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First published online 16 June 2004
doi: 10.1242/dev.01203

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FGF signaling is required for initiation of feather placode development

Markus Mandler and Annette Neubüser^*,

Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria

View larger version (97K): [in a new window]   Fig. 3. Forced expression of FGFR1-Fc and FGFR2-Fc prevents feather placode development. (A-H) Embryos were injected with the virus into the subectodermal mesenchyme of the back at HH18 and analyzed for expression of Wnt6, Shh and Bmp2 at HH29-30 by whole-mount RNA in situ hybridization. All embryos infected with RCAS-FGFR1-Fc or RCAS-FGFR2-Fc show a region devoid of any punctate expression (brackets). (I,J) Sections through the embryo shown in G stained with an antibody against the viral gag protein demonstrates viral infection in both the nude patch and adjacent bud containing regions. The asterisks in J indicate infected cells in the ectoderm. (M,M') HE stained sections through a control (M) and nude region of an RCAS-FGFR1-Fc infected embryos (M') showing failure to form ectodermal placodes (ep) and dermal condensates (dc) in infected tissue. ec, ectoderm that has not formed a placode; dd, thickened dermis. (K,L) Embryos were injected subectodermally at HH23-24 and analyzed for Bmp2 (K) and subsequently Fc expression (L, both in blue) by double whole-mount RNA in situ hybridization. Embryos showed loss of Bmp2 expression and a failure to form buds in the infected area (indicated by white dotted line). (N,O) Embryos injected at HH26 show normal bud development and Bmp2 expression in infected areas. Infection is monitored by subsequent Fc detection in Bmp2/Fc double stained embryos (indicated by white dotted circles in N and O).

View larger version (64K): [in a new window]   Fig. 4. Inhibition of FGF signaling in vitro prevents feather placode induction but not the maintenance of established feather buds. Skin explants isolated at HH28, prior to placode formation (ppf; A-H), were cultured in the presence of DMSO or Su5402 for 16 hours in vitro and analyzed by whole-mount in situ hybridization for Shh, Bmp2, ß-catenin or Fgfr1 expression. Explants isolated at HH29, when the first buds have been established (eb; I-N), were separated along the midline as indicated by the doted line in A, and halves were either fixed directly after isolation (I,K,M) or cultured for 16 hours in the presence of Su5402 (J,N) or DMSO (L), and expression of Shh (I-L) or Bmp2 (M,N) was compared in the two halves.

View larger version (50K): [in a new window]   Fig. 1. Forced expression of secreted versions of FGFR1 and 2 blocks feather development. (A) The ligand binding regions of FGFR1-IIIc and FGFR2-IIIb (red) were fused to the Fc fragment of a mouse immunoglobulin (blue) to generated secreted receptor versions. Tm, transmembrane domain; TK, tyrosine kinase domains. (B-D) Feather phenotypes in embryos injected with RCAS-AP (B), RCAS-FGFR1-Fc (C,D,F), or RCAS-FGFR2-Fc (E). Embryos in B, C and F were injected with the retrovirus at HH9. The embryos in D and E were in injected into subectodermal mesenchyme of the back at HH18. All embryos were allowed to develop until HH38. The arrowhead in F indicates fusions between adjacent feather primordia.

View larger version (46K): [in a new window]   Fig. 2. Unchanged expression of ß-catenin and Fgfr1 in RCAS-FGFR1-Fc- and RCAS-FGFR2-Fc-infected embryos at HH27-28. (A-F) Embryos were virus-injected into the subectodermal mesenchyme of the back at HH18 and analyzed for Fgfr1 and ß-catenin expression by whole-mount RNA in situ hybridization at HH27-28. (G-J) Sections through the embryos shown in B, C, E and F stained with an antibody against the viral gag protein demonstrating widespread infection of the mesenchyme. Dashed lines indicate the position of the midline of the embryos, asterisks indicate regions with infected cells in the ectoderm.

View larger version (79K): [in a new window]   Fig. 5. Expression of Fgf3 and Fgf10 in early feather development. Detection of Fgf3 (A-C) or Fgf10 (D-F,J-L) expression by whole-mount RNA in situ hybridization of embryos at HH28, before the onset of feather placode development, and at HH30. (C,F) Expression of Fgf3 and Fgf10 in embryos infected with RCAS-FGFR1-Fc and RCAS-FGFR2-Fc at HH18. The nude region devoid of Fgf3 and Fgf10 expression is included with a bracket. (G-I) Vibratome sections, at the indicated positions, through the embryos shown in B, D and E, respectively. Arrowheads in D indicate the stripes of Fgf10 expression in the region where bud development will be initiated. (J-L) Expression of Fgf10 in the alar (J), pectoral (K) and tail tract (L). Arrows indicate the feather tract, delineated by Fgf10 expression. nt, neural tube.

View larger version (65K): [in a new window]   Fig. 6. FGF10 is required for feather development. (A-F) Skin explants (ppf: A-E; eb: F) were cultured for 16 hours in the presence control IgG or 5 µg/ml anti-FGF10 blocking antibodies, or in medium containing the same amount of anti-FGF10 antibody and recombinant FGF10 protein (C) and analyzed for expression of Shh or Bmp2.

View larger version (62K): [in a new window]   Fig. 7. FGF10 is sufficient for the induction of genes essential for feather bud development. (A-G) ppf-skin explants were cultured for 16 hours in the presence of beads soaked in recombinant FGF10, recombinant FGF10 plus Noggin-Fc, or PBS, and analyzed for the expression of Bmp2, Wnt6, follistatin or Fgf10. Asterisks indicate implanted beads.