PMC

Schematic showing roles of Ephrin B2 signaling. Ephrin B2 signaling is involved in consolidating feather bud formation, feather follicle invagination and elongation and in barb ridge formation.

A). This schematic diagram shows how cells interact to form barb ridges. At step 3 the growth zone is established. Eph/ephrin signaling seems to act prior to step 4 when the growth zone becomes localized. As a result, barb ridges are of unequal size and irregular in shape. BSA, bovine serum albumen.
B) Feather branching is characterized by the formation of barb ridges within the epithelium. Cells within these structures are highly organized into forming feather barb ridges. This organization is disrupted in ephrin-B1/Fc treated explants. While periodically arranged barb ridges can still differentiate, the perturbed barb ridges lose their consistent organization. Size bar = 100um. In ephrin-B1-Fc low magnification panel, size bar = 200um.

Control (A–D) and ephrin-B1/Fc treated (A’-D’) reconstituted skin explants stained for LCAM. The feathers are growing from the left side of these panels (A, A’). LCAM positive epithelium is shown for bud (Fig. 4B, B’), invagination site (Fig. 4C, C’) and interbud region (Fig. 4D, D’). Control feathers form a more complete feather boundary at 6 days after reconstitution, than the treated samples. Control cells elongate into the base of the feather producing a higher aspect ratio (length/width) than that seen in the cells from the treated specimens (Fig. 4C, C’). E) Chart of epithelial cell aspect ratio shows a dramatic difference between the interbud areas in control vs ephrin-B1/Fc treated skin. The bud region does not show significant changes in cell aspect ratio. Data are presented as the average and standard deviation. Size bar = 10um. L = length, W = width of each cell.

A. H&E staining of sections representing three different stages of the feather (A, E7; B, E8 and C, E10). The mRNA of ephrin-B1 was detected in both the epithelial layer and mesenchymal layer. Ephrin B1 was expressed stronger in the posterior mesenchyme. EphB3 was also observed predominantly in the epithelial layer at all stages.
B. Ephrin-B1 protein is expressed toward the posterior mesenchyme at the short bud stage. The level of protein is much higher in the mesenchymal cells than the epithelial cells when compared to the transcript. At the feather follicle stage it continues to be expressed in the epithelium and mesenchyme. In barb ridge formation, expression is higher in the basal layer in regions that are to become the marginal plate epithelia.
C, C’. Expression patterns of ephrin-B1 (panel C) and EphB3 (panel C’) mRNAs in growth phase adult feather follicles. Ephrin-B1 and EphB3 are expressed in epithelial cells of the follicle. They are present in the barb ridges (red/blue boxes numbered 1–2’) and at the base of the follicle (green box numbered 3, 3’). Their patterns overlap in the rachis (yellow box numbered 4, 4’). Ephrin-B1 expression is higher than EphB3 in the rachis but Ephrin B1 is limited to the pith epithelium while EphB3 is also present in the cortical epithelium. In the barb ridges (purple box numbered 5, 5’), ephrin B1 is present in the basal epithelium and the barb plate. EphB3 is absent from these two regions.

A. The expression patterns of ephrin B1 and EphB3 were examined during early feather development. Ephrin B1 and EphB3 were expressed diffusely at E6 but were elevated in regions between feather tracts, suggesting they may be involved in tract formation. Later, at E7.5 they become expressed toward the center of the feather bud gradually diminishing toward the periphery (boundary) of the buds in the dorsal tract. This may be due to the prominent expression of ephrin B1 in mesenchymal cells at the center of the feathers as confirmed by section in situ hybridization. At E8 their expression increases and Ephrin-B1 is seen predominantly at the posterior region of the feather bud while EphB3, one of the ephrin-B receptors, was restricted to the posterior (P) part of the feather. Size bar = 750um.
B. Three stages of skin development from E9 embryos and 2 stages from E10 embryos are shown. Progressive stages of skin development are shown from the top to the bottom of the figure. The sites where the feather buds are located are shown schematically. Note, these regions are caudal to those shown in Fig. 1A. The top figure represents a region with less mature feather buds (short bud stage). Here Ephrin-B1 is in the center of younger feather buds and spreads throughout the bud at later stages. As the bud grows, Ephrin B1 expression moves to the periphery at the long bud stage. At the base of the feather where the invagination occurs the expression was in the pattern of a ring surrounding the feather base (far right of E10). Meanwhile, the expression of EphB3 at E9 was in the center of the young feather buds overlapping the expression of ephrin B1. They then were expressed throughout the bud. Expression then moved to the distal, posterior region of the buds. At E10 a ring of expression near the feather base also appeared. Size bar = 500um.

A, A’. Whole-mount view of reconstituted feather growth after 96 hours in the presence (A’) or absence (A) of ephrin-B1/Fc. Control feathers elongate with a uniform size and orientation while treated specimens are wider and are not consistently oriented.
B, B’. The ventral view of LCAM (E-cadherin) staining shows a circular staining at the periphery of the base of the feather bud shape. The control circles are more uniform and smaller than the treated skin. The staining is more diffuse in the ephrin-B1/Fc treated samples.
C, C’. Feather bud boundaries observed after 48 hours. Control feather buds have nice definitive boundaries (arrow). However, feather buds in ephrin-B1-Fc treated skin form more slowly and the boundary has not yet formed.
D, D’. Enlargements of the areas indicated in the white boxes in B, B’, respectively. 96 hrs of incubation.
E, E’. NCAM staining of dermal condensations. Control feather buds show well defined dermal condensations (E). Treated cultures show more diffuse dermal condensations with the zone of NCAM staining extending beyond the bud boundary into the adjacent interbud region (F). 96 hrs of incubation.
F, F’. Enlargements of condensations shown in E, E’.
G. Sections of feather buds 9 days after reconstitution stained for LCAM for control and ephrin-B1-Fc treated samples. Treated samples are much broader than controls. Arrows indicate the size of the feather base. LCAM staining is excluded from the basal epithelium of controls but is present in the more differentiated suprabasal layers. In contrast, LCAM staining is excluded from basal as well as suprabasal layers in the treated samples. Size bar = 100um.
H. H&E staining of feather buds 9 days after reconstitution. The invagination process was inhibited in ephrin-B1/Fc treated skin. Proliferation was increased in the epithelium of feathers treated with ephrin-B1/Fc as determined by PCNA staining. Tenascin C (TN-C) was present in the epithelium at sites of invagination in both control and treated skin (arrows); however, the control skin showed a larger region of TN-C expression and deeper invagination. Neural cell adhesion molecules (NCAM) were present at the base of the feather follicle, but the segregation between bud and interbud is not clear in the ephrin-B1/fc treated skin. Feather keratin (F-Keratin) was expressed similarly in control and ephrin-B1/fc treated feathers. Size bar = 100um.