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Activin βC(Inhbc) Gene
GENOMICS
32, 358 –366 (1996) 0130
ARTICLE NO.
Structure, Chromosomal Localization, and Expression Analysis of the Mouse Inhibin/Activin bC (Inhbc) Gene JACKY SCHMITT,*,1 GERTRUD HO¨ TTEN,† NANCY A. JENKINS,‡ DEBRA J. GILBERT,‡ NEAL G. COPELAND,‡ JENS POHL,† AND HEINRICH SCHREWE*,2 *Max-Planck-Institute of Immunobiology, Department of Developmental Biology, Stu¨beweg 51, 79108 Freiburg, Germany; †Biopharm GmbH, Czernyring 22, 69115 Heidelberg, Germany; and ‡Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received September 12, 1995; accepted December 21, 1995
The mouse inhibin/activin b C gene (Inhbc), a member of the transforming growth factor-b (TGF-b) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin b C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation. q 1996 Academic Press, Inc.
INTRODUCTION
Activins and inhibins were first identified as gonadal proteins capable of homeostatically regulating the secretion of follicle stimulating hormone (FSH) by cells of the anterior pituitary. Biochemical and molecular analyses of the proteins indicated that they are members of the transforming growth factor-b (TGF-b) superfamily (Massague´ et al., 1994). Genes coding for the inhibin and activin A and B proteins are grouped within the inhibin subfamily and are named inhibin a, inhibin bA , and inhibin bB , respectively. Their gene products are synthesized as preproprotein precursors, which are matured to an 18-kDa a subunit or to 14kDa b subunits (for review, see Vale et al., 1990). Activins (A, B, and AB), as biologically active proteins, are 1
Present address: elias GmbH, Department of Molecular and Cellular Biology, Obere Hardtstrasse 18, 79114 Freiburg, Germany 2 To whom correspondence should be addressed. Telephone: 49761-5108-580. Fax: 49-761-5108-221. E-mail: [email protected].
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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homo/heterodimers of two closely related b subunits (bAbA , bB bB , and bAbB , respectively). Inhibins are dimers composed of an a subunit associated with either of the two b chains (Vale et al., 1990). In addition to their effect on FSH production, activins have been shown to regulate growth and differentiation of embryonal carcinoma cells (Schubert and Kimura, 1991), to induce erythropoiesis (Huylebroeck et al., 1990), to stimulate insulin secretion from the pancreas (Totsuka et al., 1988), and to promote neural cell survival (Hashimoto et al., 1990). Activins have also been shown to regulate vertebrate embryonic development. They can induce formation of mesoderm in isolated Xenopus animal cap explants, and their ectopic expression in amphibian embryos results in the formation of a secondary body axis (Klein and Melton, 1994; Thomsen et al., 1990). Exogenous activin induces axial structures in chick (Mitrani et al., 1990) and zebrafish embryos (SchulteMerker et al., 1992). Activin b A and b B subunits are expressed in early pre- and postimplantation mouse embryos (Albano et al., 1993; Manova et al., 1992; van den Eijden-van Raaij et al., 1992). Functional analysis of activin bA and bB genes during mammalian development suggested, however, that they are not essential for murine embryonic development (Matzuk et al., 1995a,b; Schrewe et al., 1994; Vassalli et al., 1994). Inhibin/activins mediate their biological effects through transmembrane receptors characterized by an intracellular serine/threonine kinase domain (Kingsley, 1994; tenDijke et al., 1994). Heteromeric complexes of type I and type II receptors initiate a not yet identified signal transduction cascade leading to the activation of responsive genes (Attisano et al., 1994). We recently identified a cDNA of a new human inhibin/activin bC subunit (Ho¨tten et al., 1995). In this paper, we report the cloning, characterization, and expression analysis of the murine inhibin/activin bC gene. Chromosomal localization has been determined for both the murine and the human genes.
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MATERIALS AND METHODS Isolation of genomic and cDNA clones. A 129/SV-mouse genomic lFIX II phage library was purchased from Stratagene. One million plaques, grown on XL1-Blue MRA(P2), were lifted in duplicates on nitrocellulose BA-S 85 membranes (Schleicher & Schuell). Filters were hybridized in a solution containing 51 SSC, 51 Denhardt solution, 0.5% SDS overnight at 657C with an [a-32 P]dCTP-labeled probe from the second exon of the mouse inhibin/activin bC gene. The probe was prepared by PCR from genomic mouse DNA using oligonucleotide primers that correspond to the nucleotide sequence of the human inhibin/activin bC cDNA (positions 829 to 847 and positions 1145 to 1164, respectively) (Ho¨tten et al., 1995). Hybridizing phages were plaque purified a further two rounds, and their inserts were subcloned into pBluescript II SK(/) (Stratagene). The identity of hybridizing clones was confirmed by DNA sequencing. An oligo(dT)-primed mouse liver cDNA library, constructed in lgt10, was screened under the same hybridization conditions and with the same probe as above. DNA sequencing. DNA sequencing was performed by the dideoxy technique using the Sequenase enzyme (United States Biochemicals) or with an ALF sequencer (PudongGene, Kranenburg, Germany). Primer extension experiment. The transcriptional start sites of the mouse inhibin/activin bC were determined by primer extension analysis according to a previously described method (Schrewe et al., 1990). We used the oligonucleotide 5*-CAAAGCCTATATTCAGGGTC-3*, corresponding to positions 86 to 103 of the complementary strand of the activin bC mRNA. For the annealing reaction, we mixed 10 ng 5* [ 32P]-end-labeled oligonucleotide with 40 mg of total liver RNA and, as a control, with 40 mg of yeast RNA. Extension products were resolved on 6% polyacrylamide/8 M urea gels and detected by autoradiography. Interspecific backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J 1 Mus spretus)F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 backcross mice were used to map the inhibin/activin bC (Inhbc) locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as previously described (Jenkins et al., 1982). All blots were prepared with Hybond-N/ nylon membrane (Amersham). The probe, a 1.5-kb EcoRI mouse Inhbc cDNA fragment, was labeled with [a-32 P]dCTP by nick-translation (Boehringer Mannheim); washing was performed to a final stringency of 0.81 SSCP, 0.1% SDS, 657C. Fragments of 10.0 and 5.7 kb were detected in BglI-digested C57BL/6J DNA, and fragments of 9.4 and 6.4 kb were detected in BglI-digested M. spretus DNA. The presence or absence of the 9.4and 6.4-kb M. spretus-specific BglI fragments, which cosegregated, was followed in backcross mice. A description of the probes and RFLPs for the loci linked to Inhbc, including transformed mouse 3T3 cell double minute 2 (Mdm2), human glioma-associated oncogene (Gli), and avian erythroblastosis oncogene B-3 (Erbb3), have been reported previously (Justice et al., 1990; Ashar et al., 1994; Copeland et al., 1995). Recombination distances were calculated as described by Green (1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of double and multiple recombination events across the chromosome. Fluorescence in situ hybridization. Lymphocytes isolated from human cord blood were cultured in a-minimal essential medium (MEM) supplemented with 10% fetal calf serum and phytohemagglutanin (PHA) at 377C for 68 –72 h. The lymphocyte cultures were treated with 0.18% BrdU (Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 377C for 6 h in aMEM with 2.5 mg/ml thymidine (Sigma). Cells were harvested and slides were made by using standard procedures including hypotonic treatment, fix, and air-dry. A 1.3-kb cDNA fragment from human inhibin/activin bC cDNA (Ho¨tten et al., 1995) was biotinylated with dATP for 2 h at 157C using the BioNick labeling kit (BRL) (Heng et
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al., 1992, 1994). The procedure for fluororescence in situ hybridization (FISH) was performed according to Heng et al. (1992) and Heng and Tsui (1993). Briefly, slides were baked at 557C for 1 h. After RNase A treatment and denaturation in 70% formamide/21 SSC at 707C, the slides were dehydrated with ethanol. The probe was denatured at 757C for 5 min in a hybridization mix consisting of 50% formamide, 10% dextran sulfate, and human cot I DNA, prehybridized for 15 min at 377C, and loaded on the denatured slides. After overnight hybridization, slides were washed, FISH signals and the 4,6-diamino-2-phenylindole-dihydrochloride (DAPI) banding pattern were recorded seperately by taking photographs, and the assignment of the FISH mapping data with the chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (Heng and Tsui, 1993). Riboprobe construction. A 369-bp mouse inhibin/activin bA PCR fragment (equivalent to positions 1087 to 1455 of the rat gene (Esch et al., 1987) and a 255-bp mouse inhibin/activin bB PCR fragment (Schrewe et al., 1994) were subcloned into pGEM-4 (Promega). Resulting plasmids were linearized with HindIII and EcoRI, respectively. A 285-bp mouse inhibin/activin bC fragment (positions 886 to 1170) was subcloned into pBluescript II KS(/) (Stratagene), and the resulting construct was linearized with EcoRI. The plasmid pTRIGAPDH (Ambion), containing a mouse glyceraldehyde phosphate dehydrogenase cDNA fragment, was linearized with DdeI. Antisense riboprobes were synthesized with Sp6 RNA polymerase (bA, 436 bp), T3 RNA polymerase (bC, 345 bp), or T7 RNA polymerase (bB , 271 bp and GAPDH, 208 bp) using the riboprobe kit (Promega) and [a32 P]CTP. The labeled probes were purified on a 6% polyacrylamide/ 8 M urea gel and eluted from the gel in probe elution buffer (Ambion). Ribonuclease protection assay. Total RNA was isolated by the guanidinium thiocyanate –phenol– chloroform technique (Chomczynski and Sacchi, 1987) using RNAzol B (Cinna/Biotecx). Ribonuclease protection analysis was performed with the RPA-II kit (Ambion), using 32P-labeled antisense riboprobes. The inhibin/activin bA , bB , bC , and GAPDH probes were used together in every reaction. Total RNA samples were hybridized overnight at 457C with 105 cpm of each probe. Subsequent steps were performed according to the manufacturer’s instructions (Ambion). Protected fragments were resolved on 6% polyacrylamide/8 M urea gels and detected by autoradiography.
RESULTS
Cloning and Sequencing of the Mouse Inhibin/ Activin bC cDNA A lgt10 mouse liver cDNA library was screened with a 333-bp probe derived from the mouse inhibin/activin bC gene. Six cDNA clones were isolated and subjected to further analysis. The longest clone, designated BCE1, contains a 1565-bp insert comprising the complete coding sequence as well as the 5*- and 3*-untranslated regions (Fig. 1). Nucleotide sequence downstream of the EcoRI site at position 1565 was obtained from genomic DNA (see below). The open reading frame encodes a protein of 352 amino acids with a calculated molecular weight of 39.4 kDa. The deduced amino acid sequence reveals the typical structure of a large precursor molecule. The N-terminal hydrophobic amino acids could serve as a signal peptide for secretion. A putative basic cleavage site (amino acids 232–236) would generate a mature C-terminal polypeptide of 116 amino acids (MW 12.6 kDa). Four potential N-glycosylation sites are present in the prodomain, as indicated in Fig. 1. The preproprotein shares 76% and the mature protein
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et al., 1993) is shown in Fig. 2. The mature bC is 52 and 50% identical with the bA and bB subunits, respectively. Phylogenetic analysis (Gene Works, IntelliGenetics, Inc., CA) aligning different TGF-b family members beginning with the first invariant cysteine residue in the mature region of the proteins indicates that the bC gene product fits into the inhibin/activin subfamily (not shown). All nine conserved cysteine residues, of which seven are conserved in the entire TGF-b superfamily, are indicated by carets (Fig. 2). Genomic Organization of the Mouse Inhibin/Activin bC Gene A lFIX II genomic library from mouse strain 129/SV was screened using the same probe as that for the cDNA clone. Two genomic clones were isolated and subjected to further analysis to establish a restriction map of the corresponding gene, as shown in Fig. 3A. The murine inhibin/activin bC genomic locus (Inhbc) spans approximately 14 kb and is composed of two exons. Both exons were sequenced to map the intron and to characterize the proximal promoter region. Intron/exon boundaries are shown in Fig. 3B. Splice acceptor/donor sites are identical to the consensus sequences established for murine genes (Senapathy et al., 1990). Exon 1 encodes for amino acids 1 to 106, and exon 2 encodes for the remaining C-terminal amino acids (see Fig. 1). The size of the 11.5-kb intron was deduced from the restriction map (Fig. 3). The 3*-untranslated end (downstream of the EcoRI site, position 1565) shown in our cDNA was sequenced on genomic DNA. Use of putative polyadenylation sequences, present at position 1918 or 1935 (see Fig. 1), would yield a messenger RNA of approximately 2 kb. This correlates with our results from Northern blot analysis using total liver RNA (data not shown). Determination of the Transcriptional Start Site To determine the transcriptional start site of the mouse inhibin/activin bC gene, we performed a
FIG. 1. Nucleotide sequence of the mouse inhibin/activin bC cDNA. The figure shows the complete sequence of the BCE1 clone (GenBank/ EMBL Accession No. X90819). Sequence downstream of the EcoRI site at position 1565 has been derived from genomic DNA (GenBank/EMBL Accession No. X90842). The presumed open reading frame, from nucleotides 132 to 1190, is shown by translation into the predicted amino acids, in single-letter code. The positions of the intron (3* of nucleotide 447), of the leader cleavage site (C-terminal of amino acid 20) and of the maturation site (C-terminal of amino acid 236) are indicated by carets. Putative N-glycosylation sites are underlined in the amino acid sequence. The probable polyadenylation sites at position 1919 and 1935 are also underlined.
94% amino acid identity with the corresponding human inhibin/activin bC (not shown). Comparison of the mature bC polypeptide with the other b subunits (Albano
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FIG. 2. Comparison of mature mouse inhibin/activin b subunits. The amino acid alignment of mouse bA , bB , and bC subunits was made using CLUSTAL V (Higgins and Sharp, 1989). For bA and bB , we indicated only the amino acids that are different from those of bC . Numbering starts at the first amino acid after the basic maturation site. Conserved cysteines are indicated by carets.
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FIG. 3. Genomic structure of the mouse inhibin/activin bC gene. (A) Genomic structure and restriction map. Size and relative positions of two l clones used to map the gene are represented at the top. The positions of both exons are indicated by hatched boxes. Restriction sites are as follows: B, BglII; C, ClaI; E, EcoRI; S, SacI; X, XbaI. In the region indicated by a two-headed arrow with open heads, we found four BglII, six EcoRI, and three XbaI sites that are not shown. Below the restriction map, we show corresponding regions of exons and the open reading frame. (B) Sequence of the intron/exon boundaries. Intron sequences are shown in lowercase letters. The size of the intron is indicated in parentheses. The consensus splice donor sequence in rodents is (C/A)AG/GTUAGT, and the consensus acceptor sequence is YYYYYYYYYYNCAG/G (Y, pyrimidine; U, purine; N, any nucleotide) (Senapathy et al., 1990).
primer extension experiment (Fig. 4A). Our result indicates that transcription can be initiated at several sites located between 128 and 136 nucleotides upstream of the translational initiation site. The major transcription start site is indicated as position /1 in the sequence of the proximal inhibin/activin b C promoter (Fig. 4B). The 5*-end of our BCE1 cDNA clone is located at position /6. Genomic sequence upstream of the transcription start site does not show a TATA box sequence. Chromosomal Localization of the Mouse Inhibin/ Activin bC Gene The murine chromosomal location of mouse inhibin/activin b C (locus designated Inhbc) was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J 1 M. spretus)F1 1 C57BL/6J] mice. The interspecific backcross mapping panel has been typed for over 1900 loci that are well distributed among all mouse autosomes and the X chromosome (Copeland and Jenkins, 1991, unpublished results). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using the mouse Inhbc probe (see Materials and Methods). Two spretus-specific BglI RFLPs, which cosegregated, were used to follow the segregation of the Inhbc locus in the backcross DNAs. The mapping results indicated that Inhbc is located in the distal region of chromosome 10 (Fig. 5). Although 188 mice were analyzed for every marker shown in the haplotype analysis (Fig. 5), up to 192 mice were
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typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the combined data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere – Mdm2 – 6 / 190 – Inhbc – 0/ 191 – Gli – 1/ 192 – Erbb3. The recombination frequencies (expressed as genetic distances in centimorgans { the standard error) are centromere – Mdm2– 3.2 { 1.3 –(Inhbc, Gli) – 0.5 { 0.5– Erbb3. The Inhbc and Gli loci cosegregated in 191 animals typed in common, indicating that the two loci are within 1.6 cM of each other (95% confidence limit). Chromosomal Localization of the Human Inhibin/ Activin bC Gene Mapping of the human inhibin/activin bC gene (designated INHBC) was determined by fluorescence in situ hybridization on lymphocytes from human cord blood. Under the conditions used, the hybridization efficiency was approximately 60% for this 1.3-kb INHBC cDNA probe (among 100 checked mitotic figures, 59 of them showed signals on one pair of the chromosomes). Since DAPI banding was used to identify the specific chromosome, the assignment between signal from the probe and the long arm of chromosome 12 was obtained. The detailed position was further determined based on the summary from 10 photos (Figs. 6A and 6B). Therfore, the INHBC gene is located on human chromosome 12, region q13.1. An example of the mapping results is presented in Fig. 6C.
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FIG. 4. Analysis of the proximal promoter region of the mouse inhibin/activin bC gene. (A) Primer extension analysis. Forty micrograms of total RNA from yeast (lane 1) or from liver (lanes 2 and 3) was used in every extension reaction. The major extension product is indicated as position /1. As a size marker, DNA sequencing of the corresponding genomic region was performed using the same oligonucleotide as that for primer extensions (lanes 4– 7). (B) Sequence of the proximal promoter region. Transcription start sites, as determined by primer extension, are underlined, and the major extension product is indicated as position /1. Nucleotide numbering is according to the major transcription start site. Putative transcription factor binding sites found, using Genetics Computer Group programs, are indicated. Translation starts at nucleotide 137.
Expression Analysis of Inhibin/Activin bC in Mouse Tissues and Embryos We performed ribonuclease protection experiments to identify the inhibin/activin-b C-expressing tissues in adult mouse and to determine the stages at which it is expressed in late postimplantation mouse embryos. Probes for all three b chains, as well as for GAPDH, were used in the same reaction. Our results using adult tissues indicate that the Inhbc gene is exclusively expressed in liver (Fig. 7, lanes 12 and 13). Inhibin/activin b A message is present in the uterus, ovary, and liver (Fig. 7, lanes 6, 9, and 12, respectively). The bB gene is expressed in the uterus, testis, ovary, lung, kidney, brain, and CJ7 embryonic stem cells (Fig. 7, lanes 6, 7, 9, 11, 14, 16, and 18, respectively) and possibly in liver. Using RNA from entire embryos, we can detect inhibin/activin b C ex-
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pression from 14.5 days post coitum (d.p.c.) (Fig. 7, lanes 24 to 26); bA and bB messages are already detected in embryos at Day 12.5 d.p.c. (Fig. 7, lanes 22 to 26). DISCUSSION
In this paper, we have characterized the mouse inhibin/activin bC gene (Inhbc). This gene and its product have the characteristic features of members from the TGF-b superfamily. The mature polypeptide shares approximately 50% identity with mouse bA and bB subunits. All nine cysteine residues involved in intra- and intermolecular disulfide bridge formation are conserved. Processing at the maturation site would yield a bC protein of 116 amino acids, compared to those of 114 and 115 amino acids for bA and bB , respectively
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FIG. 5. Mapping of mouse Inhbc to chromosome 10. (Top) The segregation patterns of Inhbc with flanking markers in 188 backcross animals. For some individual pairs of loci, more than 188 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/ 6J 1 M. spretus)F1 parent. The black boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (Bottom) A partial chromosome 10 linkage map showing the location of Inhbc in relation to linked genes. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Library of The John Hopkins University (Baltimore, MD).
(Albano et al., 1993) Furthermore, the mouse inhibin/ activin bC gene structure is very similar to that of the other superfamily members, such as the human bB gene (Mason et al., 1989), the mouse bB gene (Schrewe et al., 1994), or the mouse b A gene (Matzuk et al., 1995a). In summary, these data and phylogenetic analyses strongly suggest that activin bC is a member of the inhibin/activin family within the TGF-b superfamily. Further experiments will be required to determine whether the Inhbc product also shares functional characteristics with the other b subunits. Dimerization of bC with a and/or b subunits would theoretically yield new types of inhibins or activins, respectively. We mapped, by primer extension analysis, the transcription start site of the Inhbc gene. Its promoter is TATA-less, as was found for the human (Mason et al., 1989) and mouse bB genes (Schrewe and Schmitt, unpublished). This feature in the promoter has been shown for a number of genes that are regulated during
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differentiation or development and initiate transcription at only one or a few clustered start sites (Smale and Baltimore, 1989). Inhbc maps to the distal region of mouse chromosome 10. Our placement of Inhbc on mouse chromosome 10 indicates that it is not linked to any other known mouse inhibin/activin gene. The inhibin a (Inha) and activin/ inhibin bA (Inhba) genes map to chromosome 1, while activin/inhibin bB (Inhbb) maps to mouse chromosome 13 (Barton et al., 1989). The mouse inhibin/activin bC gene was localized in close proximity to the human glioma-associated oncogene (Gli locus). Gli belongs to a linkage group common to human chromosome 12 and mouse chromosome 10. In accordance with these data, the human inhibin/activin bC gene was localized to chromosome 12q13.1. An uncloned mouse mutation, named atrichosis, that affects hair distribution and fertility in both sexes has also been mapped to the same chromosomal region as Inhbc (Handel and Eppig, 1979). The phenotype of this mutation affecting spermatogenesis and oocyte maturation could be correlated with the general function of activins and inhibins. If atrichosis or Gli map to the inhibin/activin bC gene, appearance of similar phenotypes might occur in Inhbcdeficient mice. Our expression analysis by ribonuclease protection (Fig. 7) and Northern blot technique (not shown) indicates that the inhibin/activin bC gene is predominantly expressed in adult mouse liver. This specific and high expression level of the bC gene product in liver suggests that it could have a particular function in this organ. Recent reports indeed have shown that activins have biological effects on hepatocytes. Human recombinant activin A acts as an autocrine inhibitor of liver DNA synthesis (Yasuda et al., 1993). It induces rat hepatocyte cell death in vitro and in vivo (Schwall et al., 1993), and rat hepatocytes produce glucose in response to activin (Mine et al., 1989). It remains to be determined whether other homo- or heterodimers containing bA or bB subunits have analogous activities in these systems. We observed that low amounts of activin bA message are present in mouse liver. This would allow the production of bC /bA heterodimers in addition to bC /bC homodimers. Expression of inhibin/activin bB has been detected in human liver (Tuuri et al., 1994). We could not, however, conclude from our result whether inhibin/activin bB is also expressed in mouse liver. Expression of inhibin a could not be detected in rat liver (Meunier et al., 1988; Yasuda et al., 1993). It remains questionable whether inhibins are produced and could antagonize the effects of activins on liver growth. Follistatin, a protein that binds activins and neutralizes their effects in some biological systems, is also expressed in human liver (Tuuri et al., 1994). Follistatin might bind inhibin/activin bC homo- or heterodimer combinations and could influence their function. Activin A is produced in liver after partial hepatectomy and acts as a liver chalone (Yasuda et al., 1993). The
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FIG. 6. Mapping of human INHBC to chromosome 12. (A) Fluorescence in situ hybridization was performed using a biotinylated human cDNA fragment. Arrows indicate FISH signals on chromosomes. (B) The same mitotic figures stained with DAPI to identify chromosome 12. (C) Diagram of FISH mapping results. Each dot represents the double FISH signal detected on human chromosome 12.
‘‘chalone’’ hypothesis on tissue homeostasis was proposed for the presence of stimulators or inhibitors of cell growth (Bullough, 1962; Nadal, 1979). If the inhibitor is produced by hepatocytes, partial hepatectomy would decrease inhibitor concentration and thereby allow cell division. Subsequent cell proliferation would result in an increase in the concentration of inhibitor, which would inhibit cell growth. It remains to be determined how bC expression is regulated during liver regeneration and whether it interacts with the bA subunit. The inhibin/activin bC gene is expressed during late embryonic development in the mouse (after 14.5
d.p.c.). Expression, during mouse development, of the other inhibin/activin subunits has been well documented (Albano et al., 1993, 1994; Feijen et al., 1994; Paulusma et al., 1994). In situ hybridization analyses did not, however, reveal any expression of activin ligands and/or receptors in embryonic liver (Albano et al., 1993, 1994; Feijen et al., 1994; Paulusma et al., 1994). Functional analysis of inhibin/activins bA and bB and activin receptor IIA during mammalian development suggested that they are not essential for embryogenesis in the mouse (Matzuk et al., 1995a). Discrepancies in the results between ligand- and recep-
FIG. 7. Inhibin/activin bA , bB , and bC expression. Unless otherwise indicated, 10 mg total RNA from different adult tissues (lanes 1– 18) or from whole embryos isolated at different stages of development (lanes 19 – 26) was analyzed by ribonuclease protection assay using inhibin/activin bA , bB , and bC cDNA fragments as antisense probes. As control for RNA integrity and loading, a GAPDH riboprobe was also included: lane 1, size marker; lanes 2 and 3, input probes; lane 4, no RNA; lane 5, yeast RNA; lane 6, uterus; lane 7, testis ; lane 8, spleen ; lane 9, ovary; lane 10, skeletal muscle; lane 11, lung; lane 12, liver; lane 13, liver (2 mg); lane 14, kidney; lane 15, heart; lane 16, brain; lane 17, no RNA; lane 18, CJ7 cells; lane 19, size marker; lane 20, input probes; lane 21, 10.5 days post coitum (d.p.c.); lane 22, 12.5 d.p.c.; lane 23, 13.5 d.p.c.; lane 24, 14.5 d.p.c.; lane 25, 15.5 d.p.c.; lane 26, 16.5 d.p.c. The positions of the unprotected and protected fragments are indicated to the left and right, respectively. 32P-end-labeled MspI fragments of pBR322 were used as size markers.
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tor-deficient mice could be interpreted by a functional redundancy of inhibin/activin bC with bA and bB . Production of activin-bC-deficient mice will advance the functional analysis of activins during mouse embryonic development. ACKNOWLEDGMENTS We thank Drs. Gu¨nther Schu¨tz and Wolfgang Schmid for providing us the mouse liver cDNA library. We acknowledge Renate Mielke and Debbie Barnhart for excellent technical assistance. We are grateful to our colleagues Drs. Ernst-Martin Fu¨chtbauer, Matthias Hebrok, John McLaughlin, Davor Solter, and Grace Wei for critical reading and comments about the manuscript. This research was supported, in part, by the American National Cancer Institute, DHHS, under a contract with ABL. J.S. was the recipient of a Max-PlanckSociety postdoctoral fellowship.
Albano, R. M., Arkell, R., Beddington, R. S. P., and Smith, J. C. (1994). Expression of inhibin subunits and follistatin during postimplantation mouse development: Decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain. Development 120: 803– 813. Albano, R. M., Groome, N., and Smith, J. C. (1993). Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 117: 711–723. Ashar, H. R., Benson, K. F., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Chada, K. K. (1994). Ifg, Gli, Mdm1, Mdm2 and Mdm3: Candidate genes for the mouse pg locus. Mamm. Genome 5: 608– 612. Attisano, L., Wrana, J. L., Lopez-Casillas, F., and Massague´, J. (1994). TGF-b receptors and actions. Biochim. Biophys. Acta 1222: 71–80. Barton, D. E., Yang-Feng, T. L., Mason, A. J., Seeburg, P. H., and Francke, U. (1989). Mapping of genes for inhibin subunits a, bA, and bB on human and mouse chromosomes and studies of jsd mice. Genomics 5: 91–99. Bullough, W. S. (1962). Control of mitotic activity in adult mammalian tissues. Biol. Rev. Camb. Phil. Soc. 37: 307–342. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by guanidinium thiocyanate –phenol– chloroform extraction. Anal. Biochem. 162: 156– 159. Copeland, N. G., Gilbert, D. J., Schindler, C., Zhong, Z., Wen, Z., Darnell, J. E., Jr., Mui, A. L.-F., Miyajima, A., Quelle, F. W., Ihle, J. N., and Jenkins, N. A. (1995). Distribution of the mammalian Stat gene family in mouse chromosomes. Genomics 29: 225– 228. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113– 118. Esch, F. S., Shimasaki, S., Cooksey, K., Mercado, M., Mason, A. J., Ying, S.-Y., Ueno, N., and Ling, N. (1987). Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol. Endocrinol. 5: 388 –396. Feijen, A., Goumans, M. J., and van den Eijden-van Raaij, A. J. M. (1994). Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development 120: 3621 –3637. Green, E. L. (1981). Linkage, recombination, and mapping. In ‘‘Genetics and Probability in Animal Breeding Experiments,’’ pp. 77– 113, Oxford Univ. Press, New York. Handel, M. A., and Eppig, J. J. (1979). Sertoli cell differentiation in
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the testes of mice genetically deficient in germ cells. Biol. Reprod. 20: 1031 –1038. Hashimoto, M., Kondo, S., Sakurai, T., Etoh, Y., Shibai, H., and Muramatsu, M. (1990). Activin/EDF as an inhibitor of neural differentiation. Biochem. Biophys. Res. Commun. 173: 193–200. Heng, H. H. Q., Squire, J., and Tsui, L.-C. (1992). High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. USA 89: 9509 –9513. Heng, H. H. Q., and Tsui, L.-C. (1993). Modes of DAPI banding and simultaneous in situ hybridization. Chromosoma 102: 325–332. Heng, H. H. Q., Xiao, H., Shi, X. M., Greenblatt, J., and Tsui, L.-C. (1994). Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome. Hum. Mol. Genet. 3: 61–64. Higgins, D. G., and Sharp, P. M. (1989). Fast and sensitive multiple sequence alignment on a microcomputer. Comput. Appl. Biosci. 5: 151–153. Ho¨tten, G., Neidhardt, H., Schneider, C., and Pohl, J. (1995). Cloning of a new member of the TGF-b family: A putative new activin bC chain. Biochem. Biophys. Res. Commun. 206: 608– 613.
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Huylebroeck, D., Nimmen, K. V., Waheed, A., von-Figura, K., Marmenout, A., Fransen, L., De-Waele, P., Jaspar, J. M., Franchimont, P., Stunnenberg, H., and Heuverswijn, H. v. (1990). Expression and processing of the activin-A/erythroid differentiation factor precursor: A member of the transforming growth factor-beta superfamily. Mol. Endocrinol. 4: 1153 –1165. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982). Organization, distribution and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus. J. Virol. 42: 26– 46. Justice, M. J., Siracusa, L. D., Gilbert, D. J., Heisterkampf, N., Groffen, J., Chada, K., Silan, C. M., Copeland, N. G., and Jenkins, N. A. (1990). A genetic linkage map of mouse chromosome 10: Localization of 18 molecular markers using a single interspecific backcross. Genetics 125: 855–866. Kingsley, D. M. (1994). The TGF-b superfamily: New members, new receptors and new genetic tests of function in different organisms. Genes Dev. 8: 133– 146. Klein, P. S., and Melton, D. A. (1994). Hormonal regulation of embryogenesis: The formation of mesoderm in Xenopus laevis. Endocrinol. Rev. 15: 326– 341. Manova, K., Paynton, B., and Bachvarova, R. F. (1992). Expression of activins and TGFb1 and b2 RNAs in early postimplantation mouse embryos and uterine decidua. Mech. Dev. 36: 141–152. Mason, A. J., Berkemeier, L. M., Schmelzer, C. H., and Schwall, R. H. (1989). Activin B: Precursor sequences, genomic structure and in vitro activities. Mol. Endocrinol. 3: 1352 – 1358. Massague´, J., Attisano, L., and Wrana, J. L. (1994). The TGF-b family and its composite receptors. Trends Cell Biol. 4: 172–178. Matzuk, M. M., Kumar, T. R., Vassali, A., Bickenbach, J. R., Roop, D. R., Jaenisch, R., and Bradley, A. (1995a). Functional analysis of activins during mammalian development. Nature 374: 354–356. Matzuk, M., Kumar, T. R., and Bradley, A. (1995b). Different phenotypes for mice deficient in either activins or activin type II receptor. Nature 374: 356– 360. Meunier, H., Rivier, C., Evans, R. M., and Vale, W. (1988). Gonadal and extragonadal expression of inhibin a, bA and bB subunits in various tissues predicts diverse functions. Proc. Natl. Acad. Sci. USA 85: 247– 251. Mine, T., Kojima, I., and Ogata, E. (1989). Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinology 125: 586– 591. Mitrani, E., Ziv, T., Thomsen, G., Shimoni, Y., Melton, D. A., and Bril, A. (1990). Activin can induce the formation of axial structures and is expressed in the hypoblast of the chick. Cell 63: 495–501.
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Nadal, C. (1979). Control of liver growth by growth inhibitors (chalones). Arch. Toxicol. 35: 131–142. Paulusma, C. C., Kruijssen, C. M. M., and van den Eijden-van Raaij, A. J. M. (1994). Localization of activin subunits in early murine development determined by subunit-specific antibodies. J. Immunol. Methods 169: 143– 152. Schrewe, H., Gendron-Maguire, M., Harbison, M. L., and Gridley, T. (1994). Mice homozygous for a null mutation of activin bB are viable and fertile. Mech. Dev. 47: 43– 51. Schrewe, H., Thompson, J., Bona, M., Hefta, L. J. F., Maruya, A., Hassauer, M., Shively, J. E., von Kleist, S., and Zimmermann, W. (1990). Cloning of the complete gene for carcinoembryonic antigen: Analysis of its promoter indicates a region conveying cell type specific expression. Mol. Cell. Biol. 10: 2738 – 2748. Schubert, D., and Kimura, H. (1991). Substratum-growth factor collaborations are required for the mitogenic activities of activin and FGF on embryonal carcinoma cells. J. Cell Biol. 114: 841–846. Schulte-Merker, S., Ho, S., Herrmann, B. G., and Nu¨sslein-Volhard, C. (1992). The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116: 1021 – 1032. Schwall, R. H., Robbins, K., Jardieu, P., Chang, L., Lai, C., and Terrell, T. G. (1993). Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology 18: 347– 356. Senapathy, P., Shapiro, M. B., and Harris, N. L. (1990). Splice junctions, branch point sites and exons: Sequence statistics, identification and applications to genome project. In ‘‘Methods in Enzymology’’ (R. F. Doolittle, Ed.) Vol. 183, pp. 252–278, Academic Press, New York. Smale, S. T., and Baltimore, D. (1989). The ‘‘initiator’’ as a transcription control element. Cell 57: 103–113.
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tenDijke, P., Franzen, P., Yamashita, H., Ichijo, H., Heldin, C.-H., and Miyazono, K. (1994). Serine/threonine kinase receptors. Prog. Growth Factor Res. 5: 55– 72. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W., and Melton, D. A. (1990). Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell 63: 485– 493. Totsuka, Y., Tabuchi, M., Kojima, I., Shibai, H., and Ogata, E. (1988). A novel action of activin A: Stimulation of insulin secretion in rat pancreatic islands. Biochem. Biophys. Res. Commun. 156: 335 – 339. Tuuri, T., Era¨maa, M., Hilde´ n, K., and Ritvos, O. (1994). The tissue distribution of activin bA - and bB-subunit and follistatin messenger ribonucleic acids suggests multiple sites of action for the activinfollistatin system during development. J. Clin. Endocrinol. Metab. 78: 1521 –1524. Vale, W., Hsueh, A., Rivier, C., and Yu, J. (1990). Peptide growth factors and their receptors. In ‘‘Handbook of Experimental Pharmacology’’ (M. B. Sporn and A. B. Roberts, Eds.) Vol. 95, pp. 2196 – 2203, Springer-Verlag, Berlin. Van den Eijden-van Raaij, A. J. M., Feijen, A., Lawson, K. A., and Mummery, C. L. (1992). Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development. Dev. Biol. 154: 356–365. Vassalli, A., Matzuk, M. M., Gardner, H. A. R., Lee, K.-F., and Jaenisch, R. (1994). Activin/inhibin bB subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev. 8: 414– 427. Yasuda, H., Mine, T., Shibata, H., Eto, Y., Hasegawa, Y., Takeuchi, T., Asano, S., and Kojima, I. (1993). Activin A: An autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J. Clin. Invest. 92: 1491 –1496.
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ARTICLE NO.
Structure, Chromosomal Localization, and Expression Analysis of the Mouse Inhibin/Activin bC (Inhbc) Gene JACKY SCHMITT,*,1 GERTRUD HO¨ TTEN,† NANCY A. JENKINS,‡ DEBRA J. GILBERT,‡ NEAL G. COPELAND,‡ JENS POHL,† AND HEINRICH SCHREWE*,2 *Max-Planck-Institute of Immunobiology, Department of Developmental Biology, Stu¨beweg 51, 79108 Freiburg, Germany; †Biopharm GmbH, Czernyring 22, 69115 Heidelberg, Germany; and ‡Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received September 12, 1995; accepted December 21, 1995
The mouse inhibin/activin b C gene (Inhbc), a member of the transforming growth factor-b (TGF-b) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin b C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation. q 1996 Academic Press, Inc.
INTRODUCTION
Activins and inhibins were first identified as gonadal proteins capable of homeostatically regulating the secretion of follicle stimulating hormone (FSH) by cells of the anterior pituitary. Biochemical and molecular analyses of the proteins indicated that they are members of the transforming growth factor-b (TGF-b) superfamily (Massague´ et al., 1994). Genes coding for the inhibin and activin A and B proteins are grouped within the inhibin subfamily and are named inhibin a, inhibin bA , and inhibin bB , respectively. Their gene products are synthesized as preproprotein precursors, which are matured to an 18-kDa a subunit or to 14kDa b subunits (for review, see Vale et al., 1990). Activins (A, B, and AB), as biologically active proteins, are 1
Present address: elias GmbH, Department of Molecular and Cellular Biology, Obere Hardtstrasse 18, 79114 Freiburg, Germany 2 To whom correspondence should be addressed. Telephone: 49761-5108-580. Fax: 49-761-5108-221. E-mail: [email protected].
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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homo/heterodimers of two closely related b subunits (bAbA , bB bB , and bAbB , respectively). Inhibins are dimers composed of an a subunit associated with either of the two b chains (Vale et al., 1990). In addition to their effect on FSH production, activins have been shown to regulate growth and differentiation of embryonal carcinoma cells (Schubert and Kimura, 1991), to induce erythropoiesis (Huylebroeck et al., 1990), to stimulate insulin secretion from the pancreas (Totsuka et al., 1988), and to promote neural cell survival (Hashimoto et al., 1990). Activins have also been shown to regulate vertebrate embryonic development. They can induce formation of mesoderm in isolated Xenopus animal cap explants, and their ectopic expression in amphibian embryos results in the formation of a secondary body axis (Klein and Melton, 1994; Thomsen et al., 1990). Exogenous activin induces axial structures in chick (Mitrani et al., 1990) and zebrafish embryos (SchulteMerker et al., 1992). Activin b A and b B subunits are expressed in early pre- and postimplantation mouse embryos (Albano et al., 1993; Manova et al., 1992; van den Eijden-van Raaij et al., 1992). Functional analysis of activin bA and bB genes during mammalian development suggested, however, that they are not essential for murine embryonic development (Matzuk et al., 1995a,b; Schrewe et al., 1994; Vassalli et al., 1994). Inhibin/activins mediate their biological effects through transmembrane receptors characterized by an intracellular serine/threonine kinase domain (Kingsley, 1994; tenDijke et al., 1994). Heteromeric complexes of type I and type II receptors initiate a not yet identified signal transduction cascade leading to the activation of responsive genes (Attisano et al., 1994). We recently identified a cDNA of a new human inhibin/activin bC subunit (Ho¨tten et al., 1995). In this paper, we report the cloning, characterization, and expression analysis of the murine inhibin/activin bC gene. Chromosomal localization has been determined for both the murine and the human genes.
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MATERIALS AND METHODS Isolation of genomic and cDNA clones. A 129/SV-mouse genomic lFIX II phage library was purchased from Stratagene. One million plaques, grown on XL1-Blue MRA(P2), were lifted in duplicates on nitrocellulose BA-S 85 membranes (Schleicher & Schuell). Filters were hybridized in a solution containing 51 SSC, 51 Denhardt solution, 0.5% SDS overnight at 657C with an [a-32 P]dCTP-labeled probe from the second exon of the mouse inhibin/activin bC gene. The probe was prepared by PCR from genomic mouse DNA using oligonucleotide primers that correspond to the nucleotide sequence of the human inhibin/activin bC cDNA (positions 829 to 847 and positions 1145 to 1164, respectively) (Ho¨tten et al., 1995). Hybridizing phages were plaque purified a further two rounds, and their inserts were subcloned into pBluescript II SK(/) (Stratagene). The identity of hybridizing clones was confirmed by DNA sequencing. An oligo(dT)-primed mouse liver cDNA library, constructed in lgt10, was screened under the same hybridization conditions and with the same probe as above. DNA sequencing. DNA sequencing was performed by the dideoxy technique using the Sequenase enzyme (United States Biochemicals) or with an ALF sequencer (PudongGene, Kranenburg, Germany). Primer extension experiment. The transcriptional start sites of the mouse inhibin/activin bC were determined by primer extension analysis according to a previously described method (Schrewe et al., 1990). We used the oligonucleotide 5*-CAAAGCCTATATTCAGGGTC-3*, corresponding to positions 86 to 103 of the complementary strand of the activin bC mRNA. For the annealing reaction, we mixed 10 ng 5* [ 32P]-end-labeled oligonucleotide with 40 mg of total liver RNA and, as a control, with 40 mg of yeast RNA. Extension products were resolved on 6% polyacrylamide/8 M urea gels and detected by autoradiography. Interspecific backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J 1 Mus spretus)F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 backcross mice were used to map the inhibin/activin bC (Inhbc) locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as previously described (Jenkins et al., 1982). All blots were prepared with Hybond-N/ nylon membrane (Amersham). The probe, a 1.5-kb EcoRI mouse Inhbc cDNA fragment, was labeled with [a-32 P]dCTP by nick-translation (Boehringer Mannheim); washing was performed to a final stringency of 0.81 SSCP, 0.1% SDS, 657C. Fragments of 10.0 and 5.7 kb were detected in BglI-digested C57BL/6J DNA, and fragments of 9.4 and 6.4 kb were detected in BglI-digested M. spretus DNA. The presence or absence of the 9.4and 6.4-kb M. spretus-specific BglI fragments, which cosegregated, was followed in backcross mice. A description of the probes and RFLPs for the loci linked to Inhbc, including transformed mouse 3T3 cell double minute 2 (Mdm2), human glioma-associated oncogene (Gli), and avian erythroblastosis oncogene B-3 (Erbb3), have been reported previously (Justice et al., 1990; Ashar et al., 1994; Copeland et al., 1995). Recombination distances were calculated as described by Green (1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of double and multiple recombination events across the chromosome. Fluorescence in situ hybridization. Lymphocytes isolated from human cord blood were cultured in a-minimal essential medium (MEM) supplemented with 10% fetal calf serum and phytohemagglutanin (PHA) at 377C for 68 –72 h. The lymphocyte cultures were treated with 0.18% BrdU (Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 377C for 6 h in aMEM with 2.5 mg/ml thymidine (Sigma). Cells were harvested and slides were made by using standard procedures including hypotonic treatment, fix, and air-dry. A 1.3-kb cDNA fragment from human inhibin/activin bC cDNA (Ho¨tten et al., 1995) was biotinylated with dATP for 2 h at 157C using the BioNick labeling kit (BRL) (Heng et
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al., 1992, 1994). The procedure for fluororescence in situ hybridization (FISH) was performed according to Heng et al. (1992) and Heng and Tsui (1993). Briefly, slides were baked at 557C for 1 h. After RNase A treatment and denaturation in 70% formamide/21 SSC at 707C, the slides were dehydrated with ethanol. The probe was denatured at 757C for 5 min in a hybridization mix consisting of 50% formamide, 10% dextran sulfate, and human cot I DNA, prehybridized for 15 min at 377C, and loaded on the denatured slides. After overnight hybridization, slides were washed, FISH signals and the 4,6-diamino-2-phenylindole-dihydrochloride (DAPI) banding pattern were recorded seperately by taking photographs, and the assignment of the FISH mapping data with the chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (Heng and Tsui, 1993). Riboprobe construction. A 369-bp mouse inhibin/activin bA PCR fragment (equivalent to positions 1087 to 1455 of the rat gene (Esch et al., 1987) and a 255-bp mouse inhibin/activin bB PCR fragment (Schrewe et al., 1994) were subcloned into pGEM-4 (Promega). Resulting plasmids were linearized with HindIII and EcoRI, respectively. A 285-bp mouse inhibin/activin bC fragment (positions 886 to 1170) was subcloned into pBluescript II KS(/) (Stratagene), and the resulting construct was linearized with EcoRI. The plasmid pTRIGAPDH (Ambion), containing a mouse glyceraldehyde phosphate dehydrogenase cDNA fragment, was linearized with DdeI. Antisense riboprobes were synthesized with Sp6 RNA polymerase (bA, 436 bp), T3 RNA polymerase (bC, 345 bp), or T7 RNA polymerase (bB , 271 bp and GAPDH, 208 bp) using the riboprobe kit (Promega) and [a32 P]CTP. The labeled probes were purified on a 6% polyacrylamide/ 8 M urea gel and eluted from the gel in probe elution buffer (Ambion). Ribonuclease protection assay. Total RNA was isolated by the guanidinium thiocyanate –phenol– chloroform technique (Chomczynski and Sacchi, 1987) using RNAzol B (Cinna/Biotecx). Ribonuclease protection analysis was performed with the RPA-II kit (Ambion), using 32P-labeled antisense riboprobes. The inhibin/activin bA , bB , bC , and GAPDH probes were used together in every reaction. Total RNA samples were hybridized overnight at 457C with 105 cpm of each probe. Subsequent steps were performed according to the manufacturer’s instructions (Ambion). Protected fragments were resolved on 6% polyacrylamide/8 M urea gels and detected by autoradiography.
RESULTS
Cloning and Sequencing of the Mouse Inhibin/ Activin bC cDNA A lgt10 mouse liver cDNA library was screened with a 333-bp probe derived from the mouse inhibin/activin bC gene. Six cDNA clones were isolated and subjected to further analysis. The longest clone, designated BCE1, contains a 1565-bp insert comprising the complete coding sequence as well as the 5*- and 3*-untranslated regions (Fig. 1). Nucleotide sequence downstream of the EcoRI site at position 1565 was obtained from genomic DNA (see below). The open reading frame encodes a protein of 352 amino acids with a calculated molecular weight of 39.4 kDa. The deduced amino acid sequence reveals the typical structure of a large precursor molecule. The N-terminal hydrophobic amino acids could serve as a signal peptide for secretion. A putative basic cleavage site (amino acids 232–236) would generate a mature C-terminal polypeptide of 116 amino acids (MW 12.6 kDa). Four potential N-glycosylation sites are present in the prodomain, as indicated in Fig. 1. The preproprotein shares 76% and the mature protein
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et al., 1993) is shown in Fig. 2. The mature bC is 52 and 50% identical with the bA and bB subunits, respectively. Phylogenetic analysis (Gene Works, IntelliGenetics, Inc., CA) aligning different TGF-b family members beginning with the first invariant cysteine residue in the mature region of the proteins indicates that the bC gene product fits into the inhibin/activin subfamily (not shown). All nine conserved cysteine residues, of which seven are conserved in the entire TGF-b superfamily, are indicated by carets (Fig. 2). Genomic Organization of the Mouse Inhibin/Activin bC Gene A lFIX II genomic library from mouse strain 129/SV was screened using the same probe as that for the cDNA clone. Two genomic clones were isolated and subjected to further analysis to establish a restriction map of the corresponding gene, as shown in Fig. 3A. The murine inhibin/activin bC genomic locus (Inhbc) spans approximately 14 kb and is composed of two exons. Both exons were sequenced to map the intron and to characterize the proximal promoter region. Intron/exon boundaries are shown in Fig. 3B. Splice acceptor/donor sites are identical to the consensus sequences established for murine genes (Senapathy et al., 1990). Exon 1 encodes for amino acids 1 to 106, and exon 2 encodes for the remaining C-terminal amino acids (see Fig. 1). The size of the 11.5-kb intron was deduced from the restriction map (Fig. 3). The 3*-untranslated end (downstream of the EcoRI site, position 1565) shown in our cDNA was sequenced on genomic DNA. Use of putative polyadenylation sequences, present at position 1918 or 1935 (see Fig. 1), would yield a messenger RNA of approximately 2 kb. This correlates with our results from Northern blot analysis using total liver RNA (data not shown). Determination of the Transcriptional Start Site To determine the transcriptional start site of the mouse inhibin/activin bC gene, we performed a
FIG. 1. Nucleotide sequence of the mouse inhibin/activin bC cDNA. The figure shows the complete sequence of the BCE1 clone (GenBank/ EMBL Accession No. X90819). Sequence downstream of the EcoRI site at position 1565 has been derived from genomic DNA (GenBank/EMBL Accession No. X90842). The presumed open reading frame, from nucleotides 132 to 1190, is shown by translation into the predicted amino acids, in single-letter code. The positions of the intron (3* of nucleotide 447), of the leader cleavage site (C-terminal of amino acid 20) and of the maturation site (C-terminal of amino acid 236) are indicated by carets. Putative N-glycosylation sites are underlined in the amino acid sequence. The probable polyadenylation sites at position 1919 and 1935 are also underlined.
94% amino acid identity with the corresponding human inhibin/activin bC (not shown). Comparison of the mature bC polypeptide with the other b subunits (Albano
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FIG. 2. Comparison of mature mouse inhibin/activin b subunits. The amino acid alignment of mouse bA , bB , and bC subunits was made using CLUSTAL V (Higgins and Sharp, 1989). For bA and bB , we indicated only the amino acids that are different from those of bC . Numbering starts at the first amino acid after the basic maturation site. Conserved cysteines are indicated by carets.
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FIG. 3. Genomic structure of the mouse inhibin/activin bC gene. (A) Genomic structure and restriction map. Size and relative positions of two l clones used to map the gene are represented at the top. The positions of both exons are indicated by hatched boxes. Restriction sites are as follows: B, BglII; C, ClaI; E, EcoRI; S, SacI; X, XbaI. In the region indicated by a two-headed arrow with open heads, we found four BglII, six EcoRI, and three XbaI sites that are not shown. Below the restriction map, we show corresponding regions of exons and the open reading frame. (B) Sequence of the intron/exon boundaries. Intron sequences are shown in lowercase letters. The size of the intron is indicated in parentheses. The consensus splice donor sequence in rodents is (C/A)AG/GTUAGT, and the consensus acceptor sequence is YYYYYYYYYYNCAG/G (Y, pyrimidine; U, purine; N, any nucleotide) (Senapathy et al., 1990).
primer extension experiment (Fig. 4A). Our result indicates that transcription can be initiated at several sites located between 128 and 136 nucleotides upstream of the translational initiation site. The major transcription start site is indicated as position /1 in the sequence of the proximal inhibin/activin b C promoter (Fig. 4B). The 5*-end of our BCE1 cDNA clone is located at position /6. Genomic sequence upstream of the transcription start site does not show a TATA box sequence. Chromosomal Localization of the Mouse Inhibin/ Activin bC Gene The murine chromosomal location of mouse inhibin/activin b C (locus designated Inhbc) was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J 1 M. spretus)F1 1 C57BL/6J] mice. The interspecific backcross mapping panel has been typed for over 1900 loci that are well distributed among all mouse autosomes and the X chromosome (Copeland and Jenkins, 1991, unpublished results). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using the mouse Inhbc probe (see Materials and Methods). Two spretus-specific BglI RFLPs, which cosegregated, were used to follow the segregation of the Inhbc locus in the backcross DNAs. The mapping results indicated that Inhbc is located in the distal region of chromosome 10 (Fig. 5). Although 188 mice were analyzed for every marker shown in the haplotype analysis (Fig. 5), up to 192 mice were
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typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the combined data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere – Mdm2 – 6 / 190 – Inhbc – 0/ 191 – Gli – 1/ 192 – Erbb3. The recombination frequencies (expressed as genetic distances in centimorgans { the standard error) are centromere – Mdm2– 3.2 { 1.3 –(Inhbc, Gli) – 0.5 { 0.5– Erbb3. The Inhbc and Gli loci cosegregated in 191 animals typed in common, indicating that the two loci are within 1.6 cM of each other (95% confidence limit). Chromosomal Localization of the Human Inhibin/ Activin bC Gene Mapping of the human inhibin/activin bC gene (designated INHBC) was determined by fluorescence in situ hybridization on lymphocytes from human cord blood. Under the conditions used, the hybridization efficiency was approximately 60% for this 1.3-kb INHBC cDNA probe (among 100 checked mitotic figures, 59 of them showed signals on one pair of the chromosomes). Since DAPI banding was used to identify the specific chromosome, the assignment between signal from the probe and the long arm of chromosome 12 was obtained. The detailed position was further determined based on the summary from 10 photos (Figs. 6A and 6B). Therfore, the INHBC gene is located on human chromosome 12, region q13.1. An example of the mapping results is presented in Fig. 6C.
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FIG. 4. Analysis of the proximal promoter region of the mouse inhibin/activin bC gene. (A) Primer extension analysis. Forty micrograms of total RNA from yeast (lane 1) or from liver (lanes 2 and 3) was used in every extension reaction. The major extension product is indicated as position /1. As a size marker, DNA sequencing of the corresponding genomic region was performed using the same oligonucleotide as that for primer extensions (lanes 4– 7). (B) Sequence of the proximal promoter region. Transcription start sites, as determined by primer extension, are underlined, and the major extension product is indicated as position /1. Nucleotide numbering is according to the major transcription start site. Putative transcription factor binding sites found, using Genetics Computer Group programs, are indicated. Translation starts at nucleotide 137.
Expression Analysis of Inhibin/Activin bC in Mouse Tissues and Embryos We performed ribonuclease protection experiments to identify the inhibin/activin-b C-expressing tissues in adult mouse and to determine the stages at which it is expressed in late postimplantation mouse embryos. Probes for all three b chains, as well as for GAPDH, were used in the same reaction. Our results using adult tissues indicate that the Inhbc gene is exclusively expressed in liver (Fig. 7, lanes 12 and 13). Inhibin/activin b A message is present in the uterus, ovary, and liver (Fig. 7, lanes 6, 9, and 12, respectively). The bB gene is expressed in the uterus, testis, ovary, lung, kidney, brain, and CJ7 embryonic stem cells (Fig. 7, lanes 6, 7, 9, 11, 14, 16, and 18, respectively) and possibly in liver. Using RNA from entire embryos, we can detect inhibin/activin b C ex-
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pression from 14.5 days post coitum (d.p.c.) (Fig. 7, lanes 24 to 26); bA and bB messages are already detected in embryos at Day 12.5 d.p.c. (Fig. 7, lanes 22 to 26). DISCUSSION
In this paper, we have characterized the mouse inhibin/activin bC gene (Inhbc). This gene and its product have the characteristic features of members from the TGF-b superfamily. The mature polypeptide shares approximately 50% identity with mouse bA and bB subunits. All nine cysteine residues involved in intra- and intermolecular disulfide bridge formation are conserved. Processing at the maturation site would yield a bC protein of 116 amino acids, compared to those of 114 and 115 amino acids for bA and bB , respectively
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FIG. 5. Mapping of mouse Inhbc to chromosome 10. (Top) The segregation patterns of Inhbc with flanking markers in 188 backcross animals. For some individual pairs of loci, more than 188 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/ 6J 1 M. spretus)F1 parent. The black boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (Bottom) A partial chromosome 10 linkage map showing the location of Inhbc in relation to linked genes. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Library of The John Hopkins University (Baltimore, MD).
(Albano et al., 1993) Furthermore, the mouse inhibin/ activin bC gene structure is very similar to that of the other superfamily members, such as the human bB gene (Mason et al., 1989), the mouse bB gene (Schrewe et al., 1994), or the mouse b A gene (Matzuk et al., 1995a). In summary, these data and phylogenetic analyses strongly suggest that activin bC is a member of the inhibin/activin family within the TGF-b superfamily. Further experiments will be required to determine whether the Inhbc product also shares functional characteristics with the other b subunits. Dimerization of bC with a and/or b subunits would theoretically yield new types of inhibins or activins, respectively. We mapped, by primer extension analysis, the transcription start site of the Inhbc gene. Its promoter is TATA-less, as was found for the human (Mason et al., 1989) and mouse bB genes (Schrewe and Schmitt, unpublished). This feature in the promoter has been shown for a number of genes that are regulated during
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differentiation or development and initiate transcription at only one or a few clustered start sites (Smale and Baltimore, 1989). Inhbc maps to the distal region of mouse chromosome 10. Our placement of Inhbc on mouse chromosome 10 indicates that it is not linked to any other known mouse inhibin/activin gene. The inhibin a (Inha) and activin/ inhibin bA (Inhba) genes map to chromosome 1, while activin/inhibin bB (Inhbb) maps to mouse chromosome 13 (Barton et al., 1989). The mouse inhibin/activin bC gene was localized in close proximity to the human glioma-associated oncogene (Gli locus). Gli belongs to a linkage group common to human chromosome 12 and mouse chromosome 10. In accordance with these data, the human inhibin/activin bC gene was localized to chromosome 12q13.1. An uncloned mouse mutation, named atrichosis, that affects hair distribution and fertility in both sexes has also been mapped to the same chromosomal region as Inhbc (Handel and Eppig, 1979). The phenotype of this mutation affecting spermatogenesis and oocyte maturation could be correlated with the general function of activins and inhibins. If atrichosis or Gli map to the inhibin/activin bC gene, appearance of similar phenotypes might occur in Inhbcdeficient mice. Our expression analysis by ribonuclease protection (Fig. 7) and Northern blot technique (not shown) indicates that the inhibin/activin bC gene is predominantly expressed in adult mouse liver. This specific and high expression level of the bC gene product in liver suggests that it could have a particular function in this organ. Recent reports indeed have shown that activins have biological effects on hepatocytes. Human recombinant activin A acts as an autocrine inhibitor of liver DNA synthesis (Yasuda et al., 1993). It induces rat hepatocyte cell death in vitro and in vivo (Schwall et al., 1993), and rat hepatocytes produce glucose in response to activin (Mine et al., 1989). It remains to be determined whether other homo- or heterodimers containing bA or bB subunits have analogous activities in these systems. We observed that low amounts of activin bA message are present in mouse liver. This would allow the production of bC /bA heterodimers in addition to bC /bC homodimers. Expression of inhibin/activin bB has been detected in human liver (Tuuri et al., 1994). We could not, however, conclude from our result whether inhibin/activin bB is also expressed in mouse liver. Expression of inhibin a could not be detected in rat liver (Meunier et al., 1988; Yasuda et al., 1993). It remains questionable whether inhibins are produced and could antagonize the effects of activins on liver growth. Follistatin, a protein that binds activins and neutralizes their effects in some biological systems, is also expressed in human liver (Tuuri et al., 1994). Follistatin might bind inhibin/activin bC homo- or heterodimer combinations and could influence their function. Activin A is produced in liver after partial hepatectomy and acts as a liver chalone (Yasuda et al., 1993). The
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FIG. 6. Mapping of human INHBC to chromosome 12. (A) Fluorescence in situ hybridization was performed using a biotinylated human cDNA fragment. Arrows indicate FISH signals on chromosomes. (B) The same mitotic figures stained with DAPI to identify chromosome 12. (C) Diagram of FISH mapping results. Each dot represents the double FISH signal detected on human chromosome 12.
‘‘chalone’’ hypothesis on tissue homeostasis was proposed for the presence of stimulators or inhibitors of cell growth (Bullough, 1962; Nadal, 1979). If the inhibitor is produced by hepatocytes, partial hepatectomy would decrease inhibitor concentration and thereby allow cell division. Subsequent cell proliferation would result in an increase in the concentration of inhibitor, which would inhibit cell growth. It remains to be determined how bC expression is regulated during liver regeneration and whether it interacts with the bA subunit. The inhibin/activin bC gene is expressed during late embryonic development in the mouse (after 14.5
d.p.c.). Expression, during mouse development, of the other inhibin/activin subunits has been well documented (Albano et al., 1993, 1994; Feijen et al., 1994; Paulusma et al., 1994). In situ hybridization analyses did not, however, reveal any expression of activin ligands and/or receptors in embryonic liver (Albano et al., 1993, 1994; Feijen et al., 1994; Paulusma et al., 1994). Functional analysis of inhibin/activins bA and bB and activin receptor IIA during mammalian development suggested that they are not essential for embryogenesis in the mouse (Matzuk et al., 1995a). Discrepancies in the results between ligand- and recep-
FIG. 7. Inhibin/activin bA , bB , and bC expression. Unless otherwise indicated, 10 mg total RNA from different adult tissues (lanes 1– 18) or from whole embryos isolated at different stages of development (lanes 19 – 26) was analyzed by ribonuclease protection assay using inhibin/activin bA , bB , and bC cDNA fragments as antisense probes. As control for RNA integrity and loading, a GAPDH riboprobe was also included: lane 1, size marker; lanes 2 and 3, input probes; lane 4, no RNA; lane 5, yeast RNA; lane 6, uterus; lane 7, testis ; lane 8, spleen ; lane 9, ovary; lane 10, skeletal muscle; lane 11, lung; lane 12, liver; lane 13, liver (2 mg); lane 14, kidney; lane 15, heart; lane 16, brain; lane 17, no RNA; lane 18, CJ7 cells; lane 19, size marker; lane 20, input probes; lane 21, 10.5 days post coitum (d.p.c.); lane 22, 12.5 d.p.c.; lane 23, 13.5 d.p.c.; lane 24, 14.5 d.p.c.; lane 25, 15.5 d.p.c.; lane 26, 16.5 d.p.c. The positions of the unprotected and protected fragments are indicated to the left and right, respectively. 32P-end-labeled MspI fragments of pBR322 were used as size markers.
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tor-deficient mice could be interpreted by a functional redundancy of inhibin/activin bC with bA and bB . Production of activin-bC-deficient mice will advance the functional analysis of activins during mouse embryonic development. ACKNOWLEDGMENTS We thank Drs. Gu¨nther Schu¨tz and Wolfgang Schmid for providing us the mouse liver cDNA library. We acknowledge Renate Mielke and Debbie Barnhart for excellent technical assistance. We are grateful to our colleagues Drs. Ernst-Martin Fu¨chtbauer, Matthias Hebrok, John McLaughlin, Davor Solter, and Grace Wei for critical reading and comments about the manuscript. This research was supported, in part, by the American National Cancer Institute, DHHS, under a contract with ABL. J.S. was the recipient of a Max-PlanckSociety postdoctoral fellowship.
Albano, R. M., Arkell, R., Beddington, R. S. P., and Smith, J. C. (1994). Expression of inhibin subunits and follistatin during postimplantation mouse development: Decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain. Development 120: 803– 813. Albano, R. M., Groome, N., and Smith, J. C. (1993). Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 117: 711–723. Ashar, H. R., Benson, K. F., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Chada, K. K. (1994). Ifg, Gli, Mdm1, Mdm2 and Mdm3: Candidate genes for the mouse pg locus. Mamm. Genome 5: 608– 612. Attisano, L., Wrana, J. L., Lopez-Casillas, F., and Massague´, J. (1994). TGF-b receptors and actions. Biochim. Biophys. Acta 1222: 71–80. Barton, D. E., Yang-Feng, T. L., Mason, A. J., Seeburg, P. H., and Francke, U. (1989). Mapping of genes for inhibin subunits a, bA, and bB on human and mouse chromosomes and studies of jsd mice. Genomics 5: 91–99. Bullough, W. S. (1962). Control of mitotic activity in adult mammalian tissues. Biol. Rev. Camb. Phil. Soc. 37: 307–342. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by guanidinium thiocyanate –phenol– chloroform extraction. Anal. Biochem. 162: 156– 159. Copeland, N. G., Gilbert, D. J., Schindler, C., Zhong, Z., Wen, Z., Darnell, J. E., Jr., Mui, A. L.-F., Miyajima, A., Quelle, F. W., Ihle, J. N., and Jenkins, N. A. (1995). Distribution of the mammalian Stat gene family in mouse chromosomes. Genomics 29: 225– 228. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113– 118. Esch, F. S., Shimasaki, S., Cooksey, K., Mercado, M., Mason, A. J., Ying, S.-Y., Ueno, N., and Ling, N. (1987). Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Mol. Endocrinol. 5: 388 –396. Feijen, A., Goumans, M. J., and van den Eijden-van Raaij, A. J. M. (1994). Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development 120: 3621 –3637. Green, E. L. (1981). Linkage, recombination, and mapping. In ‘‘Genetics and Probability in Animal Breeding Experiments,’’ pp. 77– 113, Oxford Univ. Press, New York. Handel, M. A., and Eppig, J. J. (1979). Sertoli cell differentiation in
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