Esrp regulated programs of alternative splicing in skin development and function

 描述（由申请人提供）：表皮执行基本屏障功能，保护身体免受环境侵害并维持液体-电解质平衡。表皮和毛囊发育缺陷导致许多疾病，如皮肤癌、牛皮癣、特应性皮炎、脱发和伤口愈合受损。尽管转录程序在皮肤的发育和疾病中已经得到了很好的研究，但选择性剪接（AS）在皮肤发育和功能中的作用基本上尚未研究。几乎所有哺乳动物的多外显子基因都产生AS mRNA，组织特异性AS因子协调AS的程序以调节生物学上连贯的途径。此外，我们的初步数据显示，AS在皮肤和毛发的发育和功能的调节程序中起着关键但未被重视的作用。在这项提案中，我们将使用我们独特的遗传小鼠模型来研究我们实验室中鉴定的剪接因子在表皮生理学和毛发生长中的关键作用。我的实验室取得了一项里程碑式的发现，上皮细胞类型特异性剪接因子Esrp 1和Esrp 2调节成纤维细胞生长因子受体2（Fgfr 2）剪接，这是一种先前与皮肤发育和表皮屏障功能有关的AS事件。为了进一步研究Esrp依赖性AS在皮肤发育中的作用，我们产生了Esrp 1和Esrp 2条件性和完全敲除等位基因的小鼠。组合的Esrp 1/Esrp 2 KO是致死性的，并导致表皮发育不全、表皮屏障功能缺陷和毛囊数量减少。我们假设这些表型反映了关键的上皮特异性剪接异构体的丢失，并将使用条件基因敲除技术来进一步表征Esrp缺失的表型，并通过以下目的来定义皮肤中Esrp调节的AS的全基因组程序：1）确定与毛囊间表皮和毛囊中Esrp消融相关的表型。我们将使用Esrp 1flox/flox/Esrp 2-/-小鼠在发育和成年表皮中有条件地减弱Esrps，并表征导致表皮和毛囊缺陷的基本细胞过程。2)定义表皮中Esrp调节的选择性剪接的综合方案。我们将使用RNA-Seq和剪接敏感的微阵列来定义体外和体内AS的全基因组程序。表皮中的Esrp调节靶将在上皮巴里测定中进行功能筛选。3)确定毛囊中的ESR调节剪接程序和毛乳头（DP）中的差异剪接。我们将使用可诱导的缺失策略来确定Esrp消融对毛囊的影响，并确定毛囊隆突干细胞和毛胚（HG）中的关键Esrp靶点。 所提出的目标构成了AS在皮肤发育和功能方面的第一个全面分析，从而为该领域引入了一个新的范式。需要这些研究来确定皮肤中Esrp消融诱导表皮屏障缺陷和脱发的分子和细胞机制，以便为未来治疗皮肤病变和脱发的疗法的开发提供信息。

 DESCRIPTION (provided by applicant): The epidermis performs essential barrier functions that protect the body from environmental insults and maintain fluid-electrolyte balance. Defects in epidermal and follicular development lead to numerous diseases such as skin cancer, psoriasis, atopic dermatitis, alopecia, and impaired wound healing. Whereas transcriptional programs have been well-studied in development and diseases of the skin, the role of alternative splicing (AS) in skin development and function is essentially unstudied. Nearly all mammalian multi- exon genes produce AS mRNAs and tissue-specific AS factors coordinate programs of AS to regulate biologically coherent pathways. Moreover, our preliminary data reveals that AS plays a critical, but unappreciated role in the regulatory programs of the development and function of both skin and hair. In this proposal, we will use our unique genetic mouse models to study the critical roles of splicing factors identified in our lab in epidermal physiology and hair growth. My laboratory made a landmark discovery that epithelial cell- type-specific splicing factors Esrp1 and Esrp2 regulate fibroblast growth factor receptor 2 (Fgfr2) splicing, an AS event previously implicated in skin development and epidermal barrier function. To further investigate the role of Esrp-dependent AS in skin development, we generated mice with conditional and complete knockout alleles for Esrp1 and Esrp2. Combined Esrp1/Esrp2 KO is lethal and results in epidermal hypoplasia, defects in epidermal barrier function, and reduced numbers of hair follicles. We hypothesize that these phenotypes reflect the loss of key epithelial-specific splice isoforms and will use conditional gene knockout technology to further characterize the phenotypes of Esrp deletion and define a genome-wide program of Esrp-regulated AS in the skin through the following aims: 1) Determine the phenotypes associated with Esrp ablation in the interfollicular epidermis and in hair follicles. We will conditionally abate the Esrps using Esrp1flox/flox/Esrp2-/- mice in developing and adult epidermis and characterize the basic cellular processes that lead to epidermal and follicular defects. 2) Define comprehensive programs of Esrp regulated alternative splicing in the epidermis. We will use RNA-Seq and splicing sensitive microarrays to define genome-wide programs of AS in vitro and in vivo. Esrp regulated targets in the epidermis will be functionally screened in epithelial barrie assays. 3) Identify Esrp-regulated splicing programs in the hair follicle and differential splicingin the dermal papilla (DP). We will use inducible deletion strategies to determine the consequence of Esrp ablation on hair follicles and identify key Esrp targets in hair follicle bulge stem cells nd the hair germ (HG). The proposed aims constitute the first comprehensive analysis of AS in skin development and function, thereby introducing a new paradigm to the field. These studies are needed to define the molecular and cellular mechanisms by which Esrp ablation in the skin induces epidermal barrier defects and hair loss in order to inform the development of future therapies to treat skin pathologies and alopecia.