Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing
Andrew M. Overmiller, Akihiko Uchiyama, Emma D Hope, Subhashree Nayak, Christopher G. O’Neill, Kowser Hasneen, Yi-Wen Chen, Faiza Naz, Stefania Dell’Orso, Stephen R. Brooks, Kan Jiang, Maria I. Morasso
Andrew M. Overmiller, Akihiko Uchiyama, Emma D Hope, Subhashree Nayak, Christopher G. O’Neill, Kowser Hasneen, Yi-Wen Chen, Faiza Naz, Stefania Dell’Orso, Stephen R. Brooks, Kan Jiang, Maria I. Morasso
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Research Article Dermatology

Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing

Abstract

Cutaneous wound healing is a slow process that often terminates with permanent scarring while oral wounds, in contrast, regenerate after damage faster. Unique molecular networks in epidermal and oral epithelial keratinocytes contribute to the tissue-specific response to wounding, but key factors that establish those networks and how the keratinocytes interact with their cellular environment remain to be elucidated. The transcription factor PITX1 is highly expressed in the oral epithelium but is undetectable in cutaneous keratinocytes. To delineate if PITX1 contributes to oral keratinocyte identity, cell-cell interactions, and the improved wound healing capabilities, we ectopically expressed PITX1 in the epidermis of murine skin. Using comparative analysis of murine skin and oral (buccal) mucosa with single-cell RNA-Seq and spatial transcriptomics, we found that PITX1 expression enhances epidermal keratinocyte migration and proliferation and alters differentiation to a quasi–oral keratinocyte state. PITX1+ keratinocytes reprogrammed intercellular communication between skin-resident cells to mirror buccal tissue while stimulating the influx of neutrophils that establish a pro-inflammatory environment. Furthermore, PITX1+ skin healed significantly faster than control skin via increased keratinocyte activation and migration and a tunable inflammatory environment. These results illustrate that PITX1 programs oral keratinocyte identity and cellular interactions while revealing critical downstream networks that promote wound closure.

Authors

Andrew M. Overmiller, Akihiko Uchiyama, Emma D Hope, Subhashree Nayak, Christopher G. O’Neill, Kowser Hasneen, Yi-Wen Chen, Faiza Naz, Stefania Dell’Orso, Stephen R. Brooks, Kan Jiang, Maria I. Morasso

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Figure 1

scRNA-Seq and Xenium in situ analysis of control skin, PITX1+ skin, and buccal mucosa.

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scRNA-Seq and Xenium in situ analysis of control skin, PITX1+ skin, and ...
(A) Representative dorsal images of male control and PITX1+ male and female mice. (B) Schematic of tissues collected (left). Representative immunofluorescent (IF) stainings for PITX1 (shown in red) and DAPI (shown in blue) in control epidermis, PITX1+ epidermis, and buccal epithelium. Dotted lines denote dermal-epidermal junction (DEJ) (top, middle). Representative H&E of tissues (bottom). Scale bars = 100 μm. (C) Uniform manifold approximation and projections (UMAPs) and cell type annotations of whole skin scRNA-Seq (left) and proportion plot of cell types in each condition (right). n = 8 control mice, 8 PITX1+ mice, 7 buccal mucosae pools. (D) Xenium in situ representative images of male FFPE skin sections with Xenium-derived cell types highlighted. n = 3 control skin, 4 PITX1+ mice, 7 buccal mucosae; scale bar = 100 μm (left). Proportion plot and total number of each cell type in each condition (right). Significance for proportion plots was assessed by proportionality testing followed by ad hoc comparisons against the corresponding cell type in control skin to derive log2 fold-change (log2FC) (*P < 0.01 & log2FC > |1.5|, ***P < 0.01 & log2FC > |4|).