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First published online 21 July 2004
doi: 10.1242/dev.01263

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Signaling dynamics of feather tract formation from the chick somatopleure

Ingrid Fliniaux, Jean P. Viallet^* and Danielle Dhouailly^*,

Equipe Biologie de la Différenciation Epithéliale, UMR CNRS 5538, LEDAC, Institut Albert Bonniot, Université Joseph Fourier, BP 53-38041 Grenoble Cedex 9, France

View larger version (65K): [in a new window]   Fig. 1. Behavior of chick proximal somatopleural cells after in ovo electroporation of a GFP plasmid at E2 (A-G) and E3 (H-N). (A) Experimental procedure at E2. (B-D) Six hours after electroporation, GFP-expressing cells are located in the proximal somatopleural mesoderm (psm). (E-G) Forty-eight hours after electroporation at E2, GFP-labeled cells are located in the dorsal part of the limbs (lb) and/or in the lateral part of the flank. (H) Experimental procedure at E3. (I-K) Twenty-four hours after electroporation at E3, GFP-expressing cells are located in a lateral compartment of the flank. (L-N) Forty-eight hours after electroporation at E3, GFP-labeled compartment has expanded slightly towards ventral direction. n, the number of embryos analyzed. (B,E,I,L) Light microscopy; (C,F,J,M) fluorescence microscopy; (D,G,K,N) schematic localization of fluorescent cells. nt, neural tube; s, somite. Scale bars: 60 µm in B,C; 250 µm in E,F,I,J; 280 µm in L,M.

View larger version (95K): [in a new window]   Fig. 2. The proximal somatopleural mesoderm is determined to form the ventral pteryla at stage HH13. (A) Diagram of the microsurgical procedure in chick embryo. The proximal somatopleural mesoderm (after peeling off the ectoderm) was grafted with a 90° rotation under the ectoderm of a host at the same stage. (B) At day 11: a supernumerary pteryla (sp) across the midventral apterium, which is rotated almost 90° compared with the endogenous ventral pteryla (vp), is formed. (C) Ventral feather macropattern of a E10 chick embryo: the pectoral (pp) and ventral pterylae (vp) are separated by a semi-apterium (sa). The midventral apterium (mva) is delineated on each side of the midventral closure (vc). (D) Ventral feather macropattern of a E10 quail embryo: there is no semi-apterium between the pectoral and ventral pterylae, and the midventral apterium is almost imperceptible. (E) The microsurgical procedure: a fragment of the right proximal somatopleure is removed from a HH13 quail embryo, the ectoderm is peeled off and the mesoderm is grafted orthotopically under the ectoderm of a HH13 chick embryo. (F) Ventral view at E10: the operated side presents a quail type feather macropattern, characterized by no distinguishable semi-apterium and a very narrow midventral apterium. ant, anterior; post, posterior; u, umbilical cord. Scale bar: 1.3 mm in B,C,D,F.

View larger version (77K): [in a new window]   Fig. 3. Induction and analysis of supplementary pterylae (sp) in the midventral apterium (mva). (A) Microsurgical procedure: fragments of 2-day-old ventral neural tube plus chord, or aggregates of cells expressing Shh or Noggin were grafted under the ectoderm of HH13 embryo in the presumptive territory of the midventral apterium, posterior to the level of the 20th somite. (B-D) Supplementary pteryla obtained 8 days after the graft of (B) ventral neural tube (VNT) plus chord (NC) fragment, (C) aggregate of Shh cells and (D) aggregate of Noggin cells. Compare with the midventral apterium of an unoperated embryo in Fig. 2C. (E) Bmp2 expression 6 days after the graft of Shh cells. A midventral stripe forms a fork around the supplementary pteryla. (F) follistatin expression 6 days after the graft of Noggin cells. A supplementary fused pteryla (sfp) appears distinct from the pectoral pteryla (pp). (G) Bmp2 expression at E8 of an embryo grafted with Noggin cells and presenting a supplementary pteryla fused to the pectoral pteryla. Bmp2 transcripts are localized in the rostral part of the placodes, which allows us to distinguish the different orientations (arrows) of the ventral (vp) and supplementary fused pteryla. (H) Section at the level of a supplementary pteryla obtained with Shh cells, labeled with QCPN antibody and revealed with the peroxidase reaction (brown). The Shh-implanted cells are found (asterisk and enlargement) far from the feather buds of the supplementary pteryla. u, umbilical cord; vc, ventral closure. (E-G) In situ hybridization. Scale bars: 1.3 mm in B,D,F; 1.2 mm in C; 2 mm in E; 1.5 mm in G; 400 µm in H.

View larger version (29K): [in a new window]   Fig. 4. Expression patterns of Noggin (A), Bmp4 (B) and Shh (C) at late stage HH13 in chick embryo. (A) Noggin expression, dorsal view. Posteriorly to the 20th somite, the transcripts are localized in the dorsal neural tube and in the intermediate mesoderm (im). (B) Bmp4 expression at the same level in transverse section. The transcripts are localized in the dorsal neural tube (nt), the entire somatopleural mesoderm (sm) and in proximal splanchnopleural mesoderm (sp). (C) Shh expression at the same level in transverse section. The transcripts are localized in the chord (nc) and in the proximal endoderm (en). ec, ectoderm. Scale bars: 200 µm in A; 60 µm. in B,C

View larger version (61K): [in a new window]   Fig. 5. Expression patterns of noggin and Bmp4 in E4.5 and E6.5 chick embryo. (A-D) E4.5; lateral view (A,B) and transversal sections (C,D) showing the expression of Noggin (A,B) and Bmp4 (C,D). The diamond-bounded line delimits a similar area in A-D. (E-H) E6.5; ventral view (E,G) and transversal section (F,H) showing the expression of noggin (E,F) and Bmp4 (G,H). Arrows delimit a similar area in F and H. Broken lines in A,C and E,G show the level of sections in B,D and F,H, respectively. (I,J) Diagram of transversal sections on one side of the ventral closure (vc) showing the exclusive expression domains of noggin and Bmp4 at E4.5 (I) and E6.5 (J). ant, anterior; post, posterior. Scale bars: 500 µm in A,C; 80 µm in B,D; 700 µm in E,G; 120 µm in F,H.

View larger version (85K): [in a new window]   Fig. 6. Effect of grafting Shh and Noggin expressing cells on Bmp4 expression in the distal somatopleure (dsm). (A) At E3, 24 hours after the graft of QT6 control cells (asterisk), the embryo shows a normal extent of Bmp4 expression. (B) At E3, 24 hours after the graft of Shh cells at the embryonic boundary, Bmp4 expression is disrupted around the graft. This was also observed (C) 48 hours after the graft. On this transversal section, there is Bmp4 expression in the dorsal (dors) mesenchyme (short arrow). (D) By contrast, no change in Bmp4 expression was seen around the two adjacent aggregates at E3, 24 hours after the graft of Noggin cells at the extra-embryonic limit. ant, anterior; ao, aorte; nc, chord; dsm, distal somatopleure; nt, neural tube; post, posterior; psm, proximal somatopleure. Scale bars:150 µm in A,B,D; 80 µm in C.

View larger version (68K): [in a new window]   Fig. 7. Msx1 expression at different stages of chick development from E2 to E8.5 (A-F), and its regulation after the graft of Noggin (G,H) or Shh (I) cell aggregates, and after treatment with cyclopamine (J-N). (A,B) Dorsal view (A) and transversal section (B) at late stage HH13 posterior to the level of the last somite formed (double arrow in A). Msx1 is expressed in the dorsal neural tube (nt), in the distal somatopleural mesoderm (dsm) and weakly in the extra-embryonic area (ee). (C-F) Msx1 expression on the lateral side at E3 (C) and E4.5 (D), and on the ventral side at E6.5 (E) and E8.5 (F). There is progressive restriction of the domain of Msx1 expression from E4.5 to E8.5 to the midventral apterium (mva), as well as expression in the amnion forming the umbilical cord (fu) at early stages, or later, the umbilical cord wall (u). (G,H) Msx1 expression is downregulated 24 hours (G) and 72 hours (H) after grafting of Noggin cells (asterisk). (I) Transverse section at E3 of an embryo showing that Msx1 expression is downregulated over the Shh cell aggregate (asterisk). (J-N) The proximal limit of the Msx1 expression domain (arrowhead, dotted line) is extended in the proximal somatopleure 72 hours (J-L) after the inhibition of Shh signaling mediated by a treatment with cyclopamine, in contrast to a control (M,N) embryo. (K) Close-up of the embryo shown in J. The domain of expression is larger in the treated embryo (K,L) than in the control (M,N). The extent of Msx1 expression can be determined by reference to the kidney (k) and to the extremity of the coelom (*). ant, anterior; lb, limb bud; post, posterior; ps, presomitic mesoderm; psm, proximal somatopleural mesoderm; so (white dotted line), somite; sp, splanchnopleure. Scale bars: 100 µm in A,I; 60 µm in B; 75 µm in C,G; 500 µm in D,H,J; 700 µm in E; 750 µm in F; 110 µm in K,L; 100 µm in M,N.

View larger version (115K): [in a new window]   Fig. 8. Production of an ectopic feathered skin in the chick amnion. (A) Microsurgical procedure: aggregates of Shh cells together with Noggin cells were grafted under the ectoderm of a HH13 embryo at the exterior of the embryonic boundary, under the level of the 20th somite. (B) At E14, the embryo shows a supplementary pteryla (arrow) in the amnion (am). (C-F) Histological sections at E11 of a supplementary pteryla and surrounding amnion. (C,D) Ectopic pteryla showing dense dermis (dd) and feather primordia (fp and arrowheads) but no Shh-producing cells. (E,F) Shh quail cells (asterisks) are found on a section located 750 µm away from the ectopic pteryla as labeled with QCPN antibody and revealed with the peroxidase reaction (brown). lf, left foot; u, umbilical cord; rf, right foot. Scale bars: 1.3 mm in B; 60 µm in C,E; 220 µm in D; 150 µm in F.

View larger version (21K): [in a new window]   Fig. 9. Proposed model for the formation of the ventral pteryla versus the mid-ventral apterium in chick embryo showing noggin, Shh, Bmp4 and Msx1 expression from E2 to E8.5 in the body wall region.