Mouse model for eyelid disease: Congenital blepharophimosis

描述（由申请人提供）：眼睑裂小眼症、上睑下垂和上眦转位综合征（BPES）是一种常染色体显性遗传疾病，其特征为主要影响眼睑发育的颅面缺陷。BPES有两种类型：I型包括小睑裂、上睑下垂、内眦赘皮和内眦赘皮四种主要特征，加上卵巢早衰（POF），导致女性不孕。II型仅包括眼睑畸形，无性别偏好。患有BPES的人患视力问题的风险增加，如近视（近视）或远视（远视）。他们也可能有眼睛不指向同一方向（斜视）和弱视（弱视）影响一只或两只眼睛。转录因子叉头盒L2（FOXL 2）结构基因的突变导致70%的BPES。FOXL 2基因提供了制造一种蛋白质的指令，这种蛋白质在出生前参与眼睑和卵巢的发育。大约30%的BPES患者没有发现FOXL 2结构基因突变;这些人的病因尚不清楚，但FOXL 2基因调控可能发生改变。 在此，我们研究了眼周间充质细胞（POMC）中Notch 1信号激活的作用，POMC有助于形成眼睑特异性结构，包括提肌平滑肌、睑板腺和睑板腺。采用多西环素（doxycycline，Dox）脉冲诱导的方法，在新的三转基因小鼠品系Kera-rtTA/tetO-Cre/R26 fN 1-ICD（KR/TC/R26 fN 1- ICD）的POMC（以下简称POMCN 1-ICD）中，条件性错表达Notch 1胞内结构域（N1-ICD）。这些三重KR/TC/R26 fN 1-ICD小鼠表现出类似于人类BPES的眼睑异常的杂色。我们的初步数据显示，N1-ICD表达导致FOXL 2和平滑肌分化标志基因α-平滑肌肌动蛋白（α-SMA）在眼睑形态发生过程中POMCs表达的特异性减少。我们的初步研究使我们能够提出这样的假设，即持续的Notch信号通过下调POMC细胞中的FoxL 2来干扰负责眼睑张开的提M？埃勒肌平滑肌的形成。在本申请中，我们将进一步表征KR/TC/R26 fN 1-ICD三重转基因小鼠作为研究先天性BPES（Aim 1）发病机制的新型动物模型。我们还将描绘一个分子途径，导致FoxL 2的下调和随后的畸形的提肌平滑肌在胚胎眼睑发育（Aim 2）。 公共卫生相关性：本研究的目的是了解正常眼睑的形态发生和人类先天性睑包茎综合征（BPES）的发病机制。具体来说，我们计划使用转基因小鼠和细胞培养物来研究Notch 1激活如何通过眼周间充质作为FoxL 2表达的上游控制机制，这是注定要用于眼睑的M？埃勒平滑肌发育的。此外，新的知识将帮助我们解开Notch通路在眼睑形态发生过程中调节FoxL 2基因表达的信号机制，FoxL 2基因表达控制提M？埃勒肌平滑肌分化。

DESCRIPTION (provided by applicant): Blephar-ophimosis, Ptosis, and Epi-canthus inversus Syndrome (BPES) is an autosomal dominant genetic disorder characterized by craniofacial defects that mainly affect the development of the eyelids. There are two types of BPES: Type I consists of the four major features of blepharophimosis, ptosis, epicanthus inversus, and telecanthus plus premature ovarian failure (POF), leading to infertility in woman. Type II consists of only the eyelid malformations without gender preference. People with BPES are at an increased risk of developing vision problems such as nearsightedness (myopia) or farsightedness (hyperopia). They may also have eyes that do not point in the same direction (strabismus) and lazy eye (amblyopia) affecting one or both eyes. Mutations in the transcription factor forkhead box L2 (FOXL2) structure gene cause 70 percent of BPES. The FOXL2 gene provides instructions for making a protein that is involved in the development of the eyelids and the ovaries before birth. Approximately 30 percent of people with BPES do not have an identified FOXL2 structure gene mutation; the cause of the condition in these people is unknown but FOXL2 gene regulation maybe altered. Herein, we have investigated the effects of Notch1 signaling activation in peri-ocular mesenchymal cells (POMC) which contribute to the formation of the lid-specific structures including levator M¿ellersmooth muscle, tarsus, and meibomian glands. Notch1 intracellular domain (N1-ICD) was conditionally mis-expressed in POMC (here refer to as POMCN1-ICD) of a novel triple transgenic mouse strain, namely Kera-rtTA/tetO-Cre/R26floxedN1-ICD (KR/TC/R26fN1- ICD), by pulse induction of doxycycline (Dox) at different developmental stages. These triple KR/TC/R26fN1-ICD mice exhibited variegation of eyelid anomalies resembling BPES in humans. Our preliminary data showed that N1-ICD expression caused specific reduction of FoxL2 and smooth muscle differentiation marker gene, alpha-smooth actin (a-SMA) expressions in POMCs during eyelid morphogenesis. Our preliminary studies allow us to propose the hypotheses that sustained Notch signaling disturbs the formation of levator M¿eller smooth muscle responsible for eyelid opening by down-regulation of FoxL2 in the POMC cells. In this application, we will further characterize KR/TC/R26fN1-ICD triple transgenic mouse as a novel animal model to study the pathogenesis of congenital BPES (Aim1). We will also delineate a molecular pathway that leads to the down-regulation of FoxL2 and subsequent malformation of the levator M¿ellersmooth muscle during embryonic eyelid development (Aim2). PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to understand the normal eyelid morphogenesis and the pathogenesis of human congenital blephrophimosis syndrome (BPES). Specifically, we plan to use transgenic mice and cell cultures to investigate the mechanism of how the Notch1 activation may serve as the upstream control of expression of FoxL2 by periocular mesenchyma, which are destined for M¿eller smooth muscle development of the eyelids. Furthermore, the new knowledge will help us unravel signaling mechanisms of the Notch pathway in regulating FoxL2 gene expression governing levator M¿eller smooth muscle differentiation during eyelid morphogenesis.