Popliteal Pterygium syndrome, IRf6, and the periderm

总结 该项目的重点是腘翼状胬肉综合征（PPS，OMIM 119500），一种罕见的疾病，发生在 1/300，000活产。据估计，97%的PPS病例可以解释为突变， 转录因子干扰素调节因子6（IRF 6）的DNA结合结构域的残基。临床 PPS的临床表现包括唇裂伴或不伴腭裂，以及各种组织粘连，包括 翼状胬肉（组织融合或带状）在膝盖后面（腘区），并指，睑粘连（融合 眼睑）和生殖器区域周围的融合。缺乏周膜，单层上皮细胞， 在胚胎发生期间覆盖发育中的表皮，被认为是这些异常组织粘连的基础。 对牙周炎的了解相对较少，旨在研究PPS的病理生理学的研究可能 为我们理解胚胎外胚层如何产生 丰富的皮肤和口腔上皮的复杂性。我们发现，与表皮生长因子相关的转录因子， 分化，包括IRF 6，在周膜中表达。然而，该领域缺乏生物标志物的可用性 这具体地反映了起源，功能和命运的周细胞，占我们的表面 了解围产期缺陷如何影响PPS。已确定的围产期标志物 在Irf 6缺陷小鼠的发育中的外胚层中不存在。在后一种情况下，发生异常组织粘连 并且在发育中的表皮中角质形成细胞的分化受到损害。我们事先 研究还揭示，Irf 6缺陷的角质形成细胞可以分层，但不能引起角质形成。虽然这些 观察结果表明，IRF 6是必不可少的规格的peridendum，它如何影响形态发生的 牙周膜以及牙周膜如何防止组织融合仍然不清楚。我们研究的前提是， 要更深入地了解PPS，需要更深入地了解IRF 6在形成中的作用， 的一部分。为此，我们在此提出定义如何形成的peridendum最初，并确定 IRF 6依赖的基因调控网络，有助于这一最初的形态发生事件。为了测试我们的中央 这一假说认为，围产期细胞在其发生和命运中呈现独特的基因表达谱， 受IRF 6水平的影响，我们将从获得的野生型和IRF 6缺陷型表皮中产生细胞文库， 从胚胎在不同的发育时间点的关键围产期形态发生和执行单细胞 RNA测序，然后进行多重原位和免疫荧光验证。这一建议具有创新性 在概念上和技术上，因为它代表了一个重大的概念范式转变， 单细胞基因组方法来了解表皮内单细胞的细胞异质性 在形态发生的早期阶段消退。我们在IRF 6生物学和皮肤方面的知识和专长 发展表明，我们已做好充分准备，能够在这一挑战的所有方面取得成功。

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.