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.

在第一阶段的努力中，我们发现在相应的小鼠基因中特异性突变的Tabby小鼠具有许多在人EDA中观察到的特征。我们证明了WNT途径直接调节EDA转录。在已发表的工作中，我们进一步发现，胚胎虎斑小鼠中编码一种胞质浮标（EDA-A1，最长的同工型）的DNA提供的提供可恢复毛囊和汗腺。通过产生由TET调节的条件转基因小鼠，我们在毛囊发育过程中解剖了EDA-A1的时空作用。我们还表征了Tabby小鼠的眼表型，包括失明和炎症敏感性，并且还通过补充相同的EDA-A1同工型来逆转它们。这项研究为眼部表面疾病提供了第一个动物模型，也进一步增加了对操纵EDA途径打击干眼症的可能性的兴趣。 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. 最近，我们进一步关注通过突变小鼠模型中表达分析鉴定的EDA和EDA靶基因的功能。我们证明了靶基因淋巴毒素（一种免疫基因）参与毛轴的形成，但不参与毛囊诱导。我们还发现，Wnt拮抗剂DKK4区分了二次毛囊形成的EDA独立机制。值得注意的是，这两种途径在下游SHH的激活下汇聚。基于这些观察结果，我们提出，毛囊的不同亚型是由变异分子机制形成的。有条件的SHH转基因小鼠和野生型和Tabby背景中的皮肤特异性SHH基因敲除小鼠表明，tabby毛轴的伸长需要SHH，但对于诱导塔比小鼠中缺少的主要毛囊的伸长所必需。 我们还研究并比较了对EDA独立的SKN附属发育途径的控制。 开发继发性淋巴器官需要类似的信号通路（TNF/NF-KB），但下游效应子非常不同。皮肤附属指甲/爪子也独立于EDA，再次受到WNT途径的调节，但通过FZD6工作，小鼠FZD6扭曲的爪形成的损失与缺乏活性基因副本的患者表现出的指甲形成的损害相一致。 现在，我们已经启动了皮肤外分泌腺的项目，再次以塔比小鼠为模型系统。在汗腺中，在发育阶段和成人阶段，FOXA1在塔比受到了显着影响的基因。皮肤特异性的FOXA1敲除小鼠表现出惊人的核虫病，在流明中大量糖蛋白积聚，而原本完整的汗腺的导管；我们进一步表明，FOXA1通过促进阴离子通道蛋白的转录最佳2。最佳2敲除小鼠还显示出严重的降低/肌瘤，揭示了FOXA1-BEST2级联反应是汗腺中的基本遗传途径，从而调节汗水分泌。因为Best2是钙活化的碳酸氢盐通道，所以我们推断出两个用于出汗的替代级联：钙K/Cl（FOXA1）额外的单价离子转运蛋白级联和钙（FOXA1）最佳2 k/cl离子转运蛋白，后者可能扮演主要角色。我们发现四个K通道和两个CL通道在汗腺中高度表达。串联估计表明，其中一个可以被钙（第一个级联）直接激活，第二个被FOXA1/BEST2（第二个级联）激活。 关于另一个外分泌皮肤附属物，我们先前表明，EDA被灭绝的塔比小鼠患有眼表疾病，EDA患者表现出极端干眼。 我们计划用梅博米亚腺作为切入点检查干眼症的病因。在第一阶段，我们表征了它们的发展。它们在Tabby小鼠中缺少，SHH淘汰小鼠，DKK4转基因小鼠和β-catenin敲除小鼠都完全缺乏梅博米亚腺体。目前，我们正在通过时间课程学和免疫组织化学分析更系统地表征这些小鼠中的梅博姆腺表型，并评估EDA在防止衰老相关的米博米亚腺功能下衰老中的营养作用。 在进一步的举措中，朝着皮肤附属的再生发展，尖锐的研究导致对毛囊或汗腺形成和维持所需的几种类型的干细胞的隔离和理解，但是这些需要非常复杂的方案以及难以润入的干细胞，以满足再生药物的需求。我们将从胚胎干细胞（ES细胞）开始，以长期但更直接的途径尝试第一步。在视黄酸和BMP4存在下，ES细胞已分化为角质形成细胞祖细胞和全厚皮肤表皮，但效率低。我们已经启动了一种方法，该方法从将其引导到角质形成细胞谱系的主转录因子开始。