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First published online 14 February 2007
doi: 10.1242/dev.000182

Development 134, 1221-1230 (2007)
Published by The Company of Biologists 2007
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BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction

Julie R. Hens1, Pamela Dann1, Jian-Ping Zhang1, Stephen Harris2, Gertraud W. Robinson3 and John Wysolmerski1,*

1 Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8020, USA.
2 Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
3 Laboratory of Genetics and Physiology, NIDDK, NIH, Bethesda, MD, USA.


Figure 1
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  		Fig. 1. BMP4 is expressed in the ventral mesenchyme during embryonic mammary gland development. (A) ß-galactosidase staining of a Bmp4-lacZneo embryo on E12.5. In these embryos ß-galatosidase is expressed under the control of the BMP4 promoter. At this stage of development, BMP4 is normally prominently expressed on the ventral surface of the embryo. (B) Cross-section of an E13.5 Bmp4-lacZneo mammary bud. BMP4 is expressed in both the mammary epithelium and the mammary mesenchyme, but more prominently within the mesenchme (arrows). Note the absence of expression in the epidermis (E).

 

Figure 2
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  		Fig. 2. PTHrP augments BMP signaling in vivo and in vitro. (A-E) Immunohistochemistry for phospho-SMAD 1, 5, 8 in sections through a PTHrP-/- mammary bud at E15.5 (A), a wild-type bud at E15.5 (B), a PTH1R-/- bud at E15.5 (C), wild-type ventral skin at E18.5 (D) and K14-PTHrP ventral skin at E18.5 (E). Note the nuclear staining for phospho-SMADs in the wild-type mammary mesenchyme (arrows in B) and its absence in the mammary mesenchyme of PTHrP-/- and PTH1R-/- buds (A,C). Also note the induction of nuclear phospho-SMAD staining in the mesenchyme of the ventral skin in K14-PTHrP embryos (arrows in E). (F) Western blot for phospho-SMAD 1, 5, 8 of whole cell lysates from C3H10T1/2 cells treated overnight with graded BMP4 concentrations (0, 10, 50, 100 ng/ml BMP4) with or without 10-7 M PTHrP. The addition of PTHrP augments phospho-SMAD expression (representative blot of five experiments).

 

Figure 3
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  		Fig. 3. PTHrP induces BMPr1a mRNA expression in vitro and in vivo. (A) BMPr1a mRNA detected by qRT-PCR in C3H10T1/2 cells treated with or without 10-7 M PTHrP overnight (n=3 experiments). (B) qRT-PCR for BMPr1a mRNA in ventral skin from cultured wild-type E13.5 embryos treated with or without 10-7 M PTHrP overnight (three experiments with two to three embryos per treatment). Note that PTHrP increases BMPr1a expression. (C) qRT-PCR for BMPr1a mRNA in the ventral skin from E18.5 K14-PTHrP transgenic and wild-type control mice. BMPr1a expression is higher in K14-PTHrP epidermis (n=4 experiments). (D) qRT-PCR for BMPr1a mRNA in mammary buds microdissected from either wild-type or PTHrP-/- embryos on E15.5. Expression is reduced in the PTHrP knockout buds compared with wild-type controls. RNA was from pooled buds (50-100 buds per sample). (E,F) Dark-field (E) and corresponding light-field (F) images of in situ hybridization for BMPr1a expression in an E15.5 wild-type mammary bud. Arrowhead points to the epithelial bud. The BMPr1a gene is expressed at a low level throughout the mesenchyme, but appears not to be expressed within the mammary epithelium.

 

Figure 4
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  		Fig. 4. BMP4 rescues outgrowth of PTHrP-/- mammary buds in organ culture. (A-D) Representative examples of bud outgrowths after 7 days in organ culture. (A) Wild-type mammary bud. (B) PTHrP-/- bud. (C) PTHrP-/- bud treated with 10-7 M PTHrP. (D) PTHrP-/- bud treated with 10 ng/ml BMP4. (E) Quantification of the frequency of bud outgrowth under different conditions as noted on the x-axis. Each experiment was performed on 15 buds over three separate experiments, except for the wild-type buds, which represent a total of 40 buds over eight separate experiments. *, P<0.05 for WT versus WT+noggin; #, P<0.05 for WT versus KO; triangle, P<0.05 for KO versus KO+PTHrP and KO versus KO+BMP4.

 

Figure 5
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  		Fig. 5. PTHrP regulates Msx2 gene expression in mammary mesenchyme. In situ hybridization for Msx2 mRNA. (A,D) PTHrP-/- mammary buds at E15.5; (B,E) wild-type mammary buds at E15.5; (C,F) PTH1R-/- buds at E15.5. (A-C) Light-field images; (D-F) corresponding dark-field images. Arrows in B,E point to the mammary mesenchyme. (G,H) Wild-type ventral skin from an E18.5 embryo. (I,J) K14-PTHrP ventral skin from an E18.5 embryo. (G,I) Dark-field images; (H,J) corresponding bright-field images. Arrows in I,J point to the underlying dermis that is induced to express Msx2 in response to overexpression of PTHrP in the overlying keratinocytes.

 

Figure 6
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  		Fig. 6. PTHrP and BMP4 interact to regulate Msx2 gene expression in C3H10T1/2 cell. qRT-PCR for Msx2 mRNA in C3H10T1/2 cells treated for 7 days with 10-7 M PTHrP, 50 ng/ml BMP4, or both together. Shown is the average level of expression represented as the fold induction over no treatment (NT). Each bar represents the average±s.e.m. of three individual experiments.

 

Figure 7
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  		Fig. 7. Loss of Msx2 rescues hair follicle induction in K14-PTHrP mice. Hematoxylin and Eosin-stained sections of wild-type ventral skin (A), K14-PTHrP transgenic ventral skin (B) and K14-PTHrP/Msx2-/- ventral skin (C). Sections were cut at 5 µm. Notice the recovery of hair follicles in the K14-PTHrP/Msx2-/- transgenic mice.

 

Figure 8
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  		Fig. 8. PTHrP and BMP signaling interact to initiate mammary bud outgrowth and nipple formation. PTHrP is secreted from mammary epithelial cells and increases BMPR1A expression in the mammary mesenchyme. This increases the sensitivity of these cells to BMPs and allows them to respond to BMP4 in a paracrine and/or autocrine fashion. BMP4 signaling in the mesenchyme, in turn, triggers epithelial outgrowth and augments MSX2 expression, which causes the mammary mesenchyme to inhibit hair follicle formation within the nipple sheath.

 

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© The Company of Biologists Ltd 2007