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

尽管有大量证据表明FGF受体（FGFR）信号传导对正常纤维细胞分化具有中心作用， 我们对关键的下游靶基因的理解，这些基因在应答中进行分化过程， FGFR刺激仍然不完全。RNA-Seq和系统生物学的创新组合使用 方法鉴定了同源框转录因子Nkx 6 -1作为FGFR诱导的细胞凋亡的潜在关键介质。 透镜纤维细胞分化。为了支持这一论点，Nkx 6 -1 mRNA在哺乳动物中的表达量是对照组的70倍。 透镜纤维细胞比透镜上皮细胞。Nkx 6 -1还与基础透镜转录共表达 在神经管和胰腺中，Pax 6和Pax 6这两种转录因子协同作用， 这些组织中的细胞类型。这些发现导致了FGF受体信号转导诱导细胞凋亡的假设。 Nkx 6 -1的表达促进次级纤维细胞分化，部分是通过拮抗PAX 6， 上皮细胞向纤维细胞的转变。将通过在透镜植入过程中专门去除Nkx 6 -1来检验该假设。 显影和表征透镜显影中任何产生的缺陷。如果透镜显影受损 在Nkx 6 -1缺陷的晶状体中，转录组分析将确定哪些转录物被解除调节。测试 该假说还将利用出生后透镜上皮外植体，其中FGFR损失对出生前透镜细胞的影响 存活率可以与FGFR损失对纤维细胞分化的影响分开。缺少FGFR的透镜外植体 丧失了响应FGF或玻璃体液而进行纤维细胞分化的能力。但 去除FGFR和PTEN恢复玻璃体液诱导的透镜上皮纤维细胞分化 外植体NKX 6 -1对玻璃体液诱导的纤维细胞分化的恢复的需要是在玻璃体液中表达的。 将测试缺乏FGFR和PTEN的透镜外植体。还将确定Nkx 6-Nkx 7的过表达是否与Nkx 6-Nkx 7的表达相关。 1在仅缺乏FGFR的透镜上皮外植体中恢复玻璃体液诱导的透镜纤维细胞分化。 几个实验还将测试在透镜纤维细胞分化期间NKX 6 -1和PAX 6之间的拮抗作用。 将确定透镜发育期间Nkx 6 -1缺失对PAX 6表达模式的影响。的 PAX 6调节的转录物的表达也将在缺乏或缺乏PAX 6的透镜上皮外植体中进行测试。 过度表达NKX 6 -1，以响应玻璃体液刺激。ChIP-Seq分析将确定哪个基因 调节序列直接与透镜中的NKX 6 -1结合，并与RNA-Seq数据结合， 确定NKX 6 -1直接调节的基因。由此产生的数据也将与以前的数据进行比较。 已发表的来自透镜的透镜ChIP-Seq数据，以确定哪些基因共享PAX 6和NKX 6的结合位点。 1.这些研究将首次全面调查NKX 6 -1在透镜开发过程中的作用 并将明确显示NKX 6 -1是否是FGFR诱导的纤维细胞分化的重要介质， 为进一步阐明NKX 6 -1用于协调透镜的分子机制奠定了基础 发展

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.