Histological structure of feather follicles from microdissection to day 13 of cultivation <i>in vitro</i>.

Formalin-fixed FF embedded in paraffin were sectioned and stained with HES. Structure after microdissection (D0), day 3 (D3), day 5 (D5), day 7 (D7), day 11 (D11) and day 13 (D13) of culture in vitro. From day 3 of cultivation, we observed necrosis of the feather pulp (*) and of the inner sheath, with pyknotic nuclei from necrotic cells scattered mainly in the dermis (black arrow). Number of necrotic cells progressively increased with time of cultivation and at day 11, most follicular cells appeared necrotic. Note the preservation of the dermal papillae (dp). At day 13, some mineral cristae were visible (white arrowhead). (TIF)</p

Minimal data set of quantitative data from Figs 6 and 8.

(PDF)</p

Expression and localization of avian markers by immunochemistry in FFs associated with growing feathers during cultivation <i>in vitro</i>.

Formalin-fixed FFs embedded in paraffin were sectioned and stained with trichrome Masson (A), or labelled with the following antibodies: anti-fibronectin (B), anti-pan-cytokeratin (C), and anti-scaffoldin (D). Staining was performed after microdissection and at 3, 5 and 7 days of cultivation ex vivo. Bar represents 500 μm.</p

Primers used for qPCR in this study.

Primers used for qPCR in this study.</p

Expression of avian cellular transcripts in FFs associated to their growing feathers at microdissection and after <i>in vitro</i> cultivation.

RNAs were extracted from FFs with a shortened feather after microdissection (D0), and after 3, 5, and 7 days of culture. Following cDNA synthesis, real-time qPCR was performed. At each time point, data from three samples (of 2 FF each), from 2 independent experiments, with qPCR technical repeats are shown. A. RNA levels of each gene at Day 0 were calculated relatively to the housekeeping gene RPS17 from freshly microdissected FF expressed in arbitrary units (A.U) and presented in the bar graph with median and interquartile range. B. RNA expression of the various markers was normalized to the housekeeping gene RPS17, and the fold-changes at Day 3, 5, and 7 relative to Day 0 are shown in a dot plot with median and interquartile range. No significant difference in the expression of NCAM, LCAM, Wnt6 and BMP4 was observed at any time point. In contrast, the expression of KRT14, HBS-1, Notch1, DKK3 and Shh changed significantly at several time points during the culture. Significance of differences was performed relatively to Day 0 by using the Kruskal-Wallis test with a Dunn correction for multiple comparison (adjusted p-value >0.5, ns; p-value <0.5, *; p-value<0.01, **; p-value <0.0001, ****).</p

Growth of feathers cultivated <i>in vitro</i> for 11 days.

The size (in mm) of 8 truncated feathers (4 from the neck and 4 from the thigh) inserted in their FF was measured after 3, 5, 7, and 11 days in culture. Note that each feather was shortened above the skin level before the dissection process. (A) Growth kinetic of individual feathers. Only the gain of growth over time is shown. Except for two feathers from the neck, all feathers weakly grew in length. (B). Total growth by origin (neck or thigh) over 11 days of cultivation.</p

Schematic representation of a feather follicle with a growing feather.

Adapted from [4]. Legends for the mesenchymal compartment appear in pink. It includes the dermis, the dermal papilla, and the feather pulp. Legends for the epidermal compartment appear in black. The epithelia (follicular wall and feather epithelium) are made of three layers: the basal layer (in blue), the intermediate layer (in orange), and the upper layer (in brown). The feather outer sheath surrounds and protects the feather germ. The feather germ epithelium is composed of four structures from the bottom to the top: i) the epidermal collar, ii) the collar bulge, iii) the epithelial ramogenic zone (ERZ), and iv) the barb ridges. Transiently amplifying cells (hatched area) are localized in the proliferative zone. The basal layer involuted into barb ridges is called the marginal plate. As shown in the enlargement (black frame) of one barb ridge, the basal layer/marginal plate proliferates at barb ridges extremity, thus providing numerous barbs and/or barbules cells. This allows the formation of the axial and barbule plates which give rise to barbs and barbules, respectively. The space between the follicular wall and the feather outer sheath is the follicular cavity. The feather follicle is irrigated by a prominent axial artery. Gene markers expressed in various zones of the FF and used in this study are indicated below each corresponding structure.</p

Steps to obtain individual feather follicles for <i>in vitro</i> culture.

In 3-week old White Leghorn chick (A), the contour feathers from two regions (blue arrowheads) (the base of the neck or the thigh) have reached a developmental stage (B, C) before becoming adult contour feathers (D). Note that feathers are heterogenous in their development (B), with feathers just popping-out the outer sheath (red arrowhead) or feathers close to their terminal size. The white zone (white arrowheads) corresponds to cornified rachis containing barbs and barbules (devoid of pulp), which are limited by the outer sheath (B, C); in contrast the pink zone (red arrow) corresponds to live tissue (B). Skin pieces (E) after cutting at the growing feathers (as shown in C, with blue dotted arrow) were soaked into supplemented William’s E medium (see text) (F), and then progressively dissected into individual growing follicles (G, H).</p

Morphology of the FF by TEM.

Images of semi-thin section of a FF after microdissection (Day 0) (A) and at Day 7 (I), stained with toluidine blue observed by optical microscopy are shown and correspond to the level of the ultra-thin sections observed at these time points. Images of FF observed by TEM at different magnifications at Day 0 (B-D), Day 5 (E-H), Day 7 (J-L). Note that J was generated by merging two pictures. Regions enlarged in another picture are indicated with black frames. On panel (B), the dermis (Ds) and the follicular wall (foll wall) are indicated as well as part of the feather. A few structures are shown on the images: feather sheath (fs; empty white triangle), basal membrane (white short arrow), mitochondria (white asterisk symbol), nucleolus (n), keratin bundles (white plain arrowhead), desmosome (black plain triangle), lipid droplet (yellow plain triangle).</p

Histological structure of feather follicles from microdissection to Day 7 of <i>in vitro</i> cultivation.

Formalin-fixed FFs embedded in paraffin were sectioned and stained with HES. (A) Overview of a typical FF with a growing feather after microdissection. bbr, barb ridges; cb, collar bulge; dermis; fs, feather sheath; follicular wall; fp, feather pulp. An enlargement of the barb ridges is shown. (B) Structure of FF after 1 (D1), 3 (D3), 6 (D6), and 7 (D7) days of cultivation in vitro. A low magnification showing the whole FF with its feather (left panels). An enlargement of the barb ridges (right panels).</p