FoxG1在小鼠耳蜗毛细胞发育及存活中的机制研究

发布时间：2018-04-29 23:33

本文选题：FoxG1 + 耳蜗　；参考：《东南大学》2016年博士论文

【摘要】：先天性听力缺陷是新生儿出生缺陷的重要方面,其发病率位居新生儿出生缺陷之首。听力损失主要分为感音神经性听力损失和传导性听力损失。目前研究表明,新生儿听力缺陷的病因主要与遗传因素尤其是耳聋相关基因突变、线粒体的遗传因素、外周的听神经病和引起传导性听力损失的疾病有关。因此对听觉相关基因功能和聋病的分子机制研究有重要的意义。FoxG1是Fox家族的一个重要成员,位于许多基因的上游,可通过与非编码序列的结合从而对多种转录因子的表达进行调控,所以FoxG1在早期神经发育中具有重要的调节作用。FoxG1与多种神经发育障碍性疾病有关,如Rett综合征、癫痫及小头畸形。在FoxG1全身性敲除小鼠中发现大脑皮质、端脑、耳、视网膜、嗅觉上皮细胞等组织的神经发育均受影响,并在出生后死亡。目前研究发现FoxG1全身性敲除小鼠的内耳发育出现严重畸形,如蜗管变短、毛细胞的排数增多、前庭缩小、轴突的生长和前庭神经元的定位出现异常、外骨壶腹缺失、毛细胞极性改变等。但是FoxG1全身性敲除是胚胎致死的,这就为研究FoxG1在出生后毛细胞发育及存活的相关机制带来了困难。我们通过Gficre小鼠与FoxG1loxp/loxp交配得到毛细胞条件性敲除FoxG1小鼠,避免了FoxG1对个体存活的影响,在此基础上进一步研究FoxG1基因在毛细胞发育及存活中发挥的功能,并对毛细胞发育相关通路因子进行检测,从而在毛细胞中对FoxG1的作用机制进行更加深入的研究。我们首先对Gficre小鼠的Cre酶活性及敲除效率进行了鉴定,结果显示Gfi-Cre可激活tdTomato的表达,同时对FoxG1loxp/-Gficre小鼠与FoxG1loxp/loxp小鼠交配所得子代耳蜗解剖后发现FoxG1被显著敲除。我们还发现在FoxG1CKO的新生鼠(p1、p7)的耳蜗顶圈及部分中圈外毛细胞出现多排分布,且这些多余的毛细胞会随着发育时间而逐渐减少。另外,我们发现FoxG1 CKO的小鼠在成年后听阈随着发育时间延长逐渐升高,并伴随着毛细胞的缺失,且缺失程度会随着小鼠年龄增长而逐渐加重,通过对不同时期耳蜗进行TUNEL染色发现FoxG1缺失后凋亡细胞会出现增加,凋亡因子的表达水平升高。我们对P1时期的小鼠进行了 RNAseq分析,发现在FoxG1缺失后多个信号通路的表达出现异常,说明FoxG1对毛细胞发育的影响是通过多个信号通路的协同调控实现的,其中Notch信号通路因FoxG1的缺失而被显著抑制,因此导致细胞的过度分化,这也是引起毛细胞增多的原因之一。此外,IGF、Wnt、EGF等信号通路相关基因表达也受到了抑制,说明FoxG1的正常表达对毛细胞的存活是非常重要的,FoxG1缺失后毛细胞的凋亡敏感性增加。以上研究为深入研究耳蜗发育机制及FoxG1基因的功能提供了新的线索及依据。
[Abstract]:Congenital hearing deficiency is an important aspect of newborn birth defect, and its incidence is the highest. Hearing loss includes sensorineural hearing loss and conductive hearing loss. Current studies have shown that the cause of neonatal hearing impairment is mainly related to genetic factors, especially deaf-related gene mutations, mitochondrial genetic factors, peripheral auditory neuropathy and diseases causing conductive hearing loss. Therefore, it is important to study the function of hearing related genes and the molecular mechanism of deafness. FoxG1 is an important member of the Fox family, located upstream of many genes. Expression of multiple transcription factors can be regulated by combining with non-coding sequences, so FoxG1 plays an important role in early neurodevelopment. FoxG1 is associated with many neurodevelopmental disorders, such as Rett syndrome. Epilepsy and microcephaly. It was found in FoxG1 knockout mice that the neural development of cerebral cortex, telencephalon, ear, retina and olfactory epithelial cells were all affected and died after birth. The present study has found that the inner ear of FoxG1 knockout mice has serious deformities, such as shorter cochlear canal, more hair cells, smaller vestibule, abnormal axon growth and location of vestibular neurons, and absence of external ampulla. Change of hair cell polarity, etc. However, systemic knockout of FoxG1 is the death of embryo, which makes it difficult to study the mechanism of hair cell development and survival in postnatal FoxG1. We obtained hair-cell conditioned knockout FoxG1 mice by mating with FoxG1loxp/loxp in Gficre mice, thus avoiding the effect of FoxG1 on individual survival. On this basis, we further studied the function of FoxG1 gene in hair cell development and survival. In order to study the mechanism of FoxG1 in hair cells, the factors related to the development of hair cells were detected. We first identified the Cre enzyme activity and knockout efficiency of Gficre mice. The results showed that Gfi-Cre could activate the expression of tdTomato. At the same time, the cochlear anatomy of the offspring of FoxG1loxp/-Gficre mice and FoxG1loxp/loxp mice showed that FoxG1 was significantly knocked out. We also found that the outer hair cells of the cochlea apical circle and part of the cochlear apical circle of the newborn mouse of FoxG1CKO were distributed in multiple rows, and the excess hair cells decreased gradually with the development time. In addition, we found that the hearing threshold of FoxG1 CKO mice gradually increased with the development time, accompanied by the loss of hair cells, and the degree of loss increased with the age of the mice. By TUNEL staining in cochlea at different stages, it was found that apoptosis cells increased and apoptotic factor expression increased after FoxG1 deletion. We analyzed the expression of multiple signal pathways after FoxG1 deletion by RNAseq analysis in P1 mice, indicating that the effect of FoxG1 on hair cell development was mediated by the co-regulation of multiple signal pathways. The Notch signaling pathway was significantly inhibited by the absence of FoxG1, which led to the over-differentiation of the cells, which was one of the reasons for the increase of hair cells. In addition, the expression of signal transduction related genes such as IGF- WntGF-EGF was also inhibited, indicating that the normal expression of FoxG1 is very important to the survival of hair cells, and the apoptosis sensitivity of hair cells increased after the deletion of FoxG1. These studies provide a new clue and basis for the further study of cochlear development mechanism and the function of FoxG1 gene.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R764

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