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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02394

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Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes

Jacqueline M. Veltmaat^1,2,*, Frédéric Relaix³, Lendy T. Le¹, Klaus Kratochwil¹, Frédéric G. Sala^1,2, Wendy van Veelen^2,, Ritva Rice⁴, Bradley Spencer-Dene⁵, Arnaud A. Mailleux^2,, David P. Rice⁴, Jean Paul Thiery² and Saverio Bellusci^1,2

¹ The Saban Research Institute of Childrens Hospital Los Angeles/University of Southern California, Developmental Biology Program, Los Angeles, CA 90027, USA.
² Institut Curie/CNRS-UMR144, Equipe de Morphogenèse Cellulaire et Progression Tumorale, 75005 Paris, France.
³ Institut Pasteur/CNRS-URA 2578, Département de Biologie Moléculaire, Génétique Moléculaire du Développement, 75015 Paris, France.
⁴ King's College, Departments of Orthodontics and Craniofacial Development, London SE1 9RT, UK.
⁵ Cancer Research UK, London Research Institute, Experimental Pathology Laboratory, and Imperial College London, Department of Histopathology, London WC2A 3PX, UK.

View larger version (37K): [in a new window]   Fig. 1. The position of mammary rudiments relative to the somites. E11 embryo with the somites (numbered) indicated as grey contours. Only the thoracic somites 11-24 possess a ventrally elongated hypaxial bud, underlying the region where mammary placodes 2, 3 and 4 develop (black dots). Mammary placodes 1 and 5 are covered by the forelimb and hindlimb, respectively, and are not indicated.

View larger version (37K): [in a new window]   Fig. 2. The absence of hypaxial Fgf10 expression correlates with a mammary phenotype in Pax3ILZ/ILZ embryos. (A-D) X-Gal staining revealing Pax3-lacZ expression; the boxed area in A and B is magnified in C and D. The hypaxial buds, indicated by black arrowheads, of the somites (numbered) are absent in the mutant. (E-J) Whole-mount in situ hybridization at E11.25 (42 somite), E11.75 (48 somite) and E12.5 for Wnt10b, revealing the mammary line (between the arrows in E,F) and placodes (numbered in G-J), demonstrates late formation of placode 3 in the mutant. (K-P) Hematoxylin-Eosin stained sections through the plane indicated by the black line in E. The vertical line in K-N through the notochord illustrates the more dorsal position of the mammary line in mutants. The horizontal line with downwards arrows (M,N) demarcates the area containing cylindrical ectodermal cells, while the area between the asterisks (O,P) contains a stratum intermedium as a hallmark of the mammary line. Both areas are narrower in mutants. (Q-R) Whole-mount in situ hybridization for Fgf10 at E11.5. The hypaxial domain with high Fgf10 expression (black arrowheads) is absent in the mutant (R). Abbreviations: s, somite stage; sg, stratum germinativum; si, stratum intermedium; p, periderm. Scale bars: 10 µm in O,P; 100 µm in C-J,M,N,Q,R; 1 mm in A,B,K,L.

View larger version (90K): [in a new window]   Fig. 3. Impaired mammary line formation precedes the absence of mammary placodes in Fgf10-/- and Fgfr2b-/- embryos. (A-F) Hematoxylin/Eosin-stained sections through a wild-type (A-D), Fgf10-/- (E) and Fgfr2b-/- embryo (F) around E11.5, through the plane indicated by the line in Fig. 2E. Boxed areas in A are magnified in B-F. First, the stratum germinativum consists of squamous cells (B) that become cuboidal and covered with a layer of periderm (C) on the Wolffian ridge or flank. Next, they become cylindrical and form an additional layer (stratum intermedium) of cylindrical cells on the ventral area of the flank (D). This indicates mammary line formation, which does not occur in either mutants (E,F). (G-I) In situ hybridization for Wnt10b expression shows formation of the mammary line (between arrows) and mammary placodes (numbered), restricted to the area of placode 4 in the mutants. (J-L) X-gal staining visualizing TOPGAL expression indicates that WNT signaling occurs in a pattern similar to Wnt10b expression. (M-O) In situ hybridization for Lef1 reveals that all placodes (numbered) except 4 are absent in the mutants. Abbreviations: s, somite stage; wt, wild type; ct, control; sg, stratum germinativum; dm, dermal mesenchyme; p, periderm; si, stratum intermedium. Scale bars: 10 µm in B-F; 100 µm in G-O; 1 mm in A.

View larger version (54K): [in a new window]   Fig. 4. Fgf10 and Fgfr2b expression patterns and rescue of the Fgf10-/- mammary defect by recombinant FGF10. (A,B) In situ hybridization for Fgf10 on an E10.0 (30 somite) wild-type embryo; whole-mount (A) and a 40 µm vibratome section (B) along the plane indicated by the line in A. Fgf10 expression is present in the lateral plate mesoderm of the flank. (C-H) Radiolabeled in situ hybridization on Hematoxylin counterstained sections shows Fgf10 expression (red) in the somites (white contours in C,D), and Fgf2b expression (red) in the surface ectoderm (E-H). The arrowed brackets in E-H indicate the Wolffian ridge (WR) or flank. The mammary line (ml) overlying the hypaxial bud of the somite (black contour in H) expresses elevated levels of Fgfr2b. (I-L) Whole-mount in situ hybridization for Wnt10b, showing mammary placode formation (arrows) in a flank of a control (I,K) or Fgf10-/- embryo (J,L), cultured with an implanted bead (circle) soaked in BSA (I,J) or rFGF10 (K,L). (M,N) Nuclear Fast Red stained section at the level of the lines through the flank in L. Boxed area in the insets are magnified; broken black lines outline the placodes. Abbreviations: LPM, lateral plate mesoderm; nt, neural tube; s, somite stage; som, somite; WR, Wolffian ridge; ml, mammary line. Panels A-H are not to scale. Scale bars: 1 mm in I-L; 100 µm in M,N.

View larger version (44K): [in a new window]   Fig. 5. Impaired mammary line formation due to reduced somitic Fgf10 expression precedes the absence of mammary bud 3 in Gli3Xt-J/Xt-J embryos. (A,B) Whole-mount in situ hybridization for Wnt10b reveals impaired mammary line formation (between arrows), the presence of placodes 2 and 4 (numbered), but not placode 3 in an E11.5 (44s) Gli3Xt-J/Xt-J embryo. (C,D) X-gal staining for TOPGAL expression indicates that WNT signaling in an E11.5 Gli3Xt-J/Xt-J embryo is reduced in a pattern similar to Wnt10b expression. (E,F) Whole-mount in situ hybridization for Lef1 reveals the absence of bud 3 in an E12.5 Gli3Xt-J/Xt-J embryo. (G,H) Hematoxylin/Eosin-stained sections through the plane indicated in Fig. 2E reveals the absence of the stratum intermedium at the mammary line of an E11.5 (45 somites) Gli3Xt-J/Xt-J mutant. (I-K) Radiolabeled in situ hybridization for Gli3 (red) on a section of an E11.0 (41 somite) wild-type embryo (I) through the plane indicated in Fig. 2E, and whole-mount in situ hybridization for Gli3 on a control (J) and Fgf10-/- (K) embryo shows unchanged somitic Gli3 expression in the Fgf10-/- mutant. (L,M) Whole-mount in situ hybridization for Fgf10 reveals that somitic Fgf10 expression (between black arrowheads) is restricted to the hypaxial domain in the Gli3Xt-J/Xt-J embryo. (N,O) Whole-mount in situ hybridization for Wnt10b on flanks of Gli3Xt-J/Xt-J embryos, cultured with an implanted bead (circle) soaked in BSA (N) or rFGF10 (O). Arrows indicate the mammary placodes or the expansion of a Wnt10b expression domain around the bead in O. (P,Q) Nuclear Fast Red stained section through the planes indicated by lines through the flank in L. Broken black lines outline the mammary epithelium. Abbreviations: s, somite stage; p, periderm; sg, stratum germinativum; si, stratum intermedium; pl #3, mammary placode 3; nt, neural tube; st, stomach; fl, forelimb; hl, hindlimb; rFGF10, recombinant FGF10. Scale bars: 10 µm in G-H,P-Q; 100 µm in A-F,I-M; 1 mm in N,O. Panel I is not to scale.

View larger version (56K): [in a new window]   Fig. 6. Hypomorphic Fgf10 mutants phenocopy the mammary defect of Gli3Xt-J/Xt-J embryos. Boxed areas in A,B,E,F are shown at high magnification in C,D,G,H. (A-D) Whole-mount in situ hybridization for Wnt10b reveals the absence of placode 3 in an E11.25 (43 somite) Fgf10 hypomorphic (Fgf10mlcv/-) mutant. (E-H) Whole-mount in situ hybridization for Lef1 reveals the absence of bud 3 in an E12.5 Fgf10 hypomorphic mutant. (I,J) Hematoxylin/Eosin-stained sections, showing the absence of the stratum intermedium in an E11.5 (45 somite) mutant. Arrows in D and F indicate morphological limb defects in Fgf10 hypomorphic mutants, while limbs are totally absent in Fgf10-/- embryos. Abbreviations: sg, stratum germinativum; p, periderm; s, somite stage. Scale bars: 10 µm in M,N; 100 µm in C,D,G,H; 1 mm in A,B,E,F.

View larger version (15K): [in a new window]   Fig. 7. A model combining ventral elongation of the somites and somitic Fgf10 expression with maturation of the surface ectoderm and the acquisition of a mammary cell fate at the position of mammary placode 3. (A) A model for the molecular interactions leading to a mammary cell fate in the surface ectoderm. GLI3 (purple dots) and an unidentified factor or factors X act upstream of Fgf10 (pink dots) in the somites. Somitic FGF10 activates ectodermal FGFR2B (green), via diffusion and/or delamination of somitic cells. Sufficient levels of activation are required for Wnt10b/TOPGAL expression (blue) and multilayering of the surface ectodermal cells as hallmarks of a mammary cell identity. (B) Schematic drawings of body halves of progressively older embryos, illustrating the correlation between somitic elongation, somitic Fgf10 expression and ectodermal maturation. For convenience, the body size is kept the same for all ages. Broken vertical lines indicate the mammary line (ml) and Wolffian ridge (WR). Increasingly darker shades of pink indicate increasingly higher levels of Fgf10 expression. The surface ectodermal cells are cuboidal (thicker black line) at the WR, whereas dorsal and ventral to the WR they are squamous (thinner black line). Ventrally on the flank, ectodermal cells become cylindrical (green) and enlarge (orange) coinciding with low levels of Wnt10b and TOPGAL expression. These cells give rise to a stratum intermedium (blue), with elevated Wnt10b and TOPGAL expression, indicating a mammary cell fate. (C) Model of cumulative FGF10/FGFR2B signaling in the flank leading to progressive maturation of the ectoderm, and eventually conversion into mammary epithelium. See main text for explanation. Color coding corresponds to color coding of B. Abbreviations: G, dorsal root ganglion; som, somite.