1 UMR144-CNRS/Institut Curie, 26 rue dUlm 75248 Paris cedex 05, France
2 Imperial Cancer Research Fund, Lincolns Inn Fields, London WC2A 3PX, UK
3 Kyoto University, Graduate School of Pharmaceutical Sciences, Yoshida-Shimoadachi, Sakyo, Kyoto 606-8501, Japan
4 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
* These authors contributed equally to this work
Author for correspondence (e-mail: saverio.bellusci{at}curie.fr)
Accepted 5 October 2001
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SUMMARY |
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Key words: Fgf10, Fgfr2b, Lef1, Mammary placode development, Mouse, Cell signaling
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INTRODUCTION |
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Little is known about the genes that regulate the initial phases of mammary placode development. The transcription factor Lef1, an effector of WNT/ß-catenin signaling, is the earliest known marker of mammary placode formation. Its inactivation in vivo leads to embryos with only a single pair of inguinal placodes (van Genderen et al., 1994). The development of mammary buds, in both female and male mouse embryos, is dependent upon signaling by parathyroid hormone related-protein (PTHrP) through its receptor (PTHR1), which are expressed in the epithelium and condensed mammary mesenchyme respectively. In the absence of PTHrP, or its receptor, mammary buds fail to elongate and branch into the primitive fat pad of female embryos, or undergo the expected androgen-mediated apoptosis in males (Wysolmerski et al., 1998; Dunbar et al., 1999).
The fibroblast growth factor (FGF) family comprises at least 22 members, many of which have been implicated in multiple aspects of vertebrate development [for review see Ornitz and Itoh (Ornitz and Itoh, 2001)]. In particular, FGF10 has been associated with instructive mesenchymal-epithelial interactions, such as those that occur during branching morphogenesis. For example, in the developing lung, Fgf10 is expressed in the distal mesenchyme at sites where prospective epithelial buds will appear. Moreover, its dynamic pattern of expression and its ability to induce epithelial expansion and budding in organ cultures have led to the hypothesis that FGF10 governs the directional outgrowth of lung buds during branching morphogenesis (Bellusci et al., 1997). Furthermore, FGF10 was shown to be a potent chemoattractant for the distal lung epithelium (Park et al., 1998; Weaver et al., 2000). Consistent with these observations, mice deficient for Fgf10 show multiple organ defects including lung agenesis (Min et al., 1998; Sekine et al., 1999; Ohuchi et al., 2000).
The mammalian Fgf receptor family comprises four genes (Fgfr1 to Fgfr4), which encode at least seven prototype receptors. Fgfr1, 2 and 3 encode two receptor isoforms (termed IIIb or IIIc) that are generated by alternative splicing, and each bind a specific repertoire of FGF ligands (Ornitz et al., 1996). FGFR2-IIIb (FGFR2b) is found mainly in epithelia and binds four known ligands (FGF1, FGF3, FGF7 and FGF10) which are primarily expressed in mesenchymal cells. While mice null for the Fgfr2 gene die early during embryogenesis, those that are null for the Fgfr2b isoform, but retain Fgfr2c, survive to birth (Arman et al., 1998; Xu et al., 1998; De Moerlooze et al., 2000; Revest et al., 2001). Mice deficient for Fgfr2b show agenesis and dysgenesis of multiple organs indicating that signaling through this receptor is critical for mesenchymal-epithelial interactions during early organogenesis.
Mammary gland development has been studied extensively in the post-natal animal, but less is known about the embryonic stages. We have investigated the initial phases of mammary placode development, and demonstrate using molecular markers and scanning electron microscopy that the placodes form asynchronously. Placode 3 is the first to appear, followed by placode 4, then placodes 1 and 5 and finally placode 2. The role of FGF10/FGFR2b signaling in the epithelial/mesenchymal interactions that characterize embryonic mammary gland development is demonstrated through the analysis of the mammary gland phenotypes of Fgf10/ and Fgfr2b/ embryos.
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MATERIALS AND METHODS |
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Scanning electron microscopy
E11.5 and E12.5 mouse embryos (C57BL/6) were extracted quickly from the uteri, washed 6 times in filtered PBS and fixed in a solution of sodium cacodylate 0.1 M pH 7.6/glutaraldehyde 2% at room temperature for 1 hour and then overnight at 4°C. They were washed three times in 0.2 M sodium cacodylate for 1 hour at room temperature and transferred in a solution of sodium cacodylate 0.1 M pH7.6/OsO4 0.1% for 1 hour at room temperature. After a 5 minute wash in distilled water, the embryos were dehydrated in graded ethanols (70% to 100%) and then in amyl acetate (30% to 100%). They were critical-point dried in liquid carbon dioxyde, mounted on aluminum stubs and coated with gold.
Mutant embryos
Fgf10/ and Fgfr2b/ embryos were generated as previously described (Sekine et al., 1999; De Moerlooze et al., 2000) and were on the C57BL/6 background. C57BL/6 or wild-type littermates mice were used as control embryos at different stages of development. The number of Fgf10/ embryos used in this study at the different stages were as follows: E11.5 (n=5), E12.5 (n=2), E13.5 (n=2), E14.5 (n=3 females), E18.5 (n=9 females). The number of Fgfr2b/ embryos used in this study were as follows: E11.5 (n=3), E12.5 (n=11), E13 (n=6), E14.5 (n=1 female), E15.5 (n=2 females), E16.5 (n=2 females).
Organotypic culture
Embryos were removed at E10.5 and E11.5. At E11.5, the heads were surgically removed and the remaining body of the embryos cut into halves along the dorsal-ventral axis. Embryos were placed on Nucleopore filters, which were then laid on the surface of 500 µl F12: DMEM medium containing 50 Units/mg penicillin and streptomycin, 1% glutamine and 10% heat-inactivated fetal calf serum in NUNCLON dishes [technique adapted from Lebeche et al. (Lebeche et al., 1999)]. We investigated the local effects of FGF10 on these cultures by implanting heparin beads (Sigma) impregnated with human recombinant FGF10 (Research and Development) (100 µg/ml) in the flanks of the embryos in the area of mammary placode formation. The embryos were usually incubated for 24 hours at 37°C under CO2 and then fixed for 2 hours in 4% PFA and processed for whole-mount in situ hybridization. BSA-impregnated beads were used as controls.
Mammary gland transplantation
The mammary gland 4 from Fgf10/ (n=3) and wild-type fetuses (n=3) at E18.5 were freshly dissected and transplanted into cleared mammary fat pads of syngenic mice (Medina, 1996). In these experiments, 21-24 days old females were used as transplant recipients. The endogenous mammary epithelium was surgically removed from the fourth inguinal glands to provide a cleared mammary fat pad. Mutant and wild-type mammary glands were transplanted separately into contralateral glands of each recipient (n=3) to ensure an identical host environment. After 4 weeks, the mice were sacrificed and the fourth inguinal mammary glands dissected. The epithelium was stained using Carmin Red as previously described (Faraldo et al., 1998).
Analysis of cell death
Apoptotic cells were detected by the incorporation of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) using the ApopTagRPlus In Situ Apoptosis Detection Kit (Oncor, USA) as recommended by the manufacturer.
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RESULTS |
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FGF10 fails to induce Lef1 expression in the epithelium
The similarity in mammary placode phenotype between Fgf10/ and Lef1/ embryos raised the interesting possibility that FGF10 induces Lef1 expression. To test this idea, FGF10-coated beads were grafted onto the flank of E10.5 and E11.5 embryos. After 30 hours of culture the FGF10-coated beads failed to induce Lef1 expression in the surrounding ectoderm of E10.5 or E11.5 embryos (Fig. 7A,B). Importantly, endogenous Lef1 expression corresponding to the normal mammary buds was detected, indicating that endogenous placode formation occurred normally (Fig. 7B). Note that the positions of the beads were dorsal, ventral and coincident with the putative mammary line. The positive control used in this experiment was the induction of Sprouty2 in the lung endoderm by FGF10-coated beads (Mailleux et al., 2001).
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DISCUSSION |
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Until recently, no molecular marker was available to monitor the emergence of mammary epithelial cells during the initial phases of placode development. The transcription factor Lef1 has been shown to participate in Wnt signaling by complexing with ß-catenin to form a transcriptional complex that modulates the expression of WNT-responsive genes (Behrens et al., 1996; Eastman and Grosschedl, 1999). Lef1 is expressed at the very early stages of placode formation (van Genderen et al., 1994). We show here that Lef1 has a very dynamic expression pattern that appears to mark the cells that aggregate to form the mammary placode. For each placode, Lef1 expression goes from a line, to a comet shape and finally to a characteristic disc. These observations would suggest that epithelial cells are recruited locally along a mammary line and migrate to a precise location to form the mammary placode.
Mammary placode formation is asynchronous
The timing of placode formation has not been addressed to date, but might be expected to occur sequentially, from anterior (placode 1) to posterior (placode 5) in line with other aspects of mouse development. However, using Lef1 expression to monitor placode formation, they were found to emerge between E11.5-E11.75 in the order 3, 4, 1 and 5 and then 2. This order of placode appearance is supported by SEM observations that show that they initially form an epidermal mound that subsides and become undetectable by E14.5 (Fig. 1, Fig. 2 and S. B. and A. de Maximy, unpublished data). One explanation for asynchrony is that placode formation is autonomous. This is consistent with placode 4 formation in Fgf10/ and the Fgfr2b/ embryos, although we cannot exclude the idea that the single inguinal placode is actually a fusion of placodes 4 and 5 which fail to separate. The autonomous nature of placode formation is also supported by our recent work on the Extratoes mice, which have a deletion in the Gli3 gene (Hui and Joyner, 1993). The Gli3 null embryos exhibit a lack of induction of placode 3 and 5, while the other placodes are induced normally (S. B. and A. de Maximy, unpublished data).
The FGFR2b/FGF10 pathway is involved in the initial phases of development of mammary glands 1, 2, 3 and 5
In contrast to the rabbit, a distinguishable mammary line seen as an elevation on the surface ectoderm has not been observed in the mouse (Bellusci and de Maximy, unpublished data; Balinsky, 1950; Propper, 1978). However, the observation of a line of transient Fgf10 expression in the dermomyotome between E10.5 and E11, prior to placode formation, may be indicative of such a mammary line. The chemoattractant properties attributed to FGF10 in the migration of lung epithelium during branching morphogenesis, suggests a similar role in directing the migration of the epithelial cells along such a hypothetical mammary line. Alternatively, FGF10 may act to specify ectodermal cells destined to form mammary placodes.
The lack of placodes 1, 2, 3 and 5 in both Fgf10/ and Fgfr2b/ mice was based on the absence of expression of the molecular markers Lef1 or Bmp4 as well as by direct histological examination. Apart from placode 4, these findings suggest a model where FGF10 might regulate Lef1 expression that in turn helps to specify the mammary epithelium. This would be consistent with Lef1/ embryos also having a similar mammary phenotype, with a single inguinal placode. The possibility that LEF1 regulates Fgf10 seemed unlikely since its expression precedes Lef1 in mammary placode development. However, FGF10-coated beads did not induce Lef1 expression when placed in the epidermis close to or within the proposed mammary line, indicating that FGF10 is either not involved in Lef1 induction, or it is required earlier to help specify the mammary epithelium. It is also possible that the epithelium is only competent to respond to FGF10 for a short period of time, or that other growth factors act in synergy with FGF10 to induce Lef1 expression.
FGFR2b ligands are involved in mammary bud 4 maintenance
In the Fgfr2b/ embryos, bud 4 is formed but undergoes apoptosis after E12.5, while in Fgf10/ embryos this bud is maintained. This finding suggests that an additional FGFR2b ligand is involved in the maintenance of the inguinal mammary bud. In situ hybridization analysis for genes encoding known FGFR2b ligands during this stage of development showed that Fgf7 was the only one detected at E12.5 in the surrounding mesenchyme of the mammary bud (Cunha and Holm, 1996), and therefore may act redundantly with Fgf10 to maintain placode integrity.
Fgf10 is not critical for mammary bud 4 epithelium ingrowth into the fat pad precursor
In female embryos between E12 and E16, the mammary bud shows a low level of proliferation termed the resting phase. At late E16 proliferation increases and the mammary bud elongates to form the mammary sprout. The sprout grows rapidly downward, penetrating the mammary fat pad precursor tissue that underlies the mammary placode [for reviews see Sakakura and Robinson et al. (Sakakura, 1987; Robinson et al., 1999)]. As the ductal epithelium penetrates the fat pad it begins to branch. PTHrP is expressed in the mammary epithelium and appears to signal to PTHR1 expressed in the surrounding mesenchyme. Disruption of the PTHrP gene leads to an absence of epithelial bud elongation and subsequent ductal branching and to the degeneration of the mammary epithelium (Wysolmerski et al., 1998). As Fgf10 is expressed in the presumptive fat pad, it is plausible that FGF10 could be a downstream target of the PTHrP/PTHR1 signaling pathway. However, our results indicate that FGF10 was not critical for the growth of the epithelium into the mammary fat pad to form the mammary sprout, although this result only applies for the mammary bud 4. FGF10 could certainly play a role in the directional growth of the other buds. Interestingly, the epithelial sprout of the mutant mammary gland did not ramify extensively after penetrating the fat pad, but this abnormality was not apparent when the Fgf10-deficient epithelium was transferred into a wild-type stroma. This suggests that the branching defect is due to a defect in the Fgf10/ fad pad that is unable to support proper branching. Consistent with this finding is a recent work demonstrating that FGF10 has a role in the development of adipose tissue, where it plays a role in the differentiation of the pre-adipocytes into adipocytes (N. Itoh unpublished data).
In conclusion, we have shown that Lef1 expression in the mammary placode is dynamic, that mammary placode formation is asynchronous and involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that one or several members of the FGF family are involved in mammary bud 4 maintenance and that Fgf10 expression is not crucial for penetration of the mammary duct of bud 4 into the fat pad precursor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K. and Lonai, P. (1998). Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc. Natl. Acad. Sci. USA 95, 5082-5087.
Balinsky, B. I. (1950). On the pre-natal growth of the mammary gland rudiment in the mouse. J. Anat. 84, 227-235.
Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996). Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382, 638-642.[Medline]
Bellusci, S., Grindley, J., Emoto, H., Itoh, N. and Hogan, B. L. M. (1997). Fibroblast Growth Factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124, 4867-4878.
Cunha, G. R. and Hom, Y. K. (1996). Role of mesenchymal-epithelail interactions in mammary gland development. J. Mamm. Gland Biol. Neoplasia 1, 21-35.[Medline]
De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini, M., Rosewell, I. and Dickson, C. (2000). An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signaling during mouse organogenesis. Development 127, 483-492.
Dunbar, M. E., Dann, P. R., Robinson, G. W., Hennighausen, L., Zhang, J. P. and Wysolmerski, J. J. (1999). Parathyroid hormone-related protein signaling is necessary for sexual dimorphism during embryonic mammary development. Development 126, 3485-3493.
Eastman, Q. and Grosschedl, R. (1999). Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr. Opin. Cell Biol. 2, 233-240.
Faraldo, M. M., Deugnier, M. A., Lukashev, M., Thiery, J. P. and Glukhova, M. A. (1998). Perturbation of beta1-integrin function alters the development of murine mammary gland. EMBO J. 17, 2139-2147.
Foley, J., Dann, P., Hong, J., Cosgrove, J., Dreyer, B., Rimm, D., Dunbar, M., Philbrick, W. and Wysolmerski, J. (2001). Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development. Development 128, 513-525.
Hui, C. C. and Joyner, A. L. (1993). A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet. 3, 241-246.[Medline]
Lebeche, D., Malpel, S. and Cardoso, W. V. (1999). Fibroblast growth factor interactions in the developing lung. Mech. Dev. 86, 125-136.[Medline]
Mailleux, A., Tefft, D., Ndiaye, D., Itoh, N., Thiery, J. P., Warburton, D. and Bellusci, S. (2001). In vivo evidence for the role of SPROUTY2 as a negative modulator of mouse embryonic lung growth and morphogenesis. Mech. Dev. 102, 81-94.[Medline]
Medina, D. (1996). The mammary gland: a unique organ for the study of development and tumorigenesis. J. Mamm. Gland Biol. Neoplasia 1, 5-19.[Medline]
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E., DeRose, M. and Simonet, W. S. (1998). Fgf-10 is required for both limb and lung development and exhibits stricking similarity to Drosophila branchless. Genes Dev. 12, 3156-3161.
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. 277, 643-649.[Medline]
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier, F., Gao, G. and Goldfarb, M. (1996). Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271, 15292-15297.
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biology 2, 1-12.
Park, W. Y., Miranda, B., Lebeche, D., Hashimoto, G., Cardoso, W. V. (1998). FGF10 as a chemotactic factor for distal epithelial buds during lung development. Dev. Biol. 201, 125-134.[Medline]
Phippard, D. J., Weber-Hall, S. J., Sharpe, P. T., Stuart Naylor, M., Jayatalake, H., Maas, R., Woo, I., Roberts-Clark, D., Francis-West, P. H., Liu et al. (1996). Regulation of Msx1, Msx2, Bmp2 and Bmp4 during foetal and postnatal mammary gland development. Development 122, 2729-2737.
Propper, A. Y. (1978). Wandering epithelial cells in the rabbit embryo milk line. Dev. Biol. 67, 225-231.[Medline]
Revest, J. M., Spencer-Dene, B., Kerr, K., De Moerlooze, L., Rosewell, I. and Dickson, C. (2001). Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev. Biol. 231, 47-62.[Medline]
Robinson, G. W., Karpf, A. B. and Kratochwil, K. (1999). Regulation of mammary gland development by tissue interaction. J. Mamm. Gland Biol. Neoplasia 4, 9-19.[Medline]
Sakakura, T. (1987). Mammary embryogenesis. In The Mammary Gland (ed. M. C. Neuville and C. W. Daniel), pp. 37-65. Plenum Publishing Corporation.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. and Kato, S. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21, 138-141.[Medline]
Turner, C. W. and Gomez, E. T. (1933). The normal development of the mammary gland of the male and female albino mouse. I. Intrauterine. Mo. Agric. Exp. Stn. Res. Bull. 182, 3-20.
van Genderen, C., Okamura, R. M., Farinas, I., Quo, R. G., Parslow, T. G., Bruhn, L. and Grosschedl, R. (1994). Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev. 8, 2691-2703.[Abstract]
Weaver, M., Dunn, N. R. and Hogan, B. L. (2000). Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis. Development 127, 2695-2704.
Wysolmerski, J. J., Philbrick, W. M., Dunbar, M. E., Lanske, B., Kronenberg, H. and Broadus, A. E. (1998). Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development. Development 125, 1285-1294.
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105-2116.[Abstract]
Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. and Deng, C. (1998). Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development 125, 753-765.