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First published online 18 March 2009
doi: 10.1242/dev.023994

Development 136, 1423-1432 (2009)
Published by The Company of Biologists 2009
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Ptch1 is required locally for mammary gland morphogenesis and systemically for ductal elongation

Ricardo C. Moraes1, Hong Chang2, Nikesha Harrington1, John D. Landua1, Jonathan T. Prigge1, Timothy F. Lane3, Brandon J. Wainwright4, Paul A. Hamel2 and Michael T. Lewis1,*

1 Lester and Sue Smith Breast Center and Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
2 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine,1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
3 UCLÁs Jonsson Comprehensive Cancer Center, 8-684 Factor Building, Box 951781, Los Angeles, CA 90095, USA.
4 Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072, Australia.


Figure 1
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  		Fig. 1. Whole-mount analysis of mammary glands from wild-type and homozygous Ptch1mes mice during postnatal virgin development. The genotype of mice from which glands were derived is shown above each column, along with the phenotype. The developmental timepoint at which glands were harvested is shown to the left. (A) Representative ductal tree and terminal end bud (TEB) array in a wild-type mouse at 5 weeks of age showing normal morphology and branching. (B) Representative stunted ductal tree in a homozygous Ptch1mes mouse. Note the lack of TEBs. (C) Representative `escape' ductal tree and TEB array in a homozygous Ptch1mes mouse showing unusual ductal morphology and branching. (D) Representative mature ductal tree in a wild-type mouse at 10 weeks of age showing normal morphology and branching. (E) The stunted ductal tree inappropriately retained in homozygous Ptch1mes mice. (F) Representative escape ductal tree and TEB array inappropriately retained in homozygous Ptch1mes mice at 10 weeks of age; note the frequently bifurcating and trifurcating TEBs. (G) Representative mature ductal tree in a wild-type mouse at 20 weeks of age. (H) The stunted ductal tree inappropriately retained in homozygous Ptch1mes mice. (I) Representative escape ductal tree that completely filled the fat pad in homozygous Ptch1mes mice at 20 weeks of age suggesting increased branching. (J) Bar chart showing the frequency of gland phenotypes in homozygous Ptch1mes mice at 10 weeks of age. (K) Bar chart showing the frequency of gland phenotypes as a function of gland position at 10 weeks of age. L, left; R, right. Scale bar: 1 mm.

 

Figure 2
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  		Fig. 2. Histological analysis of glands from wild-type and homozygous Ptch1mes mice during postnatal virgin development. The genotype of mice from which glands were derived is shown above each column, along with the phenotype. The developmental timepoint at which glands were harvested is shown to the left. (A) A TEB in a gland of a wild-type mouse showing normal histoarchitecture. (B) A duct terminus in the stunted ducts of homozygous Ptch1mes mice. (C) A TEB in a gland from homozygous Ptch1mes mice. (D) A duct in an escape gland showing a tendency towards increased cell layers and irregular lumen. (E) A mature duct in a gland of a wild-type mouse showing normal histoarchitecture. (F) A duct in a stunted gland from a homozygous Ptch1mes mouse showing multiple layers of luminal epithelial cells. (G) A TEB in an escape gland. (H) A duct in an escape gland showing generally normal histoarchitecture. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Whole gland and epithelial fragment transplantation into Rag1-/- hosts. The genotype of the epithelial fragment donor is shown above each column, and the magnification values of these inverted fluorescence images to the left. The boxed regions in A,B,E,F are shown at higher magnification in C,D,G,H. (A,C) Wild-type epithelial fragment showing complete filling of the available fat pad and normal duct morphology (A), with blunt or rounded duct termini (C). (B,D) Homozygous Ptch1mes gland showing complete fat pad filling but modestly altered duct morphology (B), with splayed duct termini (D). (E,G) Wild-type gland showing complete filling of available fat pad and normal duct morphology (E), with normal blunt or rounded duct termini (G). (F,H) Homozygous Ptch1mes fragment showing complete fat pad filling but modestly altered duct morphology (F), with excessively rounded duct termini (H). Arrows indicate aberrant termini. Scale bars: 1 mm.

 

Figure 4
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  		Fig. 4. Whole gland morphological and histological analysis of the Ptch1 conditional null allele at 10 weeks of age. (A-F) Carmine (A,C,E) and lacZ (B,D,F) staining of whole glands of mice that were either negative (Cre-) or positive (Cre+) for Cre recombinase expression; magnification, 5x. Mice were interbred with R26R mice and with a separate trangenic strain expressing Cre recombinase under the transcriptional control of the MMTV promoter region. Insets represent lacZ-stained glands from mice that were Cre negative; magnification, 1x. (G-L) Histological analysis of mice that were either negative or positive for Cre recombinase expression. Scale bars: 0.5 mm in A-F; 50 µm in G-L.

 

Figure 5
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  		Fig. 5. Effect of {Delta}Ptch1 heterozygosity in different genetic backgrounds. The mouse background is shown above each column, the genotype to the left. (A-H) Hematoxylin/Eosin-stained sections of mammary gland. (I-P) BrdU staining for evaluation of proliferation. (Q) BrdU labeling index for wild type and {Delta}Ptch1/+ in different backgrounds. *P=0.017 and *P=0.024 for DBA2 and B6D2F1, respectively (Wilcoxon rank sum test). Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Genetic polymorphism in the Ptch1 gene. (A) Partial gene sequence of Ptch1 from human, FVB, C57BL/6J and DBA2 mice. (B) Partial amino acid sequence of patched protein from human, FVB, C57BL/6J and DBA2 mice. The boxed residues indicate the A->C polymorphism (A) and resulting amino acid substitution (B) in the PTCH1 protein.

 

Figure 7
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  		Fig. 7. Ovarian hormone treatment assays to evaluate hormone responsiveness and immunohistochemical analysis for expression of ER and PR and for BrdU incorporation as a function of genotype and phenotype. (A-H) Treatment is shown above each column, the genotype of the treated mice to the left. (A) Wild type, untreated. (B) Wild type, E2-treated, showing increased branching and alveolar development. (C) Wild type, P-treated, showing increased branching and alveolar development. (D) Wild type, E2+P-treated, showing increased branching and alveolar development. (E) Homozygous Ptch1mes escape gland showing retained TEBs. (F) Homozygous Ptch1mes escape gland, E2-treated, showing a comparable response to the wild type (B). (G) Homozygous Ptch1mes escape gland, P-treated, showing a comparable response to the wild type (C). (H) Homozygous Ptch1mes escape gland, E2+P-treated, showing a comparable response to the wild type. The inset shows a stunted gland in a homozygous Ptch1mes showing no responsiveness to E2+P. (I-Q) Immunohistochemical analysis for expression of estrogen receptor (ER) and progesterone receptor (PR), and for BrdU incorporation as a function of genotype and phenotype. Scale bars: 2 mm in A-H; 50 µm in I-Q.

 

Figure 8
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  		Fig. 8. Whole-mount analysis of mammary glands from mice wild-type for Ptch1 and from Ptch1mes/Ptch1mes mice after pituitary isograft transplant. (A) Representative ductal tree in a mouse wild-type for Ptch1 (WT) at 8 weeks of age showing normal morphology and branching. (B) Representative WT ductal tree after pituitary isograft. Note the alveolar development and ducts reaching the fat pad periphery. (C) Stunted ductal tree in a sham-operated Ptch1mes/Ptch1mes mouse. (D) Representative Ptch1mes/Ptch1mes mammary gland showing ductal and alveolar development after pituitary isograft. (E) Bar chart showing the percentage of fat pad filled in Ptch1mes/Ptch1mes animals. Scale bar: 1 mm.

 

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© The Company of Biologists Ltd 2009