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
View: Text | PDF
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

×

Figure 6

PITX1 promotes cutaneous wound healing.

Options: View larger image (or click on image) Download as PowerPoint
PITX1 promotes cutaneous wound healing. (A) Schematic of full-thickness ...
(A) Schematic of full-thickness wound-healing model. (B) Representative images from control (n = 11) and PITX1+ (n = 11) mice upon wounding (day 0) until day 10 after wounding (left). Quantification of wound area remaining open during healing. Significance assessed by 2-way repeated measures ANOVA with Tukey’s post hoc testing (**P < 0.01, ***P < 0.001). (C) Representative H&E images of male day 4 wounds used for measurement of wound reepithelialization and area of granulation tissue (left). Scale bar = 100 μm. Quantification of the reepithelization distance and granulation tissue area plotted as average ± SEM (right). Significance assessed by Student’s 2-tailed t test (*P < 0.05). (D) Schematic of wound tissues used for scRNA-Seq and Xenium. (E) UMAPs and cell type annotations of control (n = 6) and PITX1+ (n = 7) skin wound scRNA-Seq (left) and proportion plot of cell types (right). Significance for proportion plot assessed by proportionality testing followed by ad hoc comparisons against the corresponding cell type in control wound skin to derive log2 fold-change (log2FC) (*P < 0.01 & log2FC > |1.5|). (F) Xenium in situ representative images of FFPE male control (n = 1) and PITX1+ (n = 2) wound sections with cell types highlighted. Dashed box highlights wound-adjacent inset image. Scale bar for whole wound = 500 μm, scale bar for wound-adjacent inset = 200 μm.