Interactions between FGF and Wnt signals and Tbx3 gene expression in mammary gland initiation in mouse embryos

Abstract

Interactions between Wnts, Fgfs and Tbx genes are involved in limb initiation and the same gene families have been implicated in mammary gland development. Here we explore how these genes act together in mammary gland initiation. We compared expression of Tbx3, the gene associated with the human condition ulnar-mammary syndrome, expression of the gene encoding the dual-specificity MAPK phosphatase Pyst1/MKP3, which is an early response to FGFR1 signalling (as judged by sensitivity to the SU5402 inhibitor), and expression of Lef1, encoding a transcription factor mediating Wnt signalling and the earliest gene so far known to be expressed in mammary gland development. We found that Tbx3 is expressed earlier than Lef1 and that Pyst1 is also expressed early but only transiently. Patterns of expression of Tbx3, Pyst1 and Lef1 in different glands suggest that the order of mammary gland initiation is 3, 4, 1, 2 and 5. Consistent with expression of Pyst1 in the mammary gland, we detected expression of Fgfr1b, Fgf8 and Fgf9 in both surface ectoderm and mammary bud epithelium, and Fgf4 and Fgf17 in mammary bud epithelium. Beads soaked in FGF-8 applied to the flank of mouse embryos, at a stage just prior to mammary bud initiation, induce expression of Pyst1 and Lef1 and maintain Tbx3 expression in flank tissue surrounding the bead. Grafting beads soaked in the FGFR1 inhibitor, SU5402, abolishes Tbx3, Pyst1 and Lef1 expression, supporting the idea that FGFR1 signalling is required for early mammary gland initiation. We also showed that blocking Wnt signalling abolishes Tbx3 expression but not Pyst1 expression. These data, taken together with previous findings, suggest a model in which Tbx3 expression is induced and maintained in early gland initiation by both Wnt and Fgf signalling through FGFR1.

Fig. 1

Expression of Tbx3, Pyst1 and Lef1 from E10.25 to E13.5. (a) Tbx3 expressed in a thin line marking what appears to be the mammary line (between two arrowheads). Tbx3 is also expressed in a graded fashion in a thicker band more dorsally with transcripts being more abundant anteriorly. (b) Tbx3 expression at E10.5 is stronger than E10.25 but the line seen at E10.25 cannot be distinguished. (c) E11.5; three pairs of mammary buds expressing Tbx3 become apparent in a dot-like pattern (Mb1, Mb3 and Mb4). (d) E12.5; five pairs of mammary buds expressing Tbx3. (e) E10.25; Pyst1 expressed as stripes marking the somites, and not yet in mammary rudiments. (f) Pyst1 expression first detected in a mammary rudiment (Mb3); at E11.25 it is also in stripes of somitic expression. (g) Pyst1 expression in Mb3 and Mb4 at E11.5. (h) Pyst1 expressed in full complement of mammary buds at E12.5 (Mb1–Mb5). (i) E11.5; Lef1, known as early marker of mammary placode formation, expressed in Mb3 in a dot-like pattern; in Mb1 and Mb4 in a comet-like pattern with a tail. (j) Lef1 expressed separately in Mb2 and Mb1 at E12. (k–l) E12.5 and E13.5; Lef1 expression detected in five pairs of mammary buds. Note that Mb5 is last to form. Mb1–5, mammary buds. Scale bars, 200 µm.

Fig. 2

Expression of Fgfr1 and FGF ligands specific for FGFR1 in mammary glands at E12. (a) Fgfr1 expression at high levels in ectoderm and mammary epithelium. (b) Fgf4 expression in centre of mammary bud, but not in epidermis. (c) Fgf8 more highly expressed in mammary epithelium than ectoderm (arrowheads). (d) Fgf9 strongly expressed in ectoderm (arrowheads) and in mammary epithelium. (e) Fgf17 expression only detected in mammary epithelium. Scale bars, 100 µm.

Fig. 3

Effects of FGF8 on gene expression in devloping mammary glands. In vitro organotypic cultures 48 h after implanting PBS (a,c,e) or FGF-8 (b,d,f) soaked beads. After whole-mount in situ hybridization for Lef1, Pyst1 and Tbx3 (a,b,c,d,e,f), cultured tissue was sectioned through the bead (a′,b′,c′,d′,e′,f′). Haematoxylin and eosin-stained sections of organ cultures (a″,b″,c″,d″,e″,f″). (a) PBS bead; Tbx3 expression confined to mammary bud. No Tbx3 expression around the bead see also (a′). Ectoderm morphology unaffected by bead (a″). (b) FGF8 bead; Tbx3 expression maintained around FGF-8 bead predominantly dorsally. (b′,b″) Section through the FGF-8 bead reveals Tbx3 in mesenchyme but no change in ectodermal morphology. (c) PBS bead; Pyst1 expression detected in mammary buds but not induced near PBS bead. (c′,c″) No Pyst1 transcripts in either epithelium and mesenchyme near bead and no change in ectoderm. (d) FGF-8 beads; strong Pyst1 expression around the beads (arrows). (d′,d″) Pyst1 expression in mesenchyme and no change in ectodermal morphology. (e) PBS bead; Lef1 expression not induced by PBS bead, but confined to mammary buds. (e′,e″) Section showing no expression of Lef1 in either epithelium and mesenchyme; ectoderm morphology normal. (f) FGF8; Lef1 induced in mesenchyme predominantly dorsal to bead (arrow). (f′,f″) Section through FGF-8 bead; Lef1 expressed in mesenchyme; no change in ectoderm morphology. Mb1–5, mammary buds; arrow, ectopic expression around FGF-8 beads; asterisk, PBS or FGF-8 soaked beads; arrowheads, mammary buds. Scale bars (a–f), 300 µm; (a′–f′, a″–f″), 150 µm.

Fig. 4

Effects of FGFR1 inhibitor (SU4502) on gene expression in mammary buds. Flank cultures 48 h after implanting either DMSO beads (a,c,e) or two SU5402 soaked beads (b,d,f). One SU4502 soaked bead applied (g,h). (a) Tbx3 expression; DMSO beads had no effect and Tbx3 expressed normally in mammary buds. (b) Tbx3 expression completely abolished in flank and mammary buds by SU5402 treatment. (c) Pyst1 expressed in mammary buds, but not around DMSO bead. (d) Expression of Pyst1 lost after SU5402 treatment. (e) Lef1 not expressed around DMSO bead. (f) Complete absence of Lef1 expression after SU5402 treatment. (g) One SU5402 bead applied for 48 h to flank; Lef1 in Mb2 remains as comet shape. (h) 72 h culture treated with one SU5402 bead. Lef1 expression in the Mb2 still remains as comet shape. Mb1–5, mammary buds; black asterisk, DMSO soaked beads; red asterisk, SU5402 soaked beads. Scale bars, 300 µm.

Fig. 5

Effects of Wnt inhibitor (CK1-7) on gene expression in mammary glands. Flank cultures 48 h after implanting either two DMSO beads (a,c,e) or two CK1-7 beads (b,d,f). (a) Lef1 expressed normally in DMSO treated tissue. (b) Lef1 expression reduced by CK1-7. Note for example weaker expression in Mb4 region. (c) Pyst1 expression; no change with PBS beads. (d) Pyst1 is also not affected by CK1-7 beads. (e) Tbx3 expression; DMSO had no effect. (f) Tbx3 expression completely lost in flank and mammary buds. Mb1–5, mammary buds; black asterisk, DMSO soaked beads; red asterisk, CK1-7 soaked beads. Scale bars, 300 µm.

Fig. 6

Genetic pathway integrating FGF, Wnt and Tbx3 in epithelial–mesenchymal interactions regulating mammary gland initiation. Fgf signalling through FgfR1, blocked by SU5402, maintains Tbx3 expression and leads to expression of Pyst1 and Lef1. Fgf10 signalling is also required for mammary gland development as shown in knock-out mice. Wnt signalling, blocked by CK1-7, also maintains Tbx3 expression. Tbx3 null mutants do not form mammary buds and expression of Wnt10b and Lef1 is absent (Davenport et al. 2003), suggesting that Tbx3 controls their expression. Fgf8, Fgf9, Fgf4, Fgf17 are expressed in ectoderm or mammary knob epithelium. Pyst1 expression depends on signalling through FgfR1.