7.1 Introduction
I examined the expression of molecules, Sonic hedgehog (Shh), Fibroblast growth factor-4 (Fgf-4), Bone morphogenetic protein-2 (Bmp-2), Bone
morphogenetic protein-4 (Bmp-4), Patched (Ptc), Wnt-7a, Cek-8 and expression of Tenascin during initiation of feather bud formation. All of these genes were
introduced in chapter 1. The earliest expression of feather patterning of back skin is expression of a epidermal stripe of Shh, Fgf-4 and Ptc in the dorsal midline. Feathers in lateral rows are initiated by discrete patches of Shh expression in the epidermis and Ptc expression which is followed closely by expression o f Bmp-2 and Bmp-4 in underlying dermal condensations.
To determine if Shh, FGFs, BMPs and Retinoic acid could themselves induce the formation of feathers, they were ectopically applied to the feather forming skin. I found that SHH and FGF-2 can induce the formation of extra buds and BMP-2 and Retinoic acid inhibit development of feathers. These data suggest that SHH and FGFs could determine the position of feather buds and BMPs could be an inhibitory signal.
7.2 Materials and methods
Beads (Shh, Retinoic Acid, Bmp-2 and Fgf-2) treatment on cultured skin
Heparin acrylic beads (H5263, Sigma), 150-200 pm in diameter, were soaked in either a 2 pi drop of 1 mg/ml FGF-2 (133-FB-025, R&D Systems) or in a 2 pi drop of 0.7 pg/pl BMP-2. Affi-gel blue CM beads, 150 pm in diameter, were soaked in a 1 pi drop of 1 mg/ml Sonic hedgehog solution, for at least 1 hr at room
temperature prior to transferring onto the cultured skin. AG1-X2 beads soaked in a solution of all-trans retinoic acid (0.7 mg/ml) in dimethyl sulphoxide were used as previously described (Tickle et al., 1985).
Antibodies
The production and characterisation of the rabbit anti-tenascin serum used in this study has been described in detail previously (Chiquet-Ehrismann et al., 1986). The antiserum was a generous gift from Dr. E. J. Mackie (Royal Veterinary College, London). The chick tenascin cDNA subclone (Tn 1-749) was a generous gift from Dr. M. Pacifici (University of Pennsylvania).
Immunocytochemistry
Chick embryos were fixed at appropriate stages in 4% paraformaldehyde in PBS, rinsed, and cryoprotected in 30% sucrose in PBS overnight. Samples were then embedded in Tissue Tek, and sections were cut at 7 to 10 pm in a cryostat. Sections were collected on pre-coated TESPA slides, allowed to air dry for 10
minutes, then rehydrated in PBS. Immunocytochemistry was conducted as described in Tucker and Matus (1988).
In-situ hybridisation
7 .3 R e s u lts
7.3.1 Gene expression
7.3.1.1 Sonic hedgehog (Shh)
Just before feather bud formation, at stage 28, a stripe of Shh transcripts is present along the midline of the back (Figure 7. 1 A). Sections show that expression of Shh is confined to the midline epidermis (data not shown). At stage 29, the midline expression breaks up into patches which form the primary row o f feather buds. In-situ hybridisation o f sections shows that Shh expression is localised to the epidermal placodes at stage 30 (Figure 7.1. B). With development of lateral rows of buds, Shh is always expressed at the time that the epidermal placodes form. At stage 31 -35, Shh expression is confined to the distal tip of the epidermis in the
developing feather buds both in the dorsal pteryla and in tail region (Figure 7.1. C).
7.3.1.2 Cek-8
At stage 28, Cek-8 transcripts could not be detected. At stage 29, Cek-8 transcripts are present as a faint thick band along the midline with greater intensity where epidermal placodes are forming (Figure 7.1. D). Sections show that expression of Cek-8 is confined to the ectoderm of the feather placodes. Cek-8 expression then spreads laterally and uniformly over the epidermis and does not show differential expression in new rows of buds (Figure 7.1. E). However, by stage 35, Cek-8 expression has become localised to the posterior region of the epidermis o f feather placodes and disappears from all other regions (Figure 7.1. F).
7.3.1.3 Fibroblast Growth Factor-4 (Fgf-4)
Fgf-4 transcripts are present in the epidermis of the feather forming region as a single stripe in the midline of the back of the embryo at stage 28 (Figure 7.1. G). Frozen sections of wholemounts show that Fgf-4 expression is localised to epidermal placodes at stage 29 (Figure 7.1. H). Between stages 30-33, Fgf-4 expression is turned off in the midline and Fgf-4 transcripts are not observed in lateral feather buds or between them. At stage 34, Fgf-4 expression returns, and now, transcripts are found in the dermis and localised to the distal epidermis of each feather bud (Figure 7 .1 .1; and sections not shown).
7.3.1.4 Bone morphogenetic proteins (BMP-2, BMP-4)
Bmp-2 is expressed at stage 29 in the dermal condensations of feather buds below the epidermal placodes (Figure 7.1. J). Bmp-2 is expressed in the dermal condensations of all feather buds in the dorsal pteryla and in wing feather buds at stage 30 (Figure 7.1. K). At this stage, faint expression can be also seen linking dermal condensations in the same row, both in back and wing skin. At stage 33, Bmp-2 transcripts become confined to the anterior region of the dermis o f midline feather buds (Figure 7.1. L) and, later, a similar localisation is progressively seen in more lateral buds in the back skin. Bmp-4 is expressed in dermal condensations as Bmp-2, but starting at a slightly later stage. However in contrast to Bmp-2, Bmp-4 is expressed throughout the epidermis of back skin at stage 30. (Figure 7.1. M). At stage 34, when the feather bud has formed, Bmp-4 transcripts are found mainly in the distal tip of the feather bud (Figure 7.1. N).
Figure 7.1 Gene expression during early development of feather buds
A) Shh transcripts are present as a line in the epidermis (arrow) along the midline at stage 28. B) In-situ hybridisation of section shows that Shh expression is localised to epidermal placodes both in midline and in a lateral row (arrow) st stage 30. C) At stage 33, Shh expression confined to distal ectodermal tip of the feather buds in the tail (arrow). D) Dorsal view of Cek-8 expression. Note Cek-8
transcripts present as a faint thick band along the midline (arrow) with greater intensity where epidermal placodes are forming. E) Transverse frozen section of back skin shows that Cek-8 expression spreads laterally and uniformly over epidermis. F) At stage 35, Cek-8 expression localised to posterior epidermis of feather placodes; shown here in tail (arrow). G) Dorsal view of Fgf-4 expression in the back of a stage 28 embryo. Note that Fgf-4 transcripts are present in epidermis of primary feather row as single stripe (arrow) in back midline. H) Fgf-4 expression is localised to epidermal placodes in back midline (arrow head) at stage 29.1) At stage 34, Fgf-4 expression is localised to dermis and distal epidermis. J) Bmp-2 gene expression at stage 29 in dermal condensation below epidermal placode (arrow head) in the midline. K) Bmp-2 in feather buds of stage 30 wing showing faint line of expression between condensations (arrow). L) Bmp-2 transcripts confined to anterior dermis of feather buds in midline at stage 33. M) Bmp-4 in dermal condensation and epidermis at stage 30. N) Stage 34, Bmp-4 transcripts throughout dermal condensations and epidermis of tail feather buds. O) Stage 29, transverse section labelled with anti-tenascin antibody showing tenascin in thin layer of dermis immediately underlying epidermis (arrow) in back midline. P) At stage 31, longitudinal section shows tenascin staining (red/brown) is intense anteriorly (arrow) in dermis at base of feather bud. Q) Tenasçîn transcripts in anterior dermis of feather buds at stage 33, midline buds arrowed.
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