Popliteal Pterygium syndrome, IRf6, and the periderm

摘要 本项目主要关注发生在中国的一种罕见的疾病--羊膜翼状胬肉综合征(PPS，OMIM 119500)。 1/300,000名活产儿。据估计，97%的PPS病例可以由影响 转录因子干扰素调节因子6(IRF6)DNA结合域的残基。临床 PPS的表现包括伴有或不伴有腭裂的唇裂，以及各种组织粘连，包括 膝后翼状胬肉(组织融合或带状)、并指、强直性眼睑(融合 眼皮)，以及生殖器区域周围的融合。没有周皮，即单层上皮细胞， 在胚胎发育过程中覆盖发育中的表皮，被认为是这些异常组织粘连的基础。 对周皮的了解相对较少，旨在揭示PPS的病理生理学基础的研究可能 为我们理解胚胎外胚层如何产生外胚层提供了新的见解和广泛的意义 丰富的皮肤和口腔上皮的复杂性。我们发现转录因子与表皮有关 分化，包括IRF6，在周皮中表达。然而，该领域缺乏生物标记物的可用性。 具体反映了周皮细胞的发生、功能和命运，解释了我们表面上的 了解周皮缺陷对PPS的影响。周皮的已建立的标记是 在IRF6基因缺陷小鼠的外胚层发育中不存在。在后一种情况下，会出现异常的组织粘连 在口腔中，发育中的表皮中角质形成细胞的分化受到影响。我们的前辈 研究还表明，IRF6缺陷的角质形成细胞可以分层，但不能形成周皮。而这些 观察表明，IRF6对于周皮的指定是必不可少的，它如何影响植物的形态发生 周皮以及周皮如何阻止组织融合仍然没有明确的定义。我们研究的前提是 更深入地理解PPS需要对IRF6在形成中的作用有更深刻的理解 周皮的。为此，我们在这里建议定义周皮最初是如何形成的，并确定 依赖于IRF6的基因调控网络促成了这一最初的形态发生事件。为了测试我们的中央 周皮细胞在其发生和命运过程中呈现独特的基因表达谱的假说 受irf6水平的影响，我们将从野生型和irf6缺陷的表皮中建立细胞库。 来自不同发育时间点的胚胎，对周皮的形态发生至关重要，并进行单细胞 RNA测序，随后进行多重原位和免疫荧光验证。这个建议很有新意 在概念和技术上都是如此，因为它代表着一种重大的概念范式转变，并将一部小说 用单细胞基因组学方法了解单细胞表皮内的细胞异质性 在形态发生的早期阶段进行分解。我们在IRF6生物学和皮肤方面的知识和专业技能 发展表明，我们已做好充分准备，能够成功应对这一挑战的所有方面。

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.