Morphogenesis of the developing mammary gland: stage-dependent impact of adipocytes - PubMed (original) (raw)

Morphogenesis of the developing mammary gland: stage-dependent impact of adipocytes

Shira Landskroner-Eiger et al. Dev Biol. 2010.

Abstract

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (apoptosis through triggered activation of caspase-8), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points and fewer terminal end buds (TEBs) and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.

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Figures

Figure 1. Ablation of adipocytes following dimerizer treatment

Mice were injected with dimerizer starting at 2-weeks of age, for either 2 or 4 weeks. A) Excised inguinal mammary glands were weighed, demonstrating a 2.2 and 2.8-fold reduction in mammary gland mass following a 2 and 4-week treatment, respectively. (*p<0.001, n=5–9/per group). B) H&E of the mammary gland of 6-week old untreated WT and FAT-ATTAC mice (Scale bar 100mm). C) Gonadal fat pads derived from same experiments as described, reveal an increase in mass following a 4-week dimerization protocol (*p<0.05, n=6–10/per group). D) A modest, yet significant, increase in the fasting glucose levels of FAT-ATTAC mice was detected (*p<0.05, n=5–13/per group). E) mRNA levels of the FAT-ATTAC transgene in the mammary gland (MG) and the gonadal fat pad (GF) was examined in untreated animals at 6-weeks of age. No significant difference was observed (n=5/per group).

Figure 2. Inhibition of ductal morphogenesis as a consequence of adipocyte depletion in the mammary stroma

A) Representative whole mounts of inguinal mammary glands treated from 2-weeks of age for 2 weeks (panel a–b) or 4-weeks (panel c–d). (Scale bar 1mm). B) Quantification of ductal extension as measured for the longest duct. C) Quantification of the number of TEBs. (B–C *p<0.05, n=5–13/per group). D) Average number of secondary branches per 0.5cm of major ducts in mice treated for 4-weeks with the dimerizer (*p<0.05, n=4–5/per group).

Figure 3. Histological examination reveals an effective loss of adipocytes in the mammary adipose stroma resulting in abnormal ductal morphology

Mice were subjected to treatment with the dimerizer from 2-weeks of age for a period of 2 or 4 weeks. A) H&E of the mammary gland (panel a–c) demonstrates that a 2- week treatment is sufficient to induce massive changes in the architecture of the adipocytes in the mammary stroma. These changes are exacerbated by 4-weeks of treatment. The ablation of mammary adipocytes resulted in irregular shape of the ducts. (Scale bar panel (a–c) 50mm, n=6–10/per group). B) Immunohistochemistry for macrophages using the F4/80 marker, as it is associated with local adipose structure disruption and apoptosis (Scale bar panel (a–b) 20mm, panel (c–d) 100mm). C) Sirius red staining (panel a–b) for collagen content at 4 weeks of age. Masson’s Trichrome staining, at 4 weeks of age, highlighting primarily collagen I and collagen III (blue) demonstrating increase in collagen deposition throughout the mammary epithelium and stromal compartment (Scale bar panel (a–b) 100mm, panel (c–d) 50mm).

Figure 4. Immunohistochemical characterization of mammary glands

Mice were subjected to treatment with the dimerizer from 2-weeks of age for a period of 4 weeks. Immunohistochemical analysis for basal cells markers such as p63, a-smooth muscle, keratin 5 and keratin 14 did not reveal any apparent expression differences among treated wild-type and FAT-ATTAC mice. Cytokeratin 8 analysis of luminal cells revealed a higher degree of epithelial clustering with multiple layers of cells within the ducts (n=5/per group, Scale bar 50mm).

Figure 5. Histological examination confirms a mild response in the gonadal fat pad

Mice were subjected to treatment with the dimerizer from 2-weeks of age for a period of 2 or 4 weeks. As observed by H&E (panel a–c), the gonadal fat pad displays a subtle response to the dimerizer with minimal disruption of adipose structure following 2 weeks of treatment and a gradual increase in alteration of adipocyte histology following 4 weeks of treatment. Panel (d–f) display immunohistochemistry staining for macrophages using the F4/80 marker. (Scale bar panel (a–c) 20mm, panel (d–f) 50mm, n=6–10/per group).

Figure 6. Ablation of mammary stromal adipocytes results in inhibition of both proliferation and apoptosis

A) BrdU immunohistochemistry was carried out on mammary glands derived from mice that were treated with the dimerizer for a 2-week period. Shown is the % BrdU positive cells per TEB field. (*p<0.05, n=4/per group, Scale bar 20mm). B) TUNEL immunohistochemistry of mice dimerized for 4 weeks. Shown is quantification of the TUNEL immunohistochemistry per TEB field (*p<0.05, n=4/per group, Scale bar 20mm). C) Mice were dimerized for 4 weeks and immunohistochemical analysis of TEBs was conducted using the luminal epithelial marker cytokeratin 8 (n=5/per group, Scale bar 50mm).

Figure 7. Normalization of mammary gland growth following cessation of dimerizer treatment

Mice were treated from 2-weeks of age for a period of 2 weeks followed by period of 2-weeks which injections of the dimerizer was ceased. At 6-weeks of age mice were sacrificed. A) Excised mammary gland were weighed, the mammary fat pad of FAT-ATTAC mice had a significantly smaller mass compared to wildtype mice (*p<0.05, n=4/per group). B) H&E of FAT-ATTAC mammary glands demonstrate extensive patches of emerging adipose tissue regeneration, with the mammary ducts gradually adopting a normal morphology. C) Circulating adiponectin levels sharply decrease during dimerizer treatment by 70–75% as compared to wildtype mice. Cessation of the dimerizer treatment partially restores adiponectin levels to approximately 60% of wildtype levels (right hand bar). (*p<0.05). D) Whole-mount analysis demonstrated that the number of TEBs, and ductal extension although slightly lower in the transgenic mice, does not mount to a significant difference when compared to wildtype littermates. In contrast, the number of secondary branches are still significantly lower in FAT-ATTAC mammary glands compared to the wildtype littermates (n=4/per group).

Figure 8. Treatment of FAT-ATTAC mice during adulthood results in lobuloalveolar differentiation

Mice were treated from 7-weeks of age for a period of 3 weeks. A) Whole-mounts and H&E are shown in panel a–b and panel c–e respectively. (Scale bar panel (a–b) 1mm, panel (c–d) 50mm, n=7–10/per group). Wild-type females are devoid of lobular-alveoli development, in contrast to FAT-ATTAC females, that display lobular-alveolar structures. B) Estradiol measurements determined in treated wild-type and FAT-ATTAC mice (n=7–8/per group). C) mRNA levels of b-casein. Total RNA was isolated from treated wild-type and FAT-ATTAC mammary glands (*p<0.05, n=3–4/per group). D) Immunohistochemistry analysis was carried for b-casein in mammary glands (Scale bar panel (a–b) 100mm, panel (c–d) 20mm, n=5/per group).

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Morphogenesis of the developing mammary gland: stage-dependent impact of adipocytes - PubMed (original) (raw)