Feather regeneration as a model for organogenesis

Abstract

In the process of organogenesis, different cell types form organized tissues and tissues are integrated into an organ. Most organs form in the developmental stage, but new organs can also form in physiological states or following injuries during adulthood. Feathers are a good model to study post-natal organogenesis because they regenerate episodically under physiological conditions and in response to injuries such as plucking. Epidermal stem cells in the collar can respond to activation signals. Dermal papilla located at the follicle base controls the regenerative process. Adhesion molecules (e.g., neural cell adhesion molecule (NCAM), tenascin), morphogens (e.g., Wnt3a, sprouty, fibroblast growth factor [FGF]10), and differentiation markers (e.g., keratins) are expressed dynamically in initiation, growth and resting phases of the feather cycle. Epidermal cells are shaped into different feather morphologies based on the molecular micro-environment at the moment of morphogenesis. Chicken feather variants provide a rich resource for us to identify genetic determinants involved in feather regeneration and morphogenesis. An example of using genome-wide single nucleotide polymorphism (SNP) analysis to identify alpha keratin 75 as the mutation in frizzled chickens is demonstrated. Due to its accessibility to experimental manipulation and observation, results of regeneration can be analyzed in a comprehensive way. The layout of time dimension along the distal (formed earlier) to proximal (formed later) feather axis makes the morphological analyses easier. Therefore feather regeneration can be a unique model for understanding organogenesis: from activation of stem cells under various physiological conditions to serving as the Rosetta stone for deciphering the language of morphogenesis.

Fig 1. Structures of feather follicle

(A) Three dimensional structure of a growing feather (Adopted from Lucas 1973). (B) Schematic drawing of the structure of a growing feather follicle. (C) Label-retaining cells (red) in downy (left) and flight feathers (right) are arranged as a horizontal ring in downy feathers, whereas they are configured as a tilted ring in flight feathers. (Adopted from Yue et al. 2005) (D) Model for the stem cell niche topology in radially symmetric feathers (left) and bilaterally symmetric feathers (right). A ring of stem cells lies parallel to the surface of the skin in radially symmetric feathers but is canted at an angle in bilaterally symmetric feathers. LRC: label-retaining cell; TA: transit amplifying. (Adopted from Yue et al. 2005).

Figure 2. Molecular expression in regenerating feather follicles

(A) Schematic diagram of the feather cycle consisting of Initiation, Growth and Resting phases (top panel). The lower panels show longitudinal sections of feathers at each stage stained for H&E, NCAM, Tenascin, FGF10, Spry4 and Ker A. Positive staining is red for immunostaining (NCAM, Tenascin) and blue for in situ hybridization (FGF10, Spry4 and Ker A). br, barb ridge; cl, collar; cm, collar mesenchyme; dPulp, degenerating pulp; dp, dermal papilla; nPulp, newly formed pulp. Bar= 1000 μm. (B) Dynamics of molecular expression patterns in semiplume feathers in cross sections. In situ hybridization shows RNA expression pattern of Wnt3a, Shh and β-keratin in the cross sections of a semiplume feather. Wnt3a is expressed in the rachises of the feather and the after-feather (arrowheads). Shh is present in the marginal plate of barbs (arrowhead). β-keratin shows up in the differentiated region of the barbs and rachises. The 2nd and third rows are magnified graphs from the boxed regions in the top left panel to highlight rachis and barb regions. Bar= 500 μm.

Figure 3. FGF/Sprouty determines the proximal-distal feather morphology and the size of the dermal papilla

(A, B) Schematic depiction of methods used for studying feather regeneration and morphogenesis. (A). Feathers are plucked to induce the initiation of a new feather. Virus carrying exogenous genes such as β–galactosidase is employed to transduce cells of the newly formed feather. Red color indicates β–galactosidase staining. (B) Classical tissue recombination studies involving microdissection and transplantation of specific components of the follicle. The dermal papilla can be microdissected from the donor follicle and transplanted to the recipient follicle where the dermal papilla has been removed. (C–E) Examples using RCAS sprouty to study the roles of signaling genes on feather morphogenesis. (C) Control feather transduced with RCAS Lac Z. (D) Regenerating feathers transduced with RCAS Spry 4 show miniaturization of the DP, which also becomes Tenascin C negative (compared with Fig. 2A). The perturbed follicle also shows an expanded pulp which is represented by empty space in the section. The follicles also show numerous ectopic branches forming within the follicle and also on the follicle sheath outside of the follicle. (E) Transduction with RCAS FGF10 induces proximal feather structures with a thickened keratinocyte collar and a diffuse dermal papilla which is positive for NCAM and laminin. aCl, abnormal collar; aBr, abnormal barb ridge; aDp, abnormal dermal papilla; aPulp, abnormal pulp.

Figure 4. Frizzle feather phenotype was caused by KRT75 mutation

(A) Adult and 1-month-old frizzle chickens. Adult frizzle chicken feathers curve away from the body. The second-generation feathers in a one-month old chick start to show a clear frizzle phenotype. (B) Comparison of body feathers from normal white leghorn and frizzle chickens in dorsal view, ventral view and side view. D, dorsal; V, ventral. (C) Shh wholemount in situ hybridization in embryonic day 12 normal and frizzle feather buds. (D) Diagram summary of PCNA and TUNEL staining at different levels of the rachis. (E) Diagram of the chicken KRT75 gene and the cryptic splice site activated by the deletion mutation that covers positions 224 of exon 5 to +59 of intron 5. Black boxes represent exon sequences; intron 5 is designated by a line. The caret designating use of the cryptic site (position 269) is shown below, and the caret designating use of the authentic site is shown above the diagram of the pre-mRNA. (F) Partial sequence of KRT75 gene. The 84-bp deletion in genomic DNA is shown in light gray letters. The additional deletion in exon 5 created by a cryptic splice site is shown in dark gray letters. The deletion in genomic DNA and use of the cryptic splice site together result in a deletion of 23-amino acids (position 311–333) in the K75 protein. Parts of exon 5 and intron 5 are shown in capital and small letters, respectively. The underlines show the authentic and cryptic mRNA splicing sites. (G) Effects of viral misexpression, as shown by qualitative changes in the rachis curvature. Without the gene mutation, the curvature from the contralateral feathers of a normal chicken were expected to exhibit mirror symmetry, which is obviously abrogated in this case.

Figure 5. Time dimension and proximal-distal feather axis during regeneration

Each feather displays its formation processes. The distal part is formed first and proximal part later. The 0–100 vertical bar is used to indicate that the signaling microenvironment in the feather follicle varies in these different arbitrary time units for feather formation. In the schematic feather on the right, numbers indicate different chronological stages during feather formation. 1) Initiation occurs in what will become the distal tip of the feather. Growth phase can be split into the 2) upper and 3) lower vane. In the typical feathers, upper and lower vane are separated by the line across the widest region of the vane. 4) This is followed by the plumulaceous region in which barbs are not attached to their neighboring barbs and remain fluffy. 5) The calamus is formed in the resting phase at the proximal end of the feather. For the molecular involvement, we know the combination of different ratios of FGF, BMP, sprouty and noggin will modulate feather stem cells into different feather forms at different time points. The quantitative amounts of these signaling molecules, however, are based on idealized speculation.