Investigating the role of NKX6-1 in secondary lens fiber cell differentiation

研究 NKX6-1 在次级晶状体纤维细胞分化中的作用

基本信息

批准号：
10087940
负责人：
MICHAEL L ROBINSON
金额：
$ 17.52万
依托单位：
MIAMI UNIVERSITY OXFORD
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10087940
关键词：
Adenovirus Vector Affect Apoptosis Binding Sites Birth Cataract Cell Differentiation process Cell Survival Cells ChIP-seq Chromatin Congenital Abnormality Data Defect Embryo Epithelial Epithelial Cells Excision Eye Development FGF2 gene Fiber Fibroblast Growth Factor Fibroblast Growth Factor Receptors Future Gene Expression Genes Genetic Transcription Glucagon Homeobox In Vitro Investigation Islets of Langerhans Knock-out Knockout Mice Lens Fiber Lens development Letters Liquid substance Mediator of activation protein Messenger RNA Mission Molecular Mus Nervous system structure Neural tube Newborn Infant Organ PTEN gene Pancreas Pattern Play Process Proteins Public Health Publishing Receptor Signaling Regulator Genes Reporting Research Role Systems Biology Testing Tissues Transcript Transgenes United States National Institutes of Health Vitreous humor cell type developmental disease differential expression endocrine pancreas development experimental study fiber cell in vivo innovation insight lens nervous system disorder neural patterning overexpression prenatal prevent promoter response restoration transcription factor transcriptome transcriptome sequencing

项目摘要

Despite abundant evidence for the centrality of FGF receptor (FGFR) signaling to normal fiber cell differentiation, our understanding of the key downstream target genes that carry out the differentiation process in response to FGFR stimulation remains incomplete. An innovative, combined use of RNA-Seq and systems biology approaches identified Nkx6-1, a homeobox transcription factor, as a potential key mediator of FGFR-induced lens fiber cell differentiation. In support of this argument, Nkx6-1 mRNA is expressed 70x more abundantly in lens fiber cells than in lens epithelial cells. Nkx6-1 is also co-expressed with the fundamental lens transcription factor, Pax6 within the neural tube and pancreas and these two transcription factors coordinately pattern different cell types in these tissues. These discoveries led to the hypothesis that FGF receptor signaling induces the expression of Nkx6-1 to promote secondary fiber cell differentiation, in part by antagonizing PAX6 during the epithelial to fiber cell transition. This hypothesis will be tested by specifically removing Nkx6-1 during lens development and characterizing any resultant defects in lens development. If lens development is compromised in Nkx6-1 deficient lenses, a transcriptome analysis will determine which transcripts are deregulated. Testing the hypothesis will also utilize post-natal lens epithelial explants where effects of FGFR loss on prenatal lens cell survival can be separated the effects of FGFR loss on fiber cell differentiation. Lens explants lacking FGFRs lose the ability to undergo fiber cell differentiation in response to either FGF or vitreous humor. However, the removal of both FGFRs and PTEN restores vitreous humor-induced fiber cell differentiation in lens epithelial explants. The requirement of NKX6-1 for this restoration of vitreous humor-induced fiber cell differentiation in lens explants lacking both FGFRs and PTEN will be tested. It will also be determined if overexpression of Nkx6- 1 restores vitreous humor-induced lens fiber cell differentiation in lens epithelial explants lacking only FGFRs. Several experiments will also test for antagonism between NKX6-1 and PAX6 during lens fiber cell differentiation. The effect of Nkx6-1 loss on the expression pattern of PAX6 during lens development will be determined. The expression of PAX6-regulated transcripts will also be tested in lens epithelial explants that either lack or overexpress NKX6-1 in response to vitreous humor stimulation. ChIP-Seq analysis will determine which gene regulatory sequences are directly bound by NKX6-1 in the lens and combined with the RNA-Seq data to determine the genes which NKX6-1 directly regulates. The resulting data will also be compared with previously published lens ChIP-Seq data from lens to determine which genes share binding sites for both PAX6 and NKX6- 1. These studies will provide the first comprehensive investigation of the role of NKX6-1 during lens development and will both definitively show if NKX6-1 is an essential mediator of FGFR-induced fiber cell differentiation and set the stage for future studies to elucidate the molecular mechanisms NKX6-1 uses to coordinate lens development.

尽管有足够的证据表明FGF受体（FGFR）信号的中心性向正常纤维细胞分化，但我们对以下分化过程的关键下游目标基因的理解 FGFR刺激仍然不完整。 RNA-seq和系统生物学的创新，联合使用方法确定了NKX6-1，一种同源词转录因子，是FGFR诱导的潜在关键介体透镜纤维细胞分化。为了支持这一论点，NKX6-1 mRNA在晶状体纤维细胞比晶状体上皮细胞。 NKX6-1也与基本镜头转录共表达因子，神经管和胰腺内的PAX6，这两个转录因子协同模式不同这些组织中的细胞类型。这些发现导致了以下假设：FGF受体信号传导诱导 NKX6-1的表达以促进次级纤维细胞分化，部分是通过对PAX6进行拮抗上皮到纤维细胞过渡。该假设将通过在镜头期间专门删除NKX6-1来检验开发和表征晶状体开发中的任何结果缺陷。如果镜头的开发受到损害在NKX6-1缺陷镜头中，转录组分析将确定哪些转录本被放松管制。测试假设还将利用产后晶状体上皮外植体，其中FGFR损失对产前晶状体细胞的影响可以将FGFR损失对纤维细胞分化的影响分开。缺乏FGFR的镜头外植体因FGF或玻璃体幽默而失去经历纤维细胞分化的能力。但是，去除FGFR和PTEN可恢复镜片上皮的玻璃体诱导的纤维细胞分化 epplants。 NKX6-1的要求恢复了玻璃体幽默诱导的纤维细胞分化缺乏FGFR和PTEN的镜头外植体将进行测试。还将确定NKX6-的过表达是否 1恢复缺乏FGFR的晶状体上皮外植体中玻璃体诱导的透镜纤维细胞分化。在晶状体纤维细胞分化过程中，NKX6-1和PAX6之间的拮抗作用还将测试一些实验。 NKX6-1损失对晶状体发育过程中PAX6表达模式的影响。这 PAX6调节的成绩单的表达也将在晶状体上皮外植体中进行测试，即缺乏或响应玻璃体幽默刺激的过表达NKX6-1。 CHIP-SEQ分析将确定哪种基因调节序列在镜头中直接由NKX6-1结合，并与RNA-Seq数据结合到确定NKX6-1直接调节的基因。结果数据也将与以前的从镜头发表的透镜芯片seq数据确定哪些基因共享PAX6和NKX6-的结合位点 1。这些研究将对NKX6-1在晶状体开发中的作用进行首次全面研究并且都将明确地表明NKX6-1是否是FGFR诱导的纤维细胞分化和为将来的研究奠定了阶段，以阐明NKX6-1用于协调晶状体的分子机制发展。