Vol. 12, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1992, p. 2418-2423

0270-7306/92/052418-06$02.00/0

Copyright X) 1992, American Society for Microbiology

Differential Regulation of the Wnt Gene Family during Pregnancy and Lactation Suggests a Role in Postnatal Development of the Mammary Gland BRIAN J. GAVIN AND ANDREW P. McMAHON* Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Received 1 July 1991/Accepted 9 February 1992

The mouse Wnt family comprises at least 10 members sharing substantial amino acid identity with the secreted glycoprotein Wnt-1/int-1. Two of these, Wnt-1 and Wnt-3, are implicated in mouse mammary tumor virus-associated adenocarcinomas, although neither member is normally expressed in the mammary gland. These results suggest the presence of active cellular pathways which mediate the action of Wnt-1 and Wnt-3 signals. An understanding of the normal role of these signalling pathways is clearly necessary to comprehend the involvement of Wnt-I and Wnt-3 in mammary tumorigenesis. We demonstrate here that five Wnt family members are expressed and differentially regulated in the normal mouse mammary gland. In addition, some of these genes are also expressed in both Wnt-l-responsive and nonresponsive mammary epithelial cell lines. We propose that Wnt-mediated signalling is involved in normal regulation of mammary development and that inappropriate expression of Wnt-1, Wnt-3, and possibly other family members can interfere with these signalling pathways.

developing central nervous system (CNS) from 8.5 to at least 16.5 days of development (48, 60, 61). Gene targeting experiments have demonstrated that mice homozygous for a Wnt-1 null allele fail to develop a large region of the brain (26, 52). Thus, the normal role of Wnt-1 lies in regional development of the CNS. Wnt-1 is only one member of a family of at least 10 related genes in the mouse (14, 28, 44, 45). Each encodes a putative secreted polypeptide that has 50 to 60% amino acid identity to Wnt-1. A second Wnt gene, Wnt-3, has recently been implicated in MMTV-induced mammary tumorigenesis. Like Wnt-1, Wnt-3 is not normally expressed in the mammary gland but is activanted by proviral insertion (45). Normal expression is associated with fetal development, particularly within the CNS (44). Two key questions arise from these observations. Why does activation of either Wnt-1 or Wnt-3 result in mammary tumors, and how does this finding relate to normal mammary development? Clearly, mammary epithelial tissue is responsive to these two gene products, despite the fact that they have no apparent normal role in the mammary gland. The hyperplasia that results from Wnt gene activation indicates that the expression of the gene perturbs normal epithelial cell growth regulation. One possible explanation is that Wnt genes other than Wnt-1 and Wnt-3 normally regulate mammary development. In this scenario, activation of either Wnt-1 or Wnt-3 may interfere with the normal regulatory pathway(s) associated with the function of Wnt genes in the mammary gland. All Wnt genes are expressed in adult tissues (14, 17, 28, 44, 45, 48, 60), but most are not widely distributed. We have investigated expression of the known mouse Wnt genes during normal mammary development and in mammary epithelial cell lines. These results support the hypothesis that Wnt genes do regulate normal mammary development. Therefore, it is reasonable to suggest that tumorigenesis results from disruption of the normal function of Wnt genes in the mammary gland.

The Wnt-1 proto-oncogene (formerly int-1 [32]) was originally identified in mouse mammary tumors induced by mouse mammary tumor virus (MMTV) (34). In many of these MMTV-associated tumors, proviral integration at the Wnt-1 locus was shown to activate transcription of the normally silent Wnt-1 gene (33, 34). As the mapping of proviral integration sites demonstrated that insertions never interrupted the Wnt-1 open reading frame, overexpression of the normal Wnt-1 protein was postulated to play a causative role in the generation of these tumors (57). Support for this conjecture has come from two experimental approaches. In cell culture, expression of Wnt-1 causes transformation of at least two mammary epithelial cell lines. C57MG, which was derived from normal mammary tissue, was partially transformed (5), whereas the nontumorigenic line RAC311C, which was derived from a virus-induced mammary tumor, exhibited a fully transformed phenotype both in vitro and in syngeneic mice (43). Interestingly, all nonmammary and several different mammary epithelial cell lines tested were nonresponsive to Wnt-1 (5). In transgenic mice, a transgene that mimics a virally activated Wnt-1 allele resulted in hyperplasia of both male and female mammary tissue, with over 80% of the females developing adenocarcinomas of the breast by 7 months of age (54). The long latency period required for the generation of tumors in these mice suggests that expression of Wnt-1 can contribute to the generation of mammary tumors but is not sufficient by itself for complete transformation. The Wnt-1 gene encodes a 44-kDa cysteine-rich secreted glycoprotein (4, 13, 37, 38) that is tightly associated with the extracellular matrix and/or the cell surface (3, 38). Normal expression of the mouse Wnt-1 gene has been shown to be spatially and temporally regulated during development. Adult expression is limited to postmeiotic spermatids of the mature testes (48), while fetal expression is restricted to the

*

Corresponding author. 2418

VOL. 12, 1992

Wnt GENE EXPRESSION IN MAMMARY GLAND DEVELOPMENT

184

Wnt-1

1105

1

Wnt-2

900

9 705

Wnt-3 .

Wnl-3a

1iI 489

1109

404

1250 1

Wnt-4

Wnt-5a

1289

1250

10i

1030

489

1203

Wnt-5b Wnt-6

Wnt-7a

Wnt-7b

1275

160

18

587

319 1522

1039

1445

FIG. 1. Probes used for Northern analysis. The positions and sizes (in nucleotides) of DNA probes for each Wnt gene are indicated relative to a schematic cDNA. The filled box indicates the coding region; the solid lines indicate 5' and 3' untranslated regions. For cDNA sequences that have been previously published, nucleotide positions in the published sequences are indicated: Wnt-1 (13), Wnt-2 (28), Wnt-3 (45), and Wnt-3a (44). Nucleotide positions for other Wnt probes refer to the sequences entered into the EMBL data base (see Materials and Methods). MATERIALS AND METHODS

RNA isolation. Thoracic and abdominal mammary glands isolated from virgin (10 to 12 weeks of age), pregnant, and lactating CD-1 female mice. The day of plug was considered day 0.5 of gestation, and the day of birth was considered day 1 of lactation. Mammary epithelial cells from three confluent 15-cm plates were trypsinized and pelleted by centrifugation. Cells were then washed with phosphatebuffered saline and recentrifuged, and the cell pellet was frozen on dry ice. Polyadenylated [poly(A)] RNA was isolated directly from fresh tissue or frozen cell pellets, using an Invitrogen Fast Trak mRNA isolation kit according to the manufacturer's instructions. Northern (RNA) analysis. Five micrograms of poly(A) RNA was electrophoretically separated on a 1.2% formaldehyde agarose gel (22), transferred to a nylon membrane (Duralon UV; Stratagene) by capillary blotting in lOx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and photo-cross-linked by UV light. RNA blots were then hybridized with 1.5 x 106 cpm of randomly primed (Amersham) 1-actin (29), glyceraldehyde 3-phosphate dehydrogenase (GAPDH [40]), ,-casein (15), or Wnt cDNA probes per ml. All probes were of similar specific activity, 2109 cpm/ ,ug. RNA blots were stripped in lx blot wash (53) and reprobed multiple times. The positions of the Wnt probes relative to a schematic cDNA are shown in Fig. 1. Hybridization was carried out at 45°C in 50% formamide-0.25 M sodium phosphate (pH 7)-0.25 M NaCl-7% sodium dodecyl sulfate (SDS)-1 mM EDTA-10% polyethylene glycol 20,000 for -16 h. Blots were then washed as follows: three times for 15 min each time in 2x SSC-1% SDS at room temperature, twice for 45 min each time in 0.2x SSC-1% SDS at 68°C, and once for 30 min in 0.1 x SSC-1% SDS at 680C. Autoradiography was performed at -70°C with intensifying screens for 1 to 7 days. Tissue culture. C57MG (55) and MMEC (51) mouse mam-

were

mary epithelial cell lines were maintained in Dulbecco modified Eagle medium (GIBCO) supplemented with 10% fetal calf serum, lx nonessential amino acids (GIBCO), 2 mM glutamine, 30 ,uM nucleosides, 50 U of penicillin per ml, and 50 jig of streptomycin per ml. Medium for C57MG cells was further supplemented with 100 ,ug of bovine insulin (Sigma) per ml. NMG and C1271 cells were obtained from the American Type Culture Collection and cultured according to supplied information. Nucleotide sequence accession numbers. The sequences reported have been entered into the EMBL data base and assigned the following accession numbers: Wnt-4, M89797; Wnt-Sa, M89798; Wnt-5b, M89799; Wnt-6; M89800; Wnt-7a, M89801; and Wnt-7b, M89802.

1435

605

406

2419

RESULTS Analysis of Wnt gene expression in mouse mammary glands. To analyze expression of the Wnt gene family during normal mammary growth and development, poly(A) RNA was isolated from adult female mammary glands at several stages of pregnancy and lactation. RNA blots were hybridized with probes for each of the 10 mouse Wnt family members (Fig. 1). Each of the probes is unique in its hybridization to Southern blots of mouse genomic DNA (data not shown), and each hybridizes to unique transcripts. Thus, there is no apparent cross-reactivity with use of these sequences. Five of the ten mouse Wnt genes, Wnt-4, -5a, -5b, -6, and -7b, were detectably expressed in mammary gland RNA (Fig. 2). All of these genes appear to be differentially regulated during pregnancy and lactation. Wnt-4 and -7b showed highest levels of expression in virgin animals. However, while Wnt-4 expression was maintained at relatively high levels up to 12.5 days of pregnancy, Wnt-7b expression decreased over this period. Wnt-5a, -5b, and -6 were all expressed at low levels in virgin animals and at increased levels during pregnancy, each with a unique pattern of transcript regulation. Wnt-Sa exhibited a modest increase in expression which peaked early in pregnancy and then quickly decayed to undetectable levels by day 17.5 of pregnancy. In contrast, Wnt-5b and -6 transcripts accumulated more gradually but to a much higher level, reaching a maximum by day 12.5 of pregnancy and then decreasing to undetectable levels by day S of lactation. Thus, individual Wnt genes showed distinct patterns of developmental regulation. In general, all five genes appeared to be down-regulated during late pregnancy or at the onset of lactation. Transcript sizes were similar to those reported for these genes in fetal and adult RNAs (14). Also as previously observed, Wnt-4, -5a, and -6 gave rise to multiple transcripts. Interestingly, the Wnt-4 transcripts exhibited differential regulation. In glands from virgins and mice at early stages of pregnancy, the 4.7- and 1.6-kb transcripts were present at similar levels. However, as Wnt-4 transcription is down-regulated during pregnancy, the larger transcript appears to be preferentially lost. To control for variations in the loading of RNA samples, the Northern blots were hybridized with a probe for the housekeeping GAPDH gene (Fig. 2). This probe did not give the uniform signal expected but instead showed a decline in intensity during pregnancy, to low but constant levels during lactation. A similar result was obtained when blots were hybridized with a probe for another constitutively expressed gene, the 1-actin gene (data not shown). However, ethidium bromide staining of the total RNA sample revealed that roughly equivalent amounts of RNA were present in all lanes

2420

MOL. CELL. BIOL.

GAVIN AND McMAHON Pregnancy Lactation

4'eF

Pregnancy Lactation

I\N