QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mailleux, A. A. Articles by Bellusci, S. Search for Related Content PubMed PubMed Citation Articles by Mailleux, A. A. Articles by Bellusci, S.

Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo

Arnaud André Mailleux^1,*, Bradley Spencer-Dene^2,*, Christian Dillon², Delphine Ndiaye¹, Catherine Savona-Baron¹, Nobuyuki Itoh³, Shigeaki Kato⁴, Clive Dickson², Jean Paul Thiery¹ and Saverio Bellusci^1,

¹ UMR144-CNRS/Institut Curie, 26 rue d’Ulm 75248 Paris cedex 05, France
² Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, UK
³ Kyoto University, Graduate School of Pharmaceutical Sciences, Yoshida-Shimoadachi, Sakyo, Kyoto 606-8501, Japan
⁴ Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
^* These authors contributed equally to this work

View larger version (74K): [in a new window]   Fig. 1. Lef1 expression in the mammary placode 3 between E11.5 and E11.75. Whole-mount in situ hybridization on embryos between E11.5 and E11.75 with dig-labeled antisense riboprobe for Lef1. (A) Lef1 is not expressed at significant level at E11.5 between the forelimb and hindlimb. (B) First evidence of Lef1 expression in the forming mammary placode (P3) occurs as a discrete line (between the 2 black arrows). (C and D) At E11.75, Lef1 is now expressed as a comet-like shape with the tail of the comet being localized caudally (between the 2 arrows) and then as a round like structure (P3). (E) Scanning electron microscopy of E11.5 embryo showing placode 3 as a knob of cells slightly elevated above the surface of the epidermis. (F) High magnification of placode 3. P: mammary placode. Scale bar, A-D, 785 µm; E, 980 µm; F, 390 µm.

View larger version (130K): [in a new window]   Fig. 2. Mammary placode development is asynchronous. Whole-mount in situ hybridization on embryos at E11.75 with dig-labeled antisense riboprobe for Lef1. (A) Lateral view of the embryo showing that placode 3 and placode 4 have formed and are seen as dot-like structures. (B) High magnification of the hindlimb area showing placode 4 as a dot- like structure and placode 5 as a line. (C) High magnification of the forelimb area showing placode 3 as a dot-like structure, placode 1 as a line and placode 2 as a diffuse line. (D-F) Scanning electron microscopy of the flank of a E12.5 wild-type embryo showing 5 mammary buds localized at precise points along the anteroposterior axis (3 thoracic and 2 inguinal). (E) High magnification of the box shown in D. Note that mammary bud 2, the last one to form, is the bud that is the most elevated above the surface of the epidermis. (F) High magnification of the mammary bud 2 showing individual cells at the surface of the knob-like structure. B1-5, mammary buds; P1-5, mammary placodes. Scale bar, A, 950 µm; B, 630 µm; C, 470 µm; D, 440 µm; E, 180 µm; F, 30 µm.

View larger version (74K): [in a new window]   Fig. 3. Fgfr2, Fgf7 and Fgf10 expression in the mammary buds between E10.5 and E15.5. Section and whole-mount in situ hybridization using 35S-labeled and dig-labeled antisense riboprobes for Fgfr2, Fgf7 and Fgf10. (A) Fgfr2 expression at E11.5 showing a high expression in the epidermis and in the epithelium of the mammary bud (arrowhead). (B) Fgfr2 expression at E15.5 showing a high expression in the basal layer of the epidermis and in the epithelium of the mammary bud. (C) Fgf10 expression at E10.5. Note the expression of Fgf10 between the fore- and hindlimb (between the two black arrows) corresponding to the area were the mammary line is forming. Fgf10 is also detected in the progress zone of the limbs. (D) High magnification of C showing Fgf10 expression as a dotted line (between the 2 arrows). Double headed arrow indicates the position of section in E. (E) Vibratome cross section (40 µm) between the fore- and hindlimb of the embryo shown in (C). Fgf10 is detected in the most ventral part of the somites. (F) High magnification of the boxed area in E showing Fgf10 expression in the epithelial part of the dermamyotome. (G) No expression of Fgf10 is detected in the mammary bud at E11.5. (H) Fgf10 expression at E15.5. Note the expression in the mammary fat pad precursor surrounding the mammary bud and in the mesenchyme of the hair follicles. (I) Fgf7 expression at E12.5. Note the high expression in the mesenchyme surrounding the epithelial bud. (J) Fgf7 expression at E15.5. Note the expression in the fat pad precursor. dm, dermamyotome; e, epithelium; fl, forelimb; fp, fat pad precursor; hf, hair follicles; hl, hindlimb; int, intestine; m, mesenchyme; my, myotome; nt, neural tube; mm, mammary mesenchyme; so, somites. Scale bar, A,B, 8 µm; C, 430 µm; D, 210 µm; E, 130 µm; F, 32 µm; G-J, 10 µm.

View larger version (61K): [in a new window]   Fig. 4. Mammary buds 1, 2, 3 and 5 are not detected in the Fgfr2b–/– embryos. Whole-mount in situ hybridization on Fgfr2b–/– and wild-type embryos at E12.5 and E13 with dig-labeled antisense riboprobe for Lef1 (A-H) and cell death analysis (I,J). B, D, F, H are high magnification views of the boxed areas in A, C, E and G, respectively. (A) Lef1 expression at E12.5 in wild-type embryo showing expression in the epithelium of the mammary bud. Note that only three mammary buds, located between the forelimb and hindlimbs, are visible. Mammary buds 1 and 5 are located behind the limbs. (B) Mammary bud 4. (C) Lef1 expression at E12.5 in Fgfr2b–/– embryo showing that only one mammary bud is detected. (E) Lef1 expression at E13 in wild-type embryo showing expression in the epithelium of the mammary bud. (F) Mammary bud 4. (G) Lef1 expression in the Fgfr2b–/– embryo is no longer detected in the area corresponding to the mammary bud 4 (dotted box). (I) Section of normal mammary bud at E12.5 showing a complete absence of cells stained by the TUNEL method. (J) Fgfr2b–/– mammary bud at E12.5 showing a high number of individual apoptotic cells labeled in the epithelium. Scale bar, A, 875 µm; B, 160 µm; C, 875 µm; D, 160 µm; E, 1220 µm; F, 235 µm; G, 1220 µm; H, 235 µm; I, J, 10 µm.

View larger version (63K): [in a new window]   Fig. 5. Mammary buds 1, 2, 3 and 5 are not detected in the Fgf10–/– embryos. Whole-mount in situ hybridization on Fgf10–/– and wild-type embryos at E11.5 and E12.5 with dig-labeled antisense riboprobe for Lef1. B, D, F and I are high magnification views of the boxed areas in A, C, E and H respectively. (A) Lef1 is expressed in the mammary placodes at E11.5. Note that only mammary placodes 3 and 4 are clearly visible. (B) Mammary placode 4. (C) Lef1 expression is not detected in mammary placodes 1, 2, 3 and 5. Note Lef1 expression as a line in the area corresponding to mammary placode 4 (dotted box). (E) Lef1 expression at E12.5 in wild-type embryo showing expression in the epithelium of the mammary bud. Note that only mammary buds 2, 3 and 4 are visible. (F) Mammary bud 4. (G) Hematoxylin-eosin staining of mammary bud 4. (H) Lef1 expression in the Fgf10–/– embryo shows that only mammary bud 4 is detected (dotted box). (J) Hematoxylin and Eosin staining of mutant mammary bud 4 showing a normal structure. Scale bar, A, 670 µm; B, 100 µm; C, 670 µm; D, 100 µm; E, 1050 µm; F, 150 µm; G, 14 µm; H, 1050 µm; I, 150 µm; J, 14 µm.

View larger version (102K): [in a new window]   Fig. 6. Mammary bud 4 from Fgf10–/– mutant female embryos is maintained and competent to ramify in wild-type stroma. Whole-mount in situ hybridization on Fgf10–/– and wild-type embryos at E13.5 with dig-labeled antisense riboprobe for Lef1 and transplantation of wild-type and mutant mammary glands in cleared fat pads. B and D are higher magnification views of the boxed areas in A and C, respectively. (A) Lef1 expression is detected in the hair follicles (small dots) and in the mammary buds (dilated dots) in wild-type embryos at E13.5. (B) Note that the mammary bud 4 (black arrowhead) is bigger than the hair follicles. (C) Lef1 expression on Fgf10–/– embryo. (D) Mammary bud 4 is still present at E13.5. (E) Transillumination picture of dissected skin corresponding to wild-type female embryo at E18.5. Note that five pairs of nipples are clearly visible. (F) Carmin Red staining of normal mammary gland 4 showing a ramified structure with numerous end buds (white arrowheads). (G) Transillumination picture of dissected skin corresponding to Fgf10–/– female embryos at E18.5. Note that only one pair of nipples is visible. (H) Carmin Red staining of mutant mammary gland 4 showing a single elongated sprout (white arrowhead). (I) Carmin Red staining of cleared fat pads transplanted with wild-type mammary gland 4 and cultured for 4 weeks. The epithelium of wild-type mammary gland 4 (black arrow) has extensively ramified into the cleared fat pad in both directions. (J) High magnification of the transplanted mammary gland (asterisk) showing a nicely ramified epithelium emerging from it. (K) The epithelium of mutant mammary gland 4 (black arrow) has also ramified into the cleared fat pad in both directions. Note the presence of numerous end buds (small black arrows). (L) High magnification of the transplanted mammary gland (asterisk) showing a ramified epithelium emerging from it and hairs (dark stripes) at the graft side. B4, mammary bud 4; fp, fat pad; G1-5, mammary glands; ln, lymph node. Scale bar, A, 800 µm; B, 309 µm; C, 800 µm; D, 309 µm; E, 1800 µm; F, 80 µm; G, 1800 µm; H, 80 µm; I, 2600 µm; J, 315 µm; K, 2100 µm; L, 270 µm.

View larger version (94K): [in a new window]   Fig. 7. FGF10-coated beads fail to induce Lef1 expression in the epithelium. Organotypic culture after FGF10-coated bead grafting was carried out for 24 hours and followed by whole-mount in situ hybridization to detect Lef1 expression. (A) Two FGF10-coated beads (asterisks) were grafted on the flank of E11.5 embryos. One bead was grafted close to the mammary line and the other one was grafted more dorsally. (B) High magnification of the boxed area in A showing endogenous Lef1 expression in the mammary placode 2 and 3 but no Lef1 induction in the ectoderm surrounding the bead. fl, forelimb; hl, hindlimb. Scale bar, A, 890 µm; B, 170 µm.