Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales

Abstract

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

Figure 1

KRT9LC2 is a marker of hard scutate scales of chicken leg skin. (A) Quantitative RT-PCR analysis of KRT9LC2 expression during differentiation of chicken leg keratinocytes (KC) in vitro. RNA was isolated from chicken KCs (n = 3, biological replicates) growing in monolayer culture and in an epidermal model. KRT9LC2 mRNA was quantified by RT-PCR and normalized to the housekeeping gene HMBS. a.u., arbitrary units. (B,C) Western blot analysis of KRT9LC2 and GAPDH in cultured cells (B) and tissues of chicks (C). Bands at predicted sizes are indicated by arrows. The positions of molecular weight markers (kD, kilo-Dalton) are shown of the right. (D,E,G) Immunohistochemical detection of KRT9LC2 (red) in scutate scales (D,E) and reticulate scales (G). A negative control (neg. con.) staining in which the anti-KRT9LC2 antiserum was replaced by non-immune serum is shown in panel (F). Scale bars, 50 µm.

Figure 2

Single-cell RNA-sequencing (scRNA-seq) analysis of scutate scales of chicks. (A) Uniform manifold approximation and projection (UMAP) of cells from chicken scutate scales according to similarity of their transcriptome. Unsupervised clustering resulted in 8 clusters that are indicated by different colors. Cell types were identified by the expression of specific markers, so that 5 unsupervised clusters (KC1-KC5) could be defined as keratinocytes. (B–D) Feature plots showing the expression level of KRT14L1 (B), KRT9L3 (C) and KRT9LC2 (D) in each cell depicted in UMAP plots. The expression levels are color-coded from grey (no expression) to purple (high expression level).

Figure 3

Volcano plots of genes differentially expressed in KRT9L3-high versus KRT14L1-high and KRT9LC2-high versus KRT14L1-high cells. Significance (-log10 of adjusted P value) was plotted against Log2 of Fold-change of gene expression levels in KRT9L3-positive versus KRT14L1-positive keratinocytes (A) and KRT9LC2-positive versus KRT14L1-positive keratinocytes (B).