Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland.

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-1 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-1-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.     

Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development

P190-B RhoGAP (p190-B, also known as ARHGAP5) has been shown to play an essential role in invasion of the terminal end buds (TEBs) into the surrounding fat pad during mammary gland ductal morphogenesis. Here we report that embryos with a homozygous p190-B gene deletion exhibit major defects in embryonic mammary bud development. Overall, p190-B-deficient buds were smaller in size, contained fewer cells, and displayed characteristics of impaired mesenchymal proliferation and differentiation. Consistent with the reported effects of p190-B deletion on IGF-1R signaling, IGF-1R-deficient embryos also displayed a similar small mammary bud phenotype. However, unlike the p190-B-deficient embryos, the IGF-1R-deficient embryos exhibited decreased epithelial proliferation and did not display mesenchymal defects. Because both IGF and p190-B signaling affect IRS-1/2, we examined IRS-1/2 double knockout embryonic mammary buds. These embryos displayed major defects similar to the p190-B-deficient embryos including smaller bud size. Importantly, like the p190-B-deficient buds, proliferation of the IRS-1/2-deficient mesenchyme was impaired. These results indicate that IGF signaling through p190-B and IRS proteins is critical for mammary bud formation and ensuing epithelial-mesenchymal interactions necessary to sustain mammary bud morphogenesis.     

Wnt-4 Expression Induces a Pregnancy-like Growth Pattern in Reconstituted Mammary Glands in Virgin Mice

Expression of Wnt-4, a member of the Wnt gene family, is induced during early pregnancy in the mouse mammary gland. To investigate the function of Wnt-4, we used a recombinant retrovirus to constitutively express the gene in transplanted mammary epithelium grown in virgin animals. In fully grown glands, Wnt-4 expression resulted in ducts that were more highly branched than normal and caused some premature alveolar development. These changes resembled those seen during pregnancy, suggesting that endogenous Wnt-4 expression may regulate epithelial branching in early pregnancy. The modified growth pattern induced by Wnt-4 expression was similar to that induced by Wnt-1, one of the members of the Wnt gene family activated by mouse mammary tumour virus. As Wnt-1 is not normally expressed in the mammary gland, it may exert its effect on the mammary gland by activating a developmental pathway normally regulated by Wnt-4.     

Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.     

TGF-beta, c-Cbl, and PDGFR-alpha the in mammary stroma

Transforming growth factor-beta (TGF-beta) is thought to regulate ductal and lobuloalveolar development as well as involution in the mammary gland. In an attempt to understand the role TGF-beta plays during normal mammary gland development, and ultimately cancer, we previously generated transgenic mice that express a dominant-negative TGF-beta type II receptor under control of the metallothionine promoter (MT-DNIIR). Upon stimulation with zinc sulfate, the transgene was expressed in the mammary stroma and resulted in an increase in ductal side branching. In this study, mammary gland transplantation experiments confirm that the increase in side branching observed was due to DNIIR activity in the stroma. Development during puberty through the end buds was also accelerated. Cbl is a multifunctional intracellular adaptor protein that regulates receptor tyrosine kinase ubiquitination and downregulation. Mice with a targeted disruption of the c-Cbl gene displayed increased side branching similar to that observed in MT-DNIIR mice; however, end bud development during puberty was normal. Transplantation experiments showed that the mammary stroma was responsible for the increased side branching observed in Cbl-null mice. Cbl expression was reduced in mammary glands from DNIIR mice compared to controls and TGF-beta stimulated expression of Cbl in cultures of primary mammary fibroblasts. In addition, both TGF-beta and Cbl regulated platelet-derived growth factor receptor-alpha (PDGFR alpha) expression in vivo and in isolated mammary fibroblasts. The hypothesis that TGF-beta mediates the levels of PDGFR alpha protein via regulation of c-Cbl was tested. We conclude that TGF-beta regulates PDGFR alpha in the mammary stroma via a c-Cbl-independent mechanism. Finally, the effects of PDGF-AA on branching were determined. Treatment in vivo with PDGF-AA did not affect branching making a functional interaction between TGF-beta and PDGF unlikely.     

Regulated expression patterns of IRX-2, an Iroquois-class homeobox gene, in the human breast

In the mouse mammary gland, homeobox gene expression patterns suggest roles in development and neoplasia. In the human breast, we now identify a family of Iroquois-class (IRX) homeobox genes. One gene, IRX-2, is expressed in discrete epithelial cell lineages being found in ductal and lobular epithelium, but not in myoepithelium. Expression is absent from associated mesenchymal adipose stroma. During gland development, expression is concentrated in terminal end buds and terminal lobules and is reduced in a subset of epithelial cells during lactation. In contrast to observations for many homeobox genes in the mouse mammary gland in which homeobox gene expression is lost on neoplastic progression, IRX-2 expression is maintained in human mammary neoplasias. Data suggest IRX-2 functions in epithelial cell differentiation and demonstrate regulated expression during ductal and lobular proliferation as well as lactation.     

Disruption of reelin signaling alters mammary gland morphogenesis

Reelin signaling is required for appropriate cell migration and ductal patterning during mammary gland morphogenesis. Dab1, an intracellular adaptor protein activated in response to reelin signaling, is expressed in the developing mammary bud and in luminal epithelial cells in the adult gland. Reelin protein is expressed in a complementary pattern, first in the epithelium overlying the mammary bud during embryogenesis and then in the myoepithelium and periductal stroma in the adult. Deletion in mouse of either reelin or Dab1 induced alterations in the development of the ductal network, including significant retardation in ductal elongation, decreased terminal branching, and thickening and disorganization of the luminal wall. At later stages, some mutant glands overcame these early delays, but went on to exhibit enlarged and chaotic ductal morphologies and decreased terminal branching: these phenotypes are suggestive of a role for reelin in spatial patterning or structural organization of the mammary epithelium. Isolated mammary epithelial cells exhibited decreased migration in response to exogenous reelin in vitro, a response that required Dab1. These observations highlight a role for reelin signaling in the directed migration of mammary epithelial cells driving ductal elongation into the mammary fat pad and provide the first evidence that reelin signaling may be crucial for regulating the migration and organization of non-neural tissues.     

Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.     

Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.