Popliteal Pterygium syndrome, IRf6, and the periderm

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SUMMARY This project focuses on popliteal pterygium syndrome (PPS, OMIM 119500), a rare disease occurring in 1/300,000 live births. It is estimated that 97% of PPS cases can be accounted for by mutations that affect residues of the DNA binding domain of the transcription factor interferon regulatory factor 6 (IRF6). Clinical manifestations of PPS include cleft lip with or without cleft palate, and a variety of tissue adhesions, including pterygium (tissue fusion or webbing) behind the knees (popliteal region), syndactyly, ankyloblepharon (fusion of eyelids), and fusions around the genital region. Absence of the periderm, a single layer of epithelial cells that covers the developing epidermis during embryogenesis, is believed to underlie these aberrant tissue adhesions. Relatively little is known about the periderm, and studies aimed at underlying the pathophysiology of PPS may provide new insight with broad significance for our understanding of how embryonic ectoderm gives rise to the rich complexity of skin and oral epithelia. We found that transcription factors implicated in epidermal differentiation, including IRF6, are expressed in the periderm. Yet, the field lacks in the availabiltity of biomarkers that specificially reflect the genesis, function and fate of periderm cells, accounting for our superficial understanding of how defects in the periderm may contribute to PPS. Established markers of the periderm are absent in the developing ectoderm of Irf6-deficient mice. In the latter setting, abnormal tissue adhesions occur in the oral cavity and differentiation of keratinocytes in the developing epidermis is compromised. Our prior studies also revealed that Irf6-deficient keratinocytes can stratify, but cannot give rise to periderm. While these observations establish that IRF6 is essential for specification of the periderm, how it affects morphogenesis of the periderm and how the periderm prevents tissue fusion remain ill-defined. The premise of our study is that a deeper understanding of PPS requires a much more profound understanding of the role of IRF6 in the formation of the periderm. Toward this end, we herein propose to define how the periderm initially forms, and identify the IRF6-dependent gene regulatory networks that contribute to this initial morphogenic event. To test our central hypothesis that periderm cells present a unique gene expression profile throughout its genesis and fate that are affected by IRF6 levels, we will generate cellular libraries from wild-type and Irf6-deficient epidermis obtained from embryos at different developmental time points critical for periderm morphogenesis and perform single-cell RNA sequencing, followed by multiplex in situ and immunofluorescence validation. This proposal is innovative both conceptually and technically as it represents a significant conceptual paradigm shift and takes a novel single-cell genomic approach to understanding cellular heterogeneity within the epidermis at single-cell resolution during early stages of morphogenesis. Our knowledge of, and expertise in IRF6 biology and skin development establish that we are well-prepared to succeed with all aspects of this challenge.