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First published online 16 August 2006
doi: 10.1242/dev.02542

Development 133, 3661-3670 (2006)
Published by The Company of Biologists 2006
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Gli3-mediated repression of Hedgehog targets is required for normal mammary development

Sarah J. Hatsell1,2 and Pamela Cowin1,2,*

1 Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
2 Department of Dermatology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.


Figure 1
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  		Fig. 1. Gli2-lacZ expression in embryonic mammary buds. (A-C) Whole-mount X-gal staining of Gli2-lacZ embryos. (A) At E11, expression is seen in an arc between the fore- and hindlimbs (arrow) and in the somites (som, arrowhead). (A, inset) A cross-section through this embryo shows staining is found within the dermal mesenchyme (mes) but not in the epithelial layer (epi, arrow). (B,C) At E14.5 strong staining is seen in both hair placodes and mammary buds (1,3,4, arrows). (D-G) Cross-sections through Gli2-lacZ embryos stained with X-gal. Staining is seen within the basal epithelial layer (epi) and surrounding stroma (str) of E14.5 mammary buds (D) and E16.5 mammary sprout (E). Epithelial Gli2-lacZ expression is restricted to a basal subset of p63-(F) and K14-(G) positive cells.

 

Figure 2
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  		Fig. 2. Detection of Gli3 mRNA in embryos by whole-mount in situ hybridization. (A) At E11 a broad band of staining is seen between the fore- and hindlimbs in the region of the developing mammary line (arrow) and in the somites (som, arrowhead). (B) At E13.5 weak staining is seen in and around all five pairs of mammary buds but expression is strongest in bud pair number 3, inset shows bud number 3 at higher magnification. (C) Immunohistochemistry for Gli3 showed nuclear staining in the mammary bud epithelium (epi) and surrounding stroma (str) in wild type. (D) Staining was not seen in the mammary bud epithelium from a Gli3xt/xt control embryo, although some background staining was present in the stroma.

 

Figure 3
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  		Fig. 3. Hh transcriptional targets are not expressed in embryonic mammary buds. Whole-mount X-gal staining of Gli1-lacZ in E14.5 (A,B) and Ptch-lacZ in E13.5 (C,D) embryos. Hair follicles show staining in these embryos but mammary buds (1-4, arrows) are unstained. B,D are higher magnification images of A,C.

 

Figure 4
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  		Fig. 4. Postnatal mammary expression of Gli2-lacZ in 4 day and 5-week-old virgins. Strong X-gal staining is seen in the stromal cells (arrows) surrounding the mammary ducts in glands of 4-day-old virgin mice (4dV) (A,B). Weaker expression is seen in the myoepithelial cells identified by p63 immunohistochemistry (arrowheads, B). In 5-week-old virgins (5wV), Gli2-lacZ is found in the stromal cells encasing the ducts and is especially strong in the condensing stroma around the terminal end bud (C,E, arrow). Expression is also found in lymphatic ducts at all stages of mammary development (E, asterisk). Cross-section through ducts (D) and terminal end buds (F) of 5-week-old mice show spikes of stromal cells (arrows) between the adipocytes and myoepithelial cells (D,F). Immunohistochemistry for p63 (G) and K14 (H) demonstrates that, at this developmental stage, all Gli2-lacZ staining lies beneath the myoepithelial layer (arrowheads) and is, thus, stromal.

 

Figure 5
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  		Fig. 5. Mammary Gli2-lacZ expression during pregnancy. At 14.5 days mid-pregnancy (14.5dP) Gli2-lacZ expression is especially strong around alveoli (arrows) (A,B). Cross-sections of glands show that at this stage of pregnancy Gli2-lacZ remains in stromal cells (arrows) and is also found in myoepithelial cells (arrowheads) that stain positive for K14 (C) and p63 (D).

 

Figure 6
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  		Fig. 6. Gli3 expression in postnatal mammary gland. (A) Gli3 mRNA was detected by northern blot analysis of mRNA isolated from mammary glands of both 7-week-old virgin (7wV) and mid-pregnant (13.5dP) mice. Keratin 18 (K18) was used as a control for mRNA integrity and loading. (B) Gli3 mRNA was detected by in situ hybridization in mammary epithelium (arrowhead) of 4-week-old virgin (4wV) mice. (D) Expression continues during pregnancy within both the luminal and myoepithelial cells (arrowhead). Sense control hybridization showed no signal in glands from 4-week-old virgins (C) or 13-day pregnant mice (E). Immunohistochemistry for Gli3 showed nuclear staining in both luminal and myoepithelial cells (arrowheads) of 4-week-old virgins (F) and 13-day pregnant mice (G). Staining was also seen in stromal cells at both stages (arrows).

 

Figure 7
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  		Fig. 7. Lymphatic expression of Gli1-lacZ in adult tissue. (A,B) Expression in mammary glands from 5-week-old virgins (5wV) is restricted to lymphatic vessels. Similar X-gal staining is seen in vessels in various organs including surrounding the heart (C) and the omentum (D).

 

Figure 8
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  		Fig. 8. Mammary bud formation is impaired in Gli3xt/xt mice and after mis-activation of the hedgehog pathway. TOP-Gal expression is seen in the developing mammary placodes number 3 and number 4 in wild-type E11 embryos (arrows) (A). TOP-Gal expression is absent from the region of bud 3 in E11 Gli3xt/xt embryos (white arrow) but is present in the developing bud 4 (black arrow) (B). In E12.5 wild-type embryos, TOP-Gal expression reveals the development of five pairs of mammary buds (C, arrows), whereas Gli3xt/xt embryos lack bud pairs 3 and 5 (D, predicted position of missing buds is marked by white arrows). E13.5 Gli21nki/1nki embryos (E) show normal development of five pairs of mammary bud, but Gli21nki/1nki; Gli3xt//+ embryos (H) show loss of buds 3 and 5 (white arrows). Higher magnification of the thoracic buds (F,I) and inguinal buds (G,J). A hypoplastic bud 3 (arrowhead) is seen in some Gli2lzki/+; Gli3xt/xt embryos (K). Misactivation of Gli1-lacZ is seen in the stroma (str) surrounding buds 1 and 4 (arrow) in Gli3xt/xt embryo wholemounts (L,M) and sections (N).

 

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© The Company of Biologists Ltd 2006