Role Of Ectodysplasin-a In Skin Appendage Formation

外胚层增生素-a 在皮肤附属器形成中的作用

• 批准号: 8736579
• 负责人: David Schlessinger
• 金额: $ 68.85万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736579
• 关键词: Adult; Affect; Anhidrosis; Anhidrotic Ectodermal Dysplasia; Animal Model; Animals; Anions; Bicarbonates; Biological Models; Blindness; Calcium; Childhood; Claw; Complex; Cosmetics; DNA; Development; Disease; Duct (organ) structure; Ectodermal Dysplasia; Embryo; Employee Strikes; Epidermis; Epithelium; Etiology; Event; Exocrine Glands; Eye; Gene Targeting; Genes; Genetic; Genetic Transcription; Germ; Gland; Glycoproteins; Goals; Hair; Hair follicle structure; Hair shaft structure; Heat Stroke; Human; Hypohidrosis; Immune; Inflammation; Ions; Knock-out; Knockout Mice; Link; Lymphoid; Maintenance; Mammary gland; Mesenchyme; Modeling; Molecular; Molecular Profiling; Molecular Target; Mus; Mutate; Mutation; NF-kappa B; Nail plate; Natural regeneration; Organ; Pathway interactions; Patients; Pattern; Phase; Phenotype; Physiological; Play; Potassium Channel; Predisposition; Production; Protein Isoforms; Proteins; Protocols documentation; Publishing; Regenerative Medicine; Regulation; Regulator Genes; Regulatory Pathway; Role; Route; Salivary; Sampling; Series; Signal Pathway; Signal Transduction; Skin; Staging; Stem cells; Supplementation; Sweat; Sweat Glands; Sweating; TNF gene; Tetanus Helper Peptide; Thick; Time; Tissues; Tooth structure; Transgenic Mice; Tretinoin; Tumor Necrosis Factor-Beta; Variant; Wild Type Mouse; Work; age effect; age related; appendage; base; beta catenin; combat; ectodysplasin; embryonic stem cell; eye dryness; genome-wide; gland development; interest; keratinocyte; lacrimal; meibomian gland; morphogens; mutant mouse model; ocular surface; prevent; progenitor; response; spatiotemporal; transcription factor

In a first phase of efforts, we discovered that the Tabby mouse, which has many of the features observed in human EDA, is specifically mutated in the corresponding mouse gene. We demonstrated that the Wnt pathway directly regulates EDA transcription. In published work, we further found that provision of DNA encoding a variant of ectodysplasin (Eda-A1, the longest isoform) in embryonic Tabby mice restores hair follicles and sweat glands. By generating Tet-regulated conditional transgenic mice, we have dissected spatiotemporal actions of Eda-A1 during hair follicle development. We also have characterized eye phenotypes of Tabby mice including blindness and inflammation susceptibility, and they are also reversed by supplementation with the same Eda-A1 isoform. This study has provided the first animal model for ocular surface disease, and also further increased the interest in the possibility of manipulating the Eda pathway to combat dry eye. By large scale genome-wide expression profiling of samples from wild-type and Tabby mice ranging from embryos to adult and from hair follicles to sweat glands and primary keratinocytes, we identified numerous downstream target genes of Eda, including lymphotoxin-, Shh, Wnt10b and Dkk4 in hair follicles and Shh and FoxA1 in sweat glands. More recently, we have further focused on the function of Eda and Eda target genes identified by expression profiling in mutant mouse models. We demonstrated that target gene lymphotoxin-, an immune gene, is involved in hair shaft formation, but not hair follicle induction. We also found that Dkk4, a Wnt antagonist, discriminates an Eda-independent mechanism of secondary hair follicle formation. Notably, both pathways converge at the activation of downstream Shh. Based on these observation, we proposed that different subtypes of hair follicles are formed by variant molecular mechanisms. Conditional Shh transgenic mice and skin-specific Shh knockout mice in wild-type and Tabby backgrounds showed that Shh is required for elongation of Tabby hair shafts, but not for the induction of the primary hair follicles that are missing in Tabby mice. We have also studied and compared the control of Eda-independent skn appendage developmental pathways. A similar signaling pathway (TNF/NF-kB) is required for development of secondary lymphoid organs, but with very different downstream effectors. Skin appendage nails/claws is also independent of EDA, again regulated by a Wnt pathway early on, but working through Fzd6, the loss of Fzd6 distorted claw formation in mice, in line with the demonstrated damage of nail formation in patients lacking an active gene copy. We have now initiated projects for skin exocrine glands, again with Tabby mice as a model system. In sweat glands, FoxA1 was strikingly affected gene in Tabby during late developmental stages and adult stage. Skin-specific FoxA1 knockout mice showed striking anhidrosis, with abundant accumulation of glycoproteins in the lumens and ducts of otherwise complete sweat glands; and we further showed that FoxA1 functions in sweat glands by promoting transcription of an anion channel protein, Best2. Best2 knockout mice also showed severe hypohidrosis/anhidrosis, revealing a FoxA1-Best2 cascade as a fundamental genetic pathway in sweat glands, regulating sweat secretion. Because Best2 is a calcium activated bicarbonate channel, we inferred two alternative cascades for sweating: calcium K/Cl (FoxA1) additional monovalent ion transporter cascade and calcium (FoxA1) Best2 K/Cl ion transporter, with the latter likely playing the major role. We found that four K channels and two Cl channels are highly expressed in sweat glands. Sublocalizations suggest that one of them may be directly activated by calcium (the first cascade), with a second activated by FoxA1/Best2 (the second cascade). Concerning another exocrine skin appendage, we previously showed that Eda-ablated Tabby mice develop ocular surface disease, and EDA patients show extreme dry eye. We plan to examine dry eye etiology with meibomian glands as an entry point. In a first phase, we characterized their development. They are missing in Tabby mice, and Shh knockout mice, Dkk4 transgenic mice, and beta-catenin knockout mice all completely lack meibomian glands. Currently we are characterizing meibomian gland phenotypes in these mice more systematically by time-course histological and immunohistochemical analyses, and assessing the possible trophic role of Eda in preventing aging-related deterioriation of Meibomian gland function. In further initiatives moving toward skin appendage regeneration, incisive studies have led to the isolation and understanding of several types of stem cells jointly required for hair follicle or sweat gland formation and maintenance, but these require very complex protocols and difficult-to-obtain quantities of stem cells to satisfy requirements of regenerative medicine. We will attempt first steps in a long-term but more direct route, starting from embryonic stem cells (ES cells). ES cells have been differentiated into keratinocyte progenitors and full thickness skin epidermis in presence of retinoic acid and Bmp4, but with low efficiency. We have initiated an approach that could be more efficient starting from a master transcription factor that directs them toward the keratinocyte lineage.

在第一阶段的工作中，我们发现猫小鼠具有人类EDA的许多特征，在相应的小鼠基因中发生了特定的突变。我们证明了Wnt途径直接调控EDA转录。在已发表的工作中，我们进一步发现，在猫小鼠胚胎中提供编码一种异构体胞外营养不良蛋白(EDA-A1，最长的异构体)的DNA可以恢复毛囊和汗腺。通过建立Tet调控的条件转基因小鼠，我们剖析了Eda-A1在毛囊发育过程中的时空作用。我们还鉴定了猫小鼠的眼睛表型，包括失明和炎症易感性，这些表型也可以通过补充相同的EDA-A1亚型而逆转。这项研究提供了第一个眼表疾病的动物模型，并进一步增加了人们对操纵EDA途径对抗干眼的可能性的兴趣。通过对野生型和猫小鼠从胚胎到成年，从毛囊到汗腺和原代角质形成细胞的大规模基因组表达谱分析，我们发现了大量的EDA下游靶基因，包括毛囊中的淋巴毒素-1、Shh、Wnt10b和Dkk4，以及汗腺中的Shh和foxA1。 最近，我们进一步关注了通过表达谱确定的EDA和EDA靶基因在突变小鼠模型中的功能。我们证明了靶基因淋巴毒素--一种免疫基因，参与了毛干的形成，但不参与毛囊的诱导。我们还发现，Wnt拮抗剂Dkk4区分了次级毛囊形成的EDA非依赖性机制。值得注意的是，这两条途径都在Shh下游的激活处汇合。基于这些观察，我们提出不同的毛囊亚型是由不同的分子机制形成的。在野生型和猫背景下的条件性Shh转基因小鼠和皮肤特异性Shh基因敲除小鼠的研究表明，Shh是猫毛干延长所必需的，但不是诱导猫小鼠所缺少的初级毛囊所必需的。 我们还研究和比较了不依赖EDA的SKN附属物发育途径的控制。次级淋巴器官的发育需要一个相似的信号通路(肿瘤坏死因子/核因子-kB)，但下游效应器非常不同。皮肤附属物指甲/爪子也独立于EDA，同样在早期受Wnt途径调控，但通过Fzd6，Fzd6扭曲的爪子形成在小鼠中的丢失，与缺乏活跃基因复制的患者指甲形成的损害是一致的。 我们现在已经启动了皮肤外分泌腺的项目，同样是以猫小鼠为模型系统。在汗腺中，FoxA1基因在发育后期和成虫期显著影响猫。皮肤特异的FoxA1基因敲除小鼠表现出显著的汗水，在其他完整的汗腺的管腔和导管中大量积累糖蛋白；我们进一步证明FoxA1通过促进阴离子通道蛋白Best2的转录在汗腺中发挥作用。Best2基因敲除小鼠也表现出严重的少汗/无汗，揭示了FoxA1-Best2级联反应是汗腺中调节汗液分泌的基本遗传途径。由于Best2是一个钙激活的碳酸氢盐通道，我们推测了两个可供选择的出汗通路：钙K/氯(FoxA1)附加单价离子转运体级联和钙(FoxA1)Best2K/Cl离子转运体，后者可能起主要作用。我们发现四个钾通道和两个氯通道在汗腺中高度表达。亚区块化表明，其中一个可能被钙直接激活(第一级联)，第二个可能被FoxA1/Best2激活(第二级联)。 关于另一种外分泌皮肤附属物，我们之前曾证明EDA消融的猫小鼠会出现眼表疾病，EDA患者表现出极度干眼。我们计划以眉板腺为切入点来研究干眼症的病因。在第一阶段，我们描述了它们的发展。这些基因在猫小鼠中缺失，Shh基因敲除小鼠、Dkk4转基因小鼠和β-连环蛋白基因敲除小鼠都完全缺乏眉毛腺。目前，我们正在通过时程组织学和免疫组织化学分析更系统地描述这些小鼠的眉板腺表型，并评估EDA在防止与衰老相关的眉板腺功能恶化方面的可能营养作用。 在皮肤附属物再生的进一步倡议中，深入的研究导致分离和理解了毛囊或汗腺形成和维护共同需要的几种类型的干细胞，但这些需要非常复杂的方案和难以获得的大量干细胞来满足再生医学的要求。我们将尝试一条长期但更直接的途径，从胚胎干细胞(ES细胞)开始。在维甲酸和Bmp4存在下，ES细胞已分化为角质形成祖细胞和全层皮肤表皮，但效率较低。我们已经启动了一种可能更有效的方法，从一种引导它们走向角质形成细胞谱系的主要转录因子开始。