Early Functions of Tbx2 and Tbx3 in inner ear development

摘要： 成熟的内耳负责哺乳动物的前庭和听觉。一个简单的耳囊 (OV)产生所有成熟的内耳结构。然而，关于控制细胞命运的基因控制 复杂的内耳结构的形态发生所需的决定是未知的。有三个T-box 转录因子基因Tbx1、Tbx2和Tbx3在卵子中表达。使用Pax2-Cre/+，I灭活 Tbx2和/或Tbx3等位基因和I在Tbx2条件性基因敲除中发现耳蜗囊缺陷 (Tbx2cKO)胚胎、半规管缺陷症(Tbx3)条件基因敲除(Tbx3cKO)胚胎及完全性 Tbx2/3cKO双突变胚胎中卵子不能发生形态发生。从这些初步的 结果提示Tbx2和Tbx3在内耳发育中具有独特而共有的功能。我有过 使用NeuroD1(神经源性分化1)生成了初步的RNAScope原位杂交数据， 标记神经元前体，Bmp4(骨形态发生蛋白4)，标记感觉前体细胞和OTX2 (Orthodenticle Homeobox 2)，标记非感觉前体细胞。我们发现了一个异位结构域 NeuroD1在OV后部的表达，Bmp4在后区的表达扩大，以及 Tbx2/3cKO胚胎腹侧区OTX2表达降低。我们的实验室之前发现 TBX1的失活导致OV的形态发生失败，神经源性结构域扩展到 后卵泡，如Tbx2/3cKO胚胎。根据这些表达式的变化，我们假设Tbx2和 Tbx3在限制感觉和神经细胞命运和促进非感觉细胞方面具有早期共享功能 命运。在目标1中，我将仔细确定Tbx2和Tbx3的时空表达模式，并将进一步 评估单个和组合Tbx2和Tbx3突变胚胎的表型以更好地了解 与这两个基因的表达有关的内耳缺陷发生的时间和地点。在《目标2》中，我会选一个 确定Tbx1、Tbx2和Tbx3在遗传中的相对位置的候选基因方法 细胞命运的层级决定形成神经元、感觉或非感觉的命运。我还将区分 细胞命运的变化和细胞群体的变化。综上所述，这个项目将阐明早期共享的 Tbx2和Tbx3的功能，并与已建立的Tbx1、Eya1(EYA转录共激活因子)进行比较 和磷酸酶1)和SIX1(Sine Oculis Homeobox Homolog 1)途径，都是形态发生所必需的 内耳。

Abstract: The mature inner ear is responsible for the vestibular and auditory senses in mammals. A simple otic vesicle (OV) gives rise to all mature inner ear structures. However, much about the genetic controls regulating cell fate decisions required for morphogenesis of the complex inner ear structures are unknown. There are three T-box transcription factor genes, Tbx1, Tbx2 and Tbx3 expressed in the OV. With the use of Pax2-Cre/+, I inactivated Tbx2 and/or Tbx3 floxed alleles and I uncovered cochlear and saccular defects in Tbx2 conditional knockout (Tbx2cKO) embryos, semicircular canal defects in Tbx3 conditional knockout (Tbx3cKO) embryos and complete failure of the OV to undergo morphogenesis in Tbx2/3cKO double mutant embryos. From these preliminary results, I suggest that Tbx2 and Tbx3 have unique and shared functions in inner ear development. I have generated preliminary RNAscope in situ hybridization data using NeuroD1 (Neurogenic Differentiation 1) that marks neuronal precursors, Bmp4 (Bone Morphogenetic Protein 4) that marks sensory precursor cells and Otx2 (Orthodenticle Homeobox 2) that marks non-sensory precursor cells. We identified an ectopic domain of NeuroD1 expression in the posterior OV, an expansion of the Bmp4 expression in the posterior domain and reduction of Otx2 expression in the ventral domain in Tbx2/3cKO embryos. Our lab previously found that inactivation of Tbx1 results in failed morphogenesis of the OV with expansion of the neurogenic domain to the posterior OV, as in Tbx2/3cKO embryos. Based upon these expression changes, we hypothesize that Tbx2 and Tbx3 have early shared functions in restricting sensory and neuronal cell fates and promoting non-sensory cell fates. In Aim 1, I will carefully determine the spatiotemporal expression patterns of Tbx2 and Tbx3 and will further assess phenotypes of individual and combined Tbx2 and Tbx3 mutant embryos to gain a better understanding of when and where inner ear defects arise with respect to expression of the two genes. In Aim 2, I will take a candidate gene approach to determine the relative position as to where Tbx1, Tbx2 and Tbx3 fit into the genetic hierarchy of cell fate decisions to form the neuronal, sensory or non-sensory fates. I will also distinguish between changes in cell fate and changes in cell populations. Taken together, this project will elucidate the early shared functions of Tbx2 and Tbx3, and compare that with established Tbx1, Eya1 (EYA Transcriptional Coactivator And Phosphatase 1), and Six1 (Sine Oculis Homeobox Homolog 1) pathways, all required for morphogenesis of the inner ear.