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。一个简单的耳泡 (OV)形成了所有成熟的内耳结构然而，关于调节细胞命运的遗传控制， 复杂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Tbx 1、Tbx 2和Tbx 3在OV中表达。使用Pax 2-Cre/+，我灭活了 Tbx 2和/或Tbx 3 floxed等位基因和I在Tbx 2条件性敲除中发现耳蜗和囊状缺陷 Tbx 3条件性敲除（Tbx 3cKO）胚胎中的半规管缺陷，以及Tbx 2cKO胚胎中的半规管缺陷。 OV在Tbx 2/3cKO双突变体胚胎中不能经历形态发生。从这些初步的 结果表明，Tbx 2和Tbx 3在内耳发育过程中具有独特和共同的功能。我有 使用NeuroD 1（神经源性分化1）生成初步RNAscope原位杂交数据， 标志神经元前体，标志感觉前体细胞的Bmp 4（骨形态发生蛋白4）和Otx 2 （Orthodenticle Homeobox 2），标记非感觉前体细胞。我们发现了一个异位结构域， NeuroD 1在后节段的表达，Bmp 4在后节段表达的扩大， Tbx 2/3cKO胚胎腹侧区域Otx 2表达减少。我们的实验室此前发现， Tbx 1的失活导致OV的形态发生失败，神经源性结构域扩展到 后OV，如在Tbx 2/3cKO胚胎中。基于这些表达变化，我们假设Tbx 2和 Tbx 3具有早期共同的功能，即限制感觉和神经细胞的命运，促进非感觉细胞的分化。 命运在目标1中，我将仔细确定Tbx 2和Tbx 3的时空表达模式，并将进一步 评估单个和组合的Tbx 2和Tbx 3突变胚胎的表型，以更好地了解 内耳缺陷的发生时间和位置与这两种基因的表达有关。在目标2中，我将采取 候选基因方法，以确定Tbx 1、Tbx 2和Tbx 3在遗传中的相对位置 细胞命运决定的层次结构形成神经元、感觉或非感觉命运。我还将区分 细胞命运的变化和细胞群体的变化。总之，这个项目将阐明早期共享的 Tbx 2和Tbx 3的功能，并与已建立的Tbx 1，Eya 1（EYA转录辅激活因子 和磷酸酶1）和Six 1（Sine Oculis同源框同源物1）途径，这些途径都是视网膜色素瘤的形态发生所必需的。 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.