Epithelial Migration and Non-adhesive Periderm Are Required for Digit Separation during Mammalian Development.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation

Epithelial mechanisms in digit separation and cutaneous wound healing

The epidermis, the outermost layer of the skin, continuously develops to form a tight epidermal barrier that is required for animal hydration and survival. Epidermal development occurs during embryogenesis via the mechanism of epidermal differentiation. We have previously described the role of the epidermal transcription factor Grainyhead like-3 (GRHL3) in epidermal differentiation and barrier formation. Additionally, we observed that mice lacking Grhl3 in the epidermis exhibit soft-tissue syndactyly between their digits, this phenomenon was also detected in Grhl3 germline knockouts (Grhl3KO). Through our work, we identified epithelial mechanisms required for normal digit separation during development. We discovered the formation of a multi-layered epithelial structure between the digits. In Grhl3KO embryos, the interdigital epithelia is fused, resulting in syndactyly. The separation of the epithelial structure depends on the anti-adhesive properties of the periderm, a transient layer located at the uppermost surface of the embryonic epidermis. We found that periderm cells in Grhl3 KO are hyper-adhesive with abnormal expression of adhesion molecules on their surfaces. Our findings reveal novel epithelial mechanisms required for digit separation; they also uncover a crucial role of Grhl3 in normal periderm development. Adult skin is normally maintained in the absence of Grhl3. Our previous work indicated that Grhl3 is required for epidermal recovery after injury. Adult mice lacking Grhl3 in the epidermis exhibit impaired full-thickness wound healing and delayed keratinocyte migration. We observed an increase in cell-cell adhesion between follower cells at the wound-edge in Grhl3 cKO, concomitant with an increase expression of the adherens junction’s protein, E-cadherin. Gene expression analysis of Grhl3 cKO wounded-keratinocytes 3 days post wounding shows a significant downregulation of F-actin protein Fscn1, a protein known to negatively regulate the expression of E-cadherin. These data indicate a novel Grhl3-Fscn1 mechanism required for cell-cell loosening in follower cells during keratinocyte migration in wounding. In addition, we observed that early after wounding, Grhl3 cKO mice exhibit a defect in dermal vasodilation and decreased blood flow. Gene expression analysis of wounded-keratinocytes 24 hrs post wounding suggest that Grhl3 regulate the expression of enzymes required for the biosynthesis of vasodilator factor prostaglandin E2. These observations are intriguing and suggest a Grhl3-mediated vasodilation during cutaneous wound healing

Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation

During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases

GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance

Differential cell composition and split epidermal differentiation in human palm, sole, and hip skin.

Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unclear. Here, we use bulk and single-cell RNA sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering differences between palmar and plantar sites. Our approach reveals an altered immune environment in palmoplantar skin, with downregulation of diverse immunological processes and decreased immune cell populations. Further, we identify specific fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated keratinocyte analysis highlights major differences in basal cell fraction among the three sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep characterization of highly adapted palmoplantar skin contributes key insights into the fundamental biology of human skin and provides a valuable data resource for further investigation