lines at the extreme 5’- and 3’-ends of the coding sequence represent the ends of the cDNA. The intron position was confirmed by sequencing the exon/intron junctions.
most of the invariant residues characteristic of this family. Notably, while all TGF-p-like molecules so far characterized are invariant in the conservation of seven cysteine residues, Vgr-2 encodes a valine at amino acid position 330, where all other TGF-8 molecules contain a cysteine. This change was confirmed when genomic clones encoding mouse Vgr-2 were sequenced, and the human homolog also encodes a valine at this position (Celeste, T., personal communication). The change could have significance in that recent crystallization studies of TGF-02 and comparison with other TGF-Plike molecules demonstrate that this residue (normally a cysteine) is involved in a disulfide linkage between two molecules (21). This disulfide bond participates in formation of a functional dimeric protein. The dimer, however, is stabilized by a variety of other interactions, including hydrophobic interactions and hydrogen bonding, in which several water molecules are involved. Further studies are required to understand the functional significance of this unique amino acid change in Vgr-2. Figure 2B illustrates the percent similarity between Vgr-2 and all other members of the Vg-l/Dpp/BMP group of the TGF-P superfamily. TGF-Pl and inhibin PA are shown for comparison. The Vgr-2 predicted protein is more closely related to Xenopus Vg-1 than to any of the mammalian genes so far analyzed. Southern Blot Analysis Localization of Vgr-2
and Chromosomal
Southern blot analysis of mouse genomic DNA using the entire Vgr-2 cDNA as a probe reveals that Vgr-2 is probably a single copy gene. Using a (C57BL/6 x Mm spretus)Fl x C57BL/6 interspecific back-cross and EcoRl digestion, a restriction fragment length polymorphism was detected, yielding a 3.9-kilobase (kb) M. spretus band and a 3.6-kb C57BL/6 band. Restriction fragment length polymorphism analysis and chromosomal localization techniques used standard procedures (22). Following the segregation of this restriction variant through 72 test animals, the gene was mapped to the distal portion of mouse chromosome 6. The gene
Genomic
. .GAPD-1.4
Organization
f 1.4 CM-Vgr-2-7.1
+ 3.0
of Vgr-2
The cDNA was used to screen a 129 mouse cosmid genomic library, and Vgr-2 genomic sequences were isolated. Restriction mapping and sequencing reveals that the coding sequence of Vgr-2 is contained on two exons (Fig. 3). The position of the exons was confirmed by sequencing the exon-intron boundaries and is designated by an arrowhead in Fig. 1. It is noteworthy that the location of the exonintron boundaries within the protein-coding sequence are in similar positions relative to the translation start as the boundaries in murine BMP-2 and BMP-4 (Lyons, K., and M. Blessing, personal communication). Expression of Vgr-2 during Mouse Embryogenesis and Differentiation of F9 Teratocarcinoma Cells Figure 4A shows that Vgr-2 is expressed at highest levels during midgestation stages of mouse development. Peak expression occurs between 12-14 dpc, after which transcript levels decline. Although originally isolated from a 6.5dpc cDNA library, within the limits of sensitivity of RNase protection, we have been unable to detect expression of the gene in embryonic RNA between 6.5-9.5 dpc. Teratocarcinoma cells, upon differentiation, are thought to mimic developmental processes occurring during early mouse embryogenesis. Northern blot analysis reveals that Vgr-2 is expressed in undifferentiated F9 cells and is represented by a single RNA species of approximately 1.4 kb (data not shown). We then proceeded to induce monolayer cultures of F9 cells to differentiate with retinoic acid (RA), (Bu)$AMP, and isobutylmethyl xanthine, which mimics parietal endoderm differentiation during embryogenesis. Vgr-2 transcripts are induced slightly at 6 and 12 h of differentiation and are then down-regulated by 2436 h after exposure to these agents. The slight downregulation of RNA levels observed at 3 h was reproducible in several different F9 differentiation experiments and could be observed by both Northern blot (not shown) and RNase protection (Fig. 48) analyses. The down-regulation of Vgr-2 RNA levels by 24 h upon F9 cell differentiation is comparable to the decrease in transcript levels observed for BMP4 using similar differentiation conditions (25). In contrast, BMP2 and -6 (Vgr-1) (26) expression are induced during the same differentiation scheme. The fact that Vgr-2 and BMP4 are down-regulated upon differentiation suggests that these molecules may be functionally associated with the undifferentiated stem cell character of these cells. On the other hand, BMP-2 and -6 expression are increased upon F9 cell differentiation, sug-
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Isolation
and Characterization
of Vgr-2
1965
mGAP
Vgr-2
mGAP Fig. 4. RNAse Protection Analysis of Vgr-2 Expression during Mouse Embryogenesis and Differentiation of F9 Teratocarcinoma Cells A, Vgr-2 expression is first detected at 10.5 dpc and increases during midgestation stages of development. Transcript levels then decline to almost undetectable levels by late gestation (17.5 dpc). B, Vgr-2 expression remains fairly constant, with a slight increase during early phases of F9 cell differentiation (6- and 12-h lanes) but then declines significantly by 24 h, and remains at low levels for extended periods of culture (at least 1 week; data not shown). The tRNA control demonstrates specificity of the hybridization, and mGAP (mouse GAPD) serves as a positive control and standard for equal RNA loading in all lanes.
gesting that these gene products are involved in promoting, or are a consequence of, the formation of parietal endoderm-like lineages. Taken together, these data show that expression of several BMP-like molecules can be modulated by RA, resulting in both increased and decreased expression. In situ hybridization studies were carried out on sections of mouse embryos between 6.5 dpc and newborn. Although the cDNA was isolated from an early embryonic cDNA library, by the sensitivity of our in situ hybridization protocol (and RNase protection assays), we have been unable to detect localized expression of Vgr-2 during early gestational stages. However, localized expression is detected in developing bone in mid-
gestation embryos (Fig. 5). Transcripts have been localized in both endochondral and intramembranous bone, including the long bones, calvarium, bones of the developing head, and in the developing ribs (Fig. 5). The highest levels of expression in embryonic bone seem to occur around 14.5 dpc, the stage when most bone in the developing mouse embryo is beginning to calcify. In contrast to the in situ localization of Vgr-2 transcripts to the osteogenic zone of newly calcifying bone, other members of the BMP group of molecules are expressed in developing bone, but at different embryonic stages and in different regions of the bone. For example, BMP2 transcripts are detected in condensing precartilagenous mesenchyme and later in the hypertropic cartilage
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MOL 1966
ENDO.
1992
Fig. 5. Localization Brightfield (A and Vgr-2 expression is Vgr-2 expression is shown). In all cases, perichondrium; pnc,
of Vgr-2 Transcripts in Developing Mouse Embryo Bone by in Situ Hybridization C) and darkfield (B and D) photomicrographs of 14.5dpc developing ribs (A and B) and calvarium (C and D). detected in the osteogenic zone (arrow, oz) of the calcifying ribs and in the ossifying calvarium (c). In addition, detected in the osteogenic zone of developing long bones and in all bones of the developing head (data not sense strand control probes showed no specific or localized hybridization (data not shown). r, Rib; pc, posterior neocortex of the developing brain. Bar = 150 pm and applies to all panels.
of developing bone, while BMPB (Vgr-1) transcripts are only detected in hypertrophic cartilage of more mature bones (27). These patterns of expression indicate that bone formation is a complex process, involving various members of the BMP family of molecules. Each exhibits a distinct spatial and temporal expression pattern within embryonic bone. Establishing a role for Vgr-2 in this process awaits further experimentation. In conclusion, molecules of the TGF-P superfamily are involved in a wide range of differentiation events during all stages of development. The amino acid sequence of Vgr-2 establishes it as a member of this gene family, and its expression during mouse development suggests that it is involved in the complex process of bone formation in the embryo.
MATERIALS
a mouse 6.5dpc cDNA library (kindly provided by David Weng, Johns Hopkins Medical School, Baltimore, MD). Randomprimed probe labeling, phage lifts, and filter hybridization were carried out using standard procedures (28). Hybridization conditions were: 20% formamide, 5x SSC (lx SSC = 0.15 M NaCI. 0.015 M Na citrate, pH 7.0), 5x Denhardt’s, 1% sodium dodecyl sulfate, and 100 pg/ml tRNA at 42 C for 16 h. Filter washing was carried out at 50 C in a solution of 5x SSC, 0.1% sodium dodecyl sulfate. Of 40 hybridizing recombinants isolated, only 1 encoded the Vgr-2 cDNA, and rescreening the library at high stringency with the isolated cDNA only resulted in isolation of the same cDNA. Isolation
Genomic
Clones
The entire Vgr-2 cDNA was used as a probe to screen a 129 mouse cosmid genomic library at high stringency. Several overlapping clones were isolated, and one, which hybridized to probes isolated from extreme 5’- and 3’-ends of the cDNA, was chosen for further analysis.
AND METHODS DNA
Isolation
of Vgr-2
of Vgr-2
Sequencing
cDNA
A 445bp Accl/BstEll restriction fragment encoding most of the 3’ TGF-P conserved region of the Xenopus Vg-1 cDNA (kindly provided by Drs. Leslie Dale and Alan Coleman, University of Birmingham, Birmingham, UK) was used to screen
DNA sequencing was carried out using the dideoxy chain termination method. The cDNA was sequenced in full on both strands, and the coding sequence and exonjintron boundaries were confirmed by sequencing fragments of the genomic clone characterized.
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Isolation
and Characterization
Culture
and
Differentiation
of Vgr-2
1967
of F9 Teratocarcinoma
Cells
F9 cells were cultured in monolayer on gelatinized tissue culture dishes and induced to differentiate with 10m7 M alltrans-RA, 10m4 M CAMP, and 10e4 M isobutylmethyl xanthine as previously described (29). RNase
Protection
and in Situ Hybridization
8.
9.
Analysis 10.
RNase protection analysis was carried out using standard methods (30) with a T3 RNA polymerase in vitro-transcribed RNA probe labeled from a linearized template containing a 260-bpPstl/Pvull fragment from the Vgr-2 cDNA in a Bluescript vector (Stratagene, La Jolla, CA). In situ hybridization was performed exactly as described (19) and used mouse embryos obtained from matings of ICR females (Harlan Sprague-Dawley, Indianapolis, IN) with Swiss-Webster males-(Taconic Farms, Germantown. NY). The time 1200 h on the dav of vaginai plug was considered 0.5 dpc.
11.
12.
13.
Acknowledgments We thank library.
Dr.
Victor
Fet
for
help
in screening
the
cosmid
Received July 14, 1992. Revision received August 20,1992. Accepted August 21,1992. Address requests for reprints to: Dr. Brigid L. M. Hogan, Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232. This work was supported by NIH Grants CA-48799 and HD-28955 (to B.L.M.H.).
14.
15.
16.
17.
REFERENCES 1. Roberts AB, Sporn MB 1990 Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, pp 419472 2. Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ing SY, Gullemin R, Niall H, Seeburg PH 1985 Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology to transforming growth factor-p. Nature 318:659-663 3. Forage RG, Ring JM, Brown RW, Mclnerney BV, Cobon GS, Gregson RP, Robertson DM, Morgan FJ, Hearn MTW, Findlay JK, Wettenhall REH, Burger HG, de Kretser DM 1986 Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Nat1 Acad Sci USA 83:3091-3095 4. Eto Y. Tsuji T, Takezawa M, Takano S, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095-l 103 5. Murata M, Eto Y, Shibai H, Sakai M, Muramatsu M 1988 Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin PA chain. Proc Nat1 Acad Sci USA 8512434-2438 6. Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AZ, Gash DJ, Chow EP, Fisher RA, Bertonis JM, Torres G, Wallner BP, Ramachandran KL, Ragin RC, Manganaro TF, Maclaughlin DT, Donahoe PK 1986 Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell 45:685-698 7. Lee S 1990 Identification of a novel member (GDF-1) of
18.
19.
20.
21.
22.
23.
24.
25.
26.
the transforming growth factor-8 superfamily. Mol Endocrinol 4:1034-l 040 Lyons KM, Jones CM, Hogan BLM 1991 The DVR gene family in embryonic development. Trends Genet 7:408412 Green JBA, Smith JC 1990 Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347:391-394 Green JBA, Smith JC 1991 Growth factors as morphogens: do gradients and thresholds establish body plan? Trends Genet 7:245-250 Padgett RW, St. Johnston RD, Gelbart WM 1987 A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-@ family. Nature 325:81-84 Reuter R, Panganiban GEF, Hoffmann FM, Scott MP 1990 Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos. Development 110:1031-l 040 Panganiban GEF, Reuter R, Scott MP, Hoffmann FM 1990 A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis. Development 110:1041-1050 lmmergluck K, Lawrence PA, Bienz M 1990 Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62:261-268 Weeks DL, Melton DA 1987 A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-fl. Cell 51:861-867 Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz R, Hewick R, Wang EA 1988 Novel regulators of bone formation: molecular clones and activities. Science 242:1528-l 534 Lyons KM, Pelton RW, Hogan BLM 1989 Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-o-like genes coordinately regulate aspects of embryonic development. Genes Dev 3:1657-l 668 Lyons KM, Pelton RW, Hogan BLM 1990 Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein2A (BMP-2A). Development 109:833-844 Jones CM, Lyons KM, Hogan BLM 1991 Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111:531-542 Kozak M 1984 Compilation analysis of sequences upstream from the translation start site in eukaryotic mRNAs. Nucleic Acids Res 12:857 Schluneger MP, Grutter MG, 1992 An unusual feature revealed by the crystal structure at 2.2 A resolution of hyman transforming growth factor+‘2. Nature 358:430434 Taylor BA 1989 Recombinant inbred strains. In: Lyon MF, Searle AG (eds) Genetic Variants and Strains of the Inbred Mouse, ed 2. Oxford University Press, Oxford Melanitou E, Simon-Chazottes D, Guenet J, Rougeon F 1991 The gene coding for the kidney androgen-regulated protein (Kap) maps between the Gapd and Kras-2 genes on mouse chromosome 6. Mammalian Genome 1:191192 Bruns GA, Pierce P, Regina VM, Gerald PS, 1978 Expression of GAPDH and TPI in dog-rodent hybrids. Cytogenet Cell Genet 22:547-551 Rogers MB, Rosen V, Wozney JM, Gudas LJ 1992 Bone morphogenetic proteins-2 and -4 are involved in the retinoic acid-induced differentiation of embryonal carcinoma cells. Mol Biol Cell 3:189-l 96 Lyons KM, Graycar JL, Lee A, Hashmi S, Lindquist PB,
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MOL 1968
27.
ENDO.
1992
Chen EY, Hogan BLM, Derynck R 1989 Vgr-1 , a mammalian gene related to Xenopus Vg-1 and a new member of the transforming growth factor p gene superfamily. Proc Nat1 Acad Sci USA 86:4554-4558 Lyons KM, Pelton RW, Hogan BLM 1989 Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-p-like genes coordinately regulate aspects of embryonic development. Genes Dev 3:1657-1668
Vo16No.11
28.
29.
30.
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor Hogan BLM, Constantini F, Lacy E 1986 Manipulating the Mouse Embryo. A Laboratory Manual, ed 1. Cold Spring Harbor Laboratory Cold Spring Harbor Gilman M 1989 Current Protocols in Molecular Biology. Greene Publishing Associates, John Wiley and Sons, New York, vol 1
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C. Michael Jones, Dominique and Brigid L. M. Hogan Department of Cell Biology Vanderbilt University Medical Nashville, Tennessee 37232
Simon-Chazottes,
Jean-Louis
Guenet,
School
lnstitut Pasteur (D.S.-C., J.-L.G.) Departement d’lmmunologie 75724 Paris Cedex 15, France
A cDNA clone, Vgr-2, with homology to certain members of the transforming growth factor-p superfamily has been isolated from a mouse embryo cDNA library. The encoded protein shows significant similarity to members of the Vg-l/decapentaplegic/bone morphogenetic protein subgroup of the transforming growth factor-p family. Within this group, Vgr-2 is more similar to Xenopus Vg-1 than to any other member so far isolated. The gene is expressed at highest levels during midgestation mouse development, and transcripts are localized by in situ hybridization to the osteogenic zone of developing bone. Vgr-2 is expressed in F9 teratocarcinoma cells, and its RNA levels are down-regulated within 24 h after differentiation with retinoic acid. The genomic organization of Vgr-2 and its location on mouse chromosome 6 are reported. (Molecular Endocrinology 6: 1961-1968, 1992)
and comprises the active TGF-@-related mature molecule. Of the TGF-P superfamily, activin has received considerable attention recently, as it has been shown to be a morphogen, instructing naive amphibian tissue to differentiate into a wide variety of mesodermal derivatives (9, 10). Another member, the product of the Drosophila Dpp gene, acts initially in establishing the dorsalventral pattern of developing larvae and later in imaginal disc (11) and midgut differentiation (12-14). Transcripts of the Xenopus Vg-1 gene are localized to the vegetal hemisphere of the frog oocyte and embryo (15) and their localization makes the protein a candidate mesoderm inducing factor. However, no function has yet been shown for the gene product. The BMPs induce de nova bone formation when injected SC into rats (16), and their expression patterns during mouse embryogenesis suggest their involvement in a variety of developmental processes (17-I 9). These include early mesodermal patterning (BMP-4) and a variety of epithelial-mesenchymal interactions, including those involved in limb, heart, and whisker follicle development (BMP-2 and -4). Additionally, BMP6 (also known as Vgr-I) is expressed in the epithelial layer of both developing skin (suprabasal layer) and forestomach (regions of stratified squamous epithelium), as well as within the developing nervous system. These expression patterns suggest that the BMPs could be key intercellular signaling molecules in many developmental pathways. In an attempt to isolate additional members of the TGF-/3 family related to dpp, Vg-1 , and the BMPs. we screened, at low stringency, a mouse embryo cDNA library with a probe encoding the C-terminal region of the Xenopus Vg-1 cDNA. We report here the isolation of a novel member of this gene family, which we have named Vgr-2 (Vg-related-2). The predicted protein is more similar to that encoded by Xenopus Vg-1 than to any other member of the TGF-P family. Expression during mouse development and differentiation of embryonal carcinoma cells is discussed. In addition, the
INTRODUCTION In recent years, proteins encoded by the transforming growth factor-p (TGF-/I) gene family have been shown to be involved in a variety of developmental processes, in both vertebrates and invertebrates. The family includes members closely related to TGF-PI, including TGF-/?2 and $3 (1) as well as more divergent members such as the activins and inhibins (2-5) and Mullerian inhibiting substance (6). A third group of the superfamily includes Drosophila decapentaplegic (DPP), Xenopus Vg-1, growth/differentiation factor-l (GDF-1) (7) and the bone morphogenetic proteins (BMPs) 2-7 (reviewed in Ref. 8). This group is characterized by the conservation of seven cysteine residues located near the carboxy-terminal end of the molecule. This C-terminal region is proteolytically released from a larger precursor 0888-8809/92/1961-1968$03 00/o Molecular Endocmology Copyright 0 1992 by The Endocme Scmety
1961
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MOL 1962
ENDO.
1992
Vo16No.11
genomic organization of Vgr-2 localization are reported.
RESULTS Cloning
AND
opus Vg-1. Several of the hybridizing clones encoded mouse homologs of BMP-2, -4, and -6 (Vgr-I), while one cDNA proved to encode a novel member of the TGF-P gene family. We have designated this new gene Vgr-2. The 1181 -base pair (bp) nucleotide sequence (Fig. 1) encodes one long open reading frame with a probable translation initiation codon in the context of a consensus translation start site (20) at position 62. Upstream of this first ATG in the cDNA is an in frame stop codon, and both of the other reading frames contain multiple stop codons throughout. No polyadenylation signal (AATAAA) or poly(A)’ tail is contained within this cDNA.
and its chromosomal
DISCUSSION
and Sequence
of Vgr-2
One million recombinants of a mouse 6.5 days post coitum (dpc) cDNA library were screened at low stringency (see Materials and Methods) using a probe encoding the C-terminal TGF-/I-conserved region of Xen-
gaattccctagaccgctgagctgcgcaccccagaggctgctctaccctggctcagacgac
60
CATGCAGCCTTATCMCGGCTTCTGGCGCTTGGCTTCCTTCTGTTMCCCTGCCCTGGGG MQPYQR
120
LLALGBLLLTLPWG
CCAGACATCCGAGTTTCMGACTCTGACCTTTTGCAGTTTCTGGGATTAGAG~GCGCC
180
QTBEFQDSDLLQPLGLEXAP TTCACCTCACAGGTTCCMCCTGTGCCTCGCGTCTT~GG~TCATCCGGGCTCGAGA 8
240
PHRFQPVPRVLRKI
I
R
A
R
E
AGCCGCTGCAGCCAGTGGGGCCTCGCAGGACTTATGCTACGTGMGGAGCTGGGTGTTCG A
A
A
A
8
300
GASQDLCYVKELGVR
TGGGAACCTGCTTCAGCTTCTCCCAGACCAG'GGTTTTTTCCTT~TACACAG~CCTTT GNLLQLLPDQGF
360
PLNTQKPF
CCMGATGGCTCCTGTCTCCAGMGGTCCTCTAAC
420
QDGSCLQKVLYPN^LSAIKEK 480
GGCAAAGTTGACCATGGCCCAGCTGACTCTAGACTTGGGGCCCAGGTCCTACTATMCCT AILTMAQL
T
L
D
L
G
P
R
S
Y
Y
N
L 340
GCGACCAGAGCTGGTGGTTGCTCTGTCTGTGGTTCAGGACCGGGGCGTGTGGGGGCGATC RPELVVAL
8
V
V
Q
DRGVWGRS
CCACCCTMGGTGCGGAGATTCGTTTTTCTGCGGTCTGTCCCTGGGCCTCMGGTCAGCT H
P
RVRRFVF
600
LRSVPGPQGQL 660
CCAGTTCAACCTGCAGGGTGCGCTTMGGATTGGAGCAGC~CCGACTGMGMTTTGGA QFNLQGALKDWSSNRLRNLD CTTACACTTAGAGATTTTGGTCAAAOAOGAGGACAGATACTCCAGGGTMCTGTCCAGCCCGA L
H
L
E
I
720
LVREDRYSRVTVQPE 780
GMCCCCTGTGACCCGCTGCTCCGCTCTCTACATGCCTCGCTGCTGGTGGTMCCCTCM N
P
CDPLLRSLHAS
LLVVTLN 840
TCCTAAACACTGTCATCCTTCTTCCAG~GGAGGGCGGCCATCTCTGTCCCC~GGG P
K
w
c
I1
P
8
SRKRRAAI
8
VP
K
G 900
TTTCTGTAGGAACTTCTGCCACCGTCATCAGCTGTTCATCMCTTCCAGGACCTGGGTTG F
CRNFCHRHQLFINFODLGW 960
GCACAAGTGGGTCATCGCCCCTMGGGGTTCATGGC~TTACTGTCATGGAGAGTGCCC H
K
w
v
IAPKGFMANYCHGECP
1020
CTTCTCMTGACCACGTATTTAAATAGTTCCMTTATCGTTTCATGCAGGCTCTGATGCA F
SNYRFMOALMR
SMTTYLN-8
TATGGCTGACCCCAAGGTCCCCAAGGCTGTCTGTGTCCCCACC~GCTCTCGCCCATCTC .M
A
D
P
K
1080
TKLSPIS
VPKAVCVP
CATGCTCTATCAGGATAGTGATAAGAACGTCATTCTCCGACATTATG~GACATGGTAGT D
MLYODS
K
NV
CGATGAGTGTGGGTGTGGGTAGtCtCgggaCtaggCtagga D
Em-C
G
C
G
I
L
R
H
Y
1140 E
D
M
V
V
1181
l
Fig. 1. Nucleotide and Deduced Amino Acid Sequence of the Vgr-2 cDNA Numbers on the right represent nucleotide positions relative to the 5’-end of the cDNA. 5’- and 3’-Untranslated represented by lower case letters. The C-terminal TGF-fl conserved region is underlined. The position of the intron the arrowhead. Two potential N-linked glycosylation sites are represented by carats. The asterisk denotes the stop
regions is depicted codon.
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are by
Isolation
and Characterization
of Vgr-2
1963
A Vgr-2
266
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. /3, TGF+l
CHRHQLFINF
-QDLGWHKWV
IAPKGFMANY
CHGECPFSMT
TYLNSS----
CKKRHLYVEF CRTRRLHVSF CRRHSLYVDF CKRHPLYVDF CARRYLKVDF CRRHSLYVDF CKKHELYVSF CKKHELYVSF CKKHELYVSF CKK-QFFVSF CVR-QLYIDF
-KDVGWQNWV -REVGWHRWV -SDVGWDDWI -SDVGWNDWI -ADIGWSEWI -SDVGWNDWI -RDLGWQDWI -QDLGWQDWI -RDLGWQDWI -KDIGWNDWI RKDLGWK-WI
IAPQGYMANY IAPRGFLANF VAPLGYDAYY VAPPGYHAPY ISPKSFDAYY VAPPGYQAFY IAPEGYAAFY IAPKGYAANY IAPEGYAAYY IAPSGYHANY HEPKGYHANF
CYGECPYPLT CQGTCALPET CHGKCPFPLA CHGECPFPLA CSGACQFPMP CHGDCPFPLA CDGECSFPLN CDGECSFPLN CEGECAFPLN CEGECPSHIA CLGPCP-YIW
EILNGS---LRGPGGPPAL DHFNST---DHLNST---KSLKPS---DHLNST---AHMNAT---AHMNAT---SYMNAT---GTSGSSL--8 ----SLD--T
l
Vgr-2
312
*
l
NYRFMQALMH
MADP-K--VP
KAECVPTKLS
PISMLYQDSD
KNVILRHYED
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. on TGF-@l
NHAILQTLVH NNAVLRALMH NHAWQTLVN NHAIVQTLVN NHATIQSIVR NHAIVQTLVN NHAIVQTLVH NHAIVQTLVH NNAIVQTLVH FHSTVINHYR QYSKVLALYN
SIEPED--1P AAAPTPG-AG NMNPGK--VP SVNS-K--1P AVGWPG-IP SVNSS---IP LMFPDH--VP LMNPEY--VP FINPET--VP MRGHSPFANL --QHNPGASA
LPCCVPTKMS SPCCVPERLS KACCVPTQLD KACCVPTELS EPCCVPEKMS KACCVPTELS KPCCAPTKLN KPCCAPTKLN KPCCAPTQLN KSCCVPTKLR APCCVPQALE
PISMLFYDNN PISVLFFDNS SVAMLYLNDQ AISMLYLDEN SLSILFFDEN AISMLYLDEY AISVLYFDDS AISVLYFDDN AISVLYFDDS PMSMLYYDDG PLPIVYYVGR
DNVVLRHYEN DNVVLRHYED STWLKNYQE EKWLKNYQD KNVVLKVYPN DKWLKNYQE SNVILKKYRN SNVILKKYRN SNVILKKYRN QNIIKKDIQN KPKV-EQLSN
Vgr-2
MVVDECGCG
360
vg-1 GDF-1 DPP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 Inh. p,, TGF-Bl
MAVDECGCR MVVDECGCR MTWGCGCR MVVEGCGCR MTVESCACR MWEGCGCR MVVRSCGCH MVVRACGCH MVVRACGCH MIVEECGCS MIVRSCKCS l
"gr-2 "g-1 GOF-1
DPP BNP-2
*
Vgr-2
Vg-1
GDP-1
DpP
100
79 100
71 75
68 77 63 100
100
BMP-2 BHP-3 15
a3
64 71
68
66
a7 100
IMP-3
BMP-4 BMP-5 BW-6 BMP-7 Inh B,
TGF-Bl
B"P-4 74 62
67 68 100
69
88
97 69 100
w-5 67 77
68 lb lb 66 lb 100
w-6
w-7
Inhg,TGF-,?l
72 16
69
61
50
78
69
50 47
lb
78
67 62
78 64
57
46
93 91 100
67 62 59 62
49 50 50
67 77 66 lb
93 100
78
it
100
48
4a
48
48 100
Fig. 2. Comparison of Vgr-2 Amino Acid Sequence with Other Members of the TGF-P Gene Family A, Alignment of the C-terminal region of Vgr-2 with the corresponding region of all other characterized members of the Vg-1 /Dpp group. TGF-pl and inhibin PA sequences are included for comparison. Alignment begins at the first conserved cysteine of each molecule and extends to the C-terminus. Numbers represent amino acid positions encoded by the Vgr-2 cDNA. The positions of the seven conserved cysteines characteristic of the family are marked by asterisks, and the residues are shaded. Double underlining marks the valine contained in the Vgr-2 cDNA, where all other TGF-P-like molecules contain a cysteine (see text). Dashes represent gaps introduced to optimize alignments. B, Percent amino acid similarity between members of the TGF-0 family of molecules. Numbers represent percent similarity considering conservative amino acid changes using the Gap program from the sequence comparison software of Genetics Computer Group (University of Wisconsin, Madison, WI): gap wt = 5; length wt = 0.3.
The predicted protein sequence of Vgr-2 encodes a polypeptide of 366 amino acids. The amino terminus contains a core stretch of 13 hydrophobic amino acids characteristic of a leader sequence, suggesting that Vgr-2 is a secreted molecule. Two potential N-linked glycosylation sites are present at amino acid positions
113 and 308. Comparison of the C-terminal 118 amino acids of Vgr-2 with other TGF+like proteins reveals significant homology to members of the Vg-@p-related group of molecules (Fig. 2A). The predicted protein encodes a potential dibasic cleavage site preceding the presumed mature region of the molecule which contains
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Vo16No.11
MOL ENDO. 1992 1964
is located between kidney androgen-regulated protein (KAP) (23) and glyceraldehyde 3-phosphate dehydrogenase (GAPD/) (24), with calculated centimorgan (CM) distances as follows: Centromere. CM-KAP. . Fig. 3. Genomic Organization and Restriction Map of the Vgr2 Gene The protein coding sequence is contained on two exons (boxed regions). The stippled area represents the TGF-8 mature region of the encoded protein. The dashed lines at the extreme 5’- and 3’-ends of the coding sequence represent the ends of the cDNA. The intron position was confirmed by sequencing the exon/intron junctions.
most of the invariant residues characteristic of this family. Notably, while all TGF-p-like molecules so far characterized are invariant in the conservation of seven cysteine residues, Vgr-2 encodes a valine at amino acid position 330, where all other TGF-8 molecules contain a cysteine. This change was confirmed when genomic clones encoding mouse Vgr-2 were sequenced, and the human homolog also encodes a valine at this position (Celeste, T., personal communication). The change could have significance in that recent crystallization studies of TGF-02 and comparison with other TGF-Plike molecules demonstrate that this residue (normally a cysteine) is involved in a disulfide linkage between two molecules (21). This disulfide bond participates in formation of a functional dimeric protein. The dimer, however, is stabilized by a variety of other interactions, including hydrophobic interactions and hydrogen bonding, in which several water molecules are involved. Further studies are required to understand the functional significance of this unique amino acid change in Vgr-2. Figure 2B illustrates the percent similarity between Vgr-2 and all other members of the Vg-l/Dpp/BMP group of the TGF-P superfamily. TGF-Pl and inhibin PA are shown for comparison. The Vgr-2 predicted protein is more closely related to Xenopus Vg-1 than to any of the mammalian genes so far analyzed. Southern Blot Analysis Localization of Vgr-2
and Chromosomal
Southern blot analysis of mouse genomic DNA using the entire Vgr-2 cDNA as a probe reveals that Vgr-2 is probably a single copy gene. Using a (C57BL/6 x Mm spretus)Fl x C57BL/6 interspecific back-cross and EcoRl digestion, a restriction fragment length polymorphism was detected, yielding a 3.9-kilobase (kb) M. spretus band and a 3.6-kb C57BL/6 band. Restriction fragment length polymorphism analysis and chromosomal localization techniques used standard procedures (22). Following the segregation of this restriction variant through 72 test animals, the gene was mapped to the distal portion of mouse chromosome 6. The gene
Genomic
. .GAPD-1.4
Organization
f 1.4 CM-Vgr-2-7.1
+ 3.0
of Vgr-2
The cDNA was used to screen a 129 mouse cosmid genomic library, and Vgr-2 genomic sequences were isolated. Restriction mapping and sequencing reveals that the coding sequence of Vgr-2 is contained on two exons (Fig. 3). The position of the exons was confirmed by sequencing the exon-intron boundaries and is designated by an arrowhead in Fig. 1. It is noteworthy that the location of the exonintron boundaries within the protein-coding sequence are in similar positions relative to the translation start as the boundaries in murine BMP-2 and BMP-4 (Lyons, K., and M. Blessing, personal communication). Expression of Vgr-2 during Mouse Embryogenesis and Differentiation of F9 Teratocarcinoma Cells Figure 4A shows that Vgr-2 is expressed at highest levels during midgestation stages of mouse development. Peak expression occurs between 12-14 dpc, after which transcript levels decline. Although originally isolated from a 6.5dpc cDNA library, within the limits of sensitivity of RNase protection, we have been unable to detect expression of the gene in embryonic RNA between 6.5-9.5 dpc. Teratocarcinoma cells, upon differentiation, are thought to mimic developmental processes occurring during early mouse embryogenesis. Northern blot analysis reveals that Vgr-2 is expressed in undifferentiated F9 cells and is represented by a single RNA species of approximately 1.4 kb (data not shown). We then proceeded to induce monolayer cultures of F9 cells to differentiate with retinoic acid (RA), (Bu)$AMP, and isobutylmethyl xanthine, which mimics parietal endoderm differentiation during embryogenesis. Vgr-2 transcripts are induced slightly at 6 and 12 h of differentiation and are then down-regulated by 2436 h after exposure to these agents. The slight downregulation of RNA levels observed at 3 h was reproducible in several different F9 differentiation experiments and could be observed by both Northern blot (not shown) and RNase protection (Fig. 48) analyses. The down-regulation of Vgr-2 RNA levels by 24 h upon F9 cell differentiation is comparable to the decrease in transcript levels observed for BMP4 using similar differentiation conditions (25). In contrast, BMP2 and -6 (Vgr-1) (26) expression are induced during the same differentiation scheme. The fact that Vgr-2 and BMP4 are down-regulated upon differentiation suggests that these molecules may be functionally associated with the undifferentiated stem cell character of these cells. On the other hand, BMP-2 and -6 expression are increased upon F9 cell differentiation, sug-
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Isolation
and Characterization
of Vgr-2
1965
mGAP
Vgr-2
mGAP Fig. 4. RNAse Protection Analysis of Vgr-2 Expression during Mouse Embryogenesis and Differentiation of F9 Teratocarcinoma Cells A, Vgr-2 expression is first detected at 10.5 dpc and increases during midgestation stages of development. Transcript levels then decline to almost undetectable levels by late gestation (17.5 dpc). B, Vgr-2 expression remains fairly constant, with a slight increase during early phases of F9 cell differentiation (6- and 12-h lanes) but then declines significantly by 24 h, and remains at low levels for extended periods of culture (at least 1 week; data not shown). The tRNA control demonstrates specificity of the hybridization, and mGAP (mouse GAPD) serves as a positive control and standard for equal RNA loading in all lanes.
gesting that these gene products are involved in promoting, or are a consequence of, the formation of parietal endoderm-like lineages. Taken together, these data show that expression of several BMP-like molecules can be modulated by RA, resulting in both increased and decreased expression. In situ hybridization studies were carried out on sections of mouse embryos between 6.5 dpc and newborn. Although the cDNA was isolated from an early embryonic cDNA library, by the sensitivity of our in situ hybridization protocol (and RNase protection assays), we have been unable to detect localized expression of Vgr-2 during early gestational stages. However, localized expression is detected in developing bone in mid-
gestation embryos (Fig. 5). Transcripts have been localized in both endochondral and intramembranous bone, including the long bones, calvarium, bones of the developing head, and in the developing ribs (Fig. 5). The highest levels of expression in embryonic bone seem to occur around 14.5 dpc, the stage when most bone in the developing mouse embryo is beginning to calcify. In contrast to the in situ localization of Vgr-2 transcripts to the osteogenic zone of newly calcifying bone, other members of the BMP group of molecules are expressed in developing bone, but at different embryonic stages and in different regions of the bone. For example, BMP2 transcripts are detected in condensing precartilagenous mesenchyme and later in the hypertropic cartilage
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MOL 1966
ENDO.
1992
Fig. 5. Localization Brightfield (A and Vgr-2 expression is Vgr-2 expression is shown). In all cases, perichondrium; pnc,
of Vgr-2 Transcripts in Developing Mouse Embryo Bone by in Situ Hybridization C) and darkfield (B and D) photomicrographs of 14.5dpc developing ribs (A and B) and calvarium (C and D). detected in the osteogenic zone (arrow, oz) of the calcifying ribs and in the ossifying calvarium (c). In addition, detected in the osteogenic zone of developing long bones and in all bones of the developing head (data not sense strand control probes showed no specific or localized hybridization (data not shown). r, Rib; pc, posterior neocortex of the developing brain. Bar = 150 pm and applies to all panels.
of developing bone, while BMPB (Vgr-1) transcripts are only detected in hypertrophic cartilage of more mature bones (27). These patterns of expression indicate that bone formation is a complex process, involving various members of the BMP family of molecules. Each exhibits a distinct spatial and temporal expression pattern within embryonic bone. Establishing a role for Vgr-2 in this process awaits further experimentation. In conclusion, molecules of the TGF-P superfamily are involved in a wide range of differentiation events during all stages of development. The amino acid sequence of Vgr-2 establishes it as a member of this gene family, and its expression during mouse development suggests that it is involved in the complex process of bone formation in the embryo.
MATERIALS
a mouse 6.5dpc cDNA library (kindly provided by David Weng, Johns Hopkins Medical School, Baltimore, MD). Randomprimed probe labeling, phage lifts, and filter hybridization were carried out using standard procedures (28). Hybridization conditions were: 20% formamide, 5x SSC (lx SSC = 0.15 M NaCI. 0.015 M Na citrate, pH 7.0), 5x Denhardt’s, 1% sodium dodecyl sulfate, and 100 pg/ml tRNA at 42 C for 16 h. Filter washing was carried out at 50 C in a solution of 5x SSC, 0.1% sodium dodecyl sulfate. Of 40 hybridizing recombinants isolated, only 1 encoded the Vgr-2 cDNA, and rescreening the library at high stringency with the isolated cDNA only resulted in isolation of the same cDNA. Isolation
Genomic
Clones
The entire Vgr-2 cDNA was used as a probe to screen a 129 mouse cosmid genomic library at high stringency. Several overlapping clones were isolated, and one, which hybridized to probes isolated from extreme 5’- and 3’-ends of the cDNA, was chosen for further analysis.
AND METHODS DNA
Isolation
of Vgr-2
of Vgr-2
Sequencing
cDNA
A 445bp Accl/BstEll restriction fragment encoding most of the 3’ TGF-P conserved region of the Xenopus Vg-1 cDNA (kindly provided by Drs. Leslie Dale and Alan Coleman, University of Birmingham, Birmingham, UK) was used to screen
DNA sequencing was carried out using the dideoxy chain termination method. The cDNA was sequenced in full on both strands, and the coding sequence and exonjintron boundaries were confirmed by sequencing fragments of the genomic clone characterized.
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Isolation
and Characterization
Culture
and
Differentiation
of Vgr-2
1967
of F9 Teratocarcinoma
Cells
F9 cells were cultured in monolayer on gelatinized tissue culture dishes and induced to differentiate with 10m7 M alltrans-RA, 10m4 M CAMP, and 10e4 M isobutylmethyl xanthine as previously described (29). RNase
Protection
and in Situ Hybridization
8.
9.
Analysis 10.
RNase protection analysis was carried out using standard methods (30) with a T3 RNA polymerase in vitro-transcribed RNA probe labeled from a linearized template containing a 260-bpPstl/Pvull fragment from the Vgr-2 cDNA in a Bluescript vector (Stratagene, La Jolla, CA). In situ hybridization was performed exactly as described (19) and used mouse embryos obtained from matings of ICR females (Harlan Sprague-Dawley, Indianapolis, IN) with Swiss-Webster males-(Taconic Farms, Germantown. NY). The time 1200 h on the dav of vaginai plug was considered 0.5 dpc.
11.
12.
13.
Acknowledgments We thank library.
Dr.
Victor
Fet
for
help
in screening
the
cosmid
Received July 14, 1992. Revision received August 20,1992. Accepted August 21,1992. Address requests for reprints to: Dr. Brigid L. M. Hogan, Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232. This work was supported by NIH Grants CA-48799 and HD-28955 (to B.L.M.H.).
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