Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty - PubMed (original) (raw)

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty

Teresa Elo et al. Sci Rep. 2017.

Abstract

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1

Fgf20 is induced by Eda and is expressed in embryonic mammary glands. (a) qRT-PCR analysis of Fgf4 (n = 4), Fgf 9 (n = 4), Fgf17 (n = 6) and Fgf20 (n = 7) expression in E13.5 Eda −/− mammary buds after 4 h treatment with Eda protein ex vivo. Values represent mean ± SD. (b,c) X-gal_-_stained whole mounts of Fgf20 LacZ/+ embryos at E11.5 (b) and E13.5 (c) showing positive staining in the developing mammary buds (numbered). (d) In situ hybridization of a WT embryo with an Fgf20 specific probe at E13.5. (e,f) X-Gal stained whole mount of E15.5 whole embryo (e) and dissected skin of E16.5 embryo (f) showing staining in the developing mammary buds (numbered) and hair follicles. (g,h) Representative figures of histological sections of X-Gal whole mount-stained mammary glands of Fgf20 LacZ/+ embryos at E16.5 (g) and E18.5 (h). *p < 0.05. At least two litters of Fgf20 LacZ/+ embryos per stage were analyzed. *p < 0.05. mb, mammary bud.

Figure 2

Figure 2

The influence of loss (Eda −/−) and gain of Eda (K14-Eda) on the expression of Fgf20-LacZ in embryonic mammary glands. (a,b) Whole-mount X-Gal staining of Eda −/−;Fgf20 LacZ/+; Eda +/+;Fgf20 LacZ/+, and K14-Eda;Fgf20 LacZ/+ embryos at E11.5 and E12.5. (c–e) Histological sections of whole mount X-Gal stained mammary buds at E13.5 (mb2), E14.5 (mb2), and E15.5 (mb3).

Figure 3

Figure 3

Fgf20 deficiency does not impede placode induction but compromises bud growth. (a) Expression of Wnt10b at somite stage 46–48 (Fgf20 LacZ/+, n = 7; Fgf20 LacZ/LacZ, n = 7) and (b) E12.5 (Fgf20 LacZ/+, n = 6; Fgf20 LacZ/LacZ, n = 6), and (b’) quantification of Wnt10b expression area (mammary bud 3) at E12.5. (d,e) 3D images and volume quantifications of EpCAM-stained mammary bud 3 at E13.5 (Fgf20_LacZ_/+, n = 24; Fgf20 LacZ/LacZ, n = 28), and E15.5 (Fgf20_LacZ_/+, n = 8; Fgf20 LacZ/LacZ, n = 13). The bud contours were outlined manually (purple) for volume quantification. ***p < 0.001; ****p < 0.0001.

Figure 4

Figure 4

Fgf20 deficiency delays induction of supernumerary buds in K14-Eda mice. (a) Expression of PTHrP at E13.5 (Fgf20 LacZ/+, n = 4; Fgf20 LacZ/LacZ, n = 4; K14-Eda;Fgf20 LacZ/+, n = 7; K14-Eda;Fgf20 LacZ/LacZ, n = 5), and (b) X-gal staining of Fgf20-LacZ at E13.5 (Fgf20 LacZ/+, n = 4; Fgf20 LacZ/LacZ, n = 6; K14-Eda;Fgf20 LacZ/+, n = 11; K14-Eda;Fgf20 LacZ/LacZ, n = 8). Supernumerary placodes (stars) were detected between buds 3 and 4 in K14-Eda;Fgf20_LacZ/+_ embryos at E13.5, but not in K14-Eda;Fgf20_LacZ/LacZ_ embryos.

Figure 5

Figure 5

Fgf20 deficiency compromises TEB formation and ductal invasion. (a–c) Carmine alum stained ductal trees of the 4th mammary gland (a,b) and histology of TEBs (c) of WT and Fgf20 LacZ/LacZ mice at 5 weeks of age. (d–g) Quantification of the ductal ends (d), TEBs (e), ductal outgrowth (measured as the distance of furthest grown ductal end from the center of the lymph node) (f), and width of five biggest ductal ends in each gland (f) in WT (n = 6) and Fgf20_LacZ/LacZ_ (n = 10) mice. (h, i) Immunohistochemical staining and quantification of Ki-67 -positive cells in TEBs of WT (n = 4) and Fgf20 LacZ/LacZ (n = 3) mice. Total number of TEBs analyzed was n = 15 (WT), n = 9 (Fgf20 LacZ/LacZ). Bars show mean ± SD. *p < 0.05; **p < 0.01.

Figure 6

Figure 6

Loss of Fgf20 attenuates the K14-Eda hyperbranching phenotype. (a–d) Carmine alum stained 4th mammary gland of WT, Fgf20 LacZ/LacZ, K14-Eda, and K14-Eda;Fgf20 LacZ/LacZ mice at E18 (a), 3 weeks (b), 7 weeks (c), and 12 weeks of age (d). (a’–d’) Quantification of the total number of end ducts (a’,b’) or end ducts past the lymph node (c’,d’) in 4th mammary gland. Number of glands analyzed were: WT (nE18 = 5, n3wk = 18, n7wk = 12, n12wk = 15) Fgf20 LacZ/LacZ (nE18 = 8, n3wk = 16, n7wk = 28, n12wk = 5), K14-Eda (nE18 = 7, n3wk = 8, n7wk = 7, n12wk = 13) and K14-Eda;Fgf20 LacZ/LacZ (nE18 = 6, n3wk = 8, n7wk = 9, n12wk = 10) (e) Ductal outgrowth (mm) measured from center of the lymph node in Fgf20 +/+ (nglands = 7) and Fgf20 LacZ/LacZ (nglands = 23). Data are shown as mean ± SD. ***p < 0.001; **p < 0.01; *p < 0.05; NS, not significant.

Figure 7

Figure 7

Analysis of terminal end buds of Fgf20 LacZ/LacZ mice at 7 weeks of age. (a) Hematoxylin Eosin -stained sections of WT and Fgf20 LacZ/LacZ TEBs. (b) Quantification of TEB area from Carmine alum stained mammary glands of WT (n = 9) and Fgf20 LacZ/LacZ (n = 12) mice. (c,c’) Immunohistochemical staining and quantification of Ki-67 –positive cells in TEBs of WT (n = 4) and Fgf20 LacZ/LacZ (n = 5) mice. Total number of TEBs analyzed was n = 26 (WT), n = 30 (Fgf20 LacZ/LacZ). (d,d’) Immunohistochemical staining and quantification of cleaved caspase-3 –positive cells in WT (n = 4) and Fgf20 LacZ/LacZ mice (n = 4). Total number of TEBs analyzed was n = 34 (WT), n = 29 (Fgf20 LacZ/LacZ). (e–i) Immunohistochemical staining of ERα (e), PR (f), K8 and K14 (g), SMAα (h), and p63 (i) in the TEBs of WT and Fgf20 LacZ/LacZ mice. Minimum of 4 mice per genotype were analyzed. Values represent mean ± SD. **p < 0.01; *p < 0.05; NS, not significant.

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