Hair Cell Development in the Mammalian Cochlea

听觉和前庭功能依赖于功能性内耳的形成。虽然这两个系统都有多个组成部分，但本实验室专注于感觉上皮的发育，其中包含机械感觉毛细胞和相关细胞，称为支持细胞，以及来自VIIIth(听前庭)脑神经的神经元对这些毛细胞的神经支配。这三种细胞类型都来自耳囊，耳囊是一种胎盘结构，在发育早期形成于后脑附近。确定指定每一种细胞类型的因素，然后将它们组装成功能单位是发育神经科学科的一个关键目标。在过去的一年里，实验室的不同成员检查了这些发育过程的几个不同方面。 随着所有剩余的COVID限制的取消，实验室的生产力在很大程度上恢复到大流行前的水平。 2023年11月，一个使用单细胞RNA测序来描述螺旋神经节神经元发育的项目发表在了PNAS上。这项研究使用单细胞RNA测序来探索当神经元在螺旋神经节内发育时发生的转录变化。通过收集不同时间点的发育中神经元，然后使用单细胞RNA-Seq生成转录图谱，我们能够组装出存在于功能螺旋神经节中的四种神经元亚型的每一种发育轨迹。此外，我们还能够确定可能影响不同亚型形成的候选转录因子。作为下一步，我们现在正在开发/生成针对其中几个因素的条件小鼠突变体。初步数据表明，在我们的单细胞研究中发现的Tle4基因的缺失，会导致具有几种已知表型的神经元比例下降，这表明该因子在这些特定细胞表型的特异性中发挥作用。 2023年7月，我们发表了一项研究结果，该研究探讨了富含亮氨酸重复序列的神经元1(Lrrn1)在耳蜗感觉上皮构型中的作用。用单细胞RNAseq对耳蜗发育的分析发现，Lrrn1表达在内耳感觉上皮的一侧。为了研究Lrrn1的作用，我们产生了Lrrn1突变小鼠。对他们耳朵的分析表明，耳朵内的细胞模式发生了破坏。在随后的研究中，我们证明了Lrrn1的作用是增强位于感觉上皮附近的细胞中Notch1的激活，防止它们成为毛细胞。 POU4F3是一种人类耳聋基因，据报道，它是毛细胞生存所必需的。然而，实验室的一名研究员发现，Pou4f3突变小鼠的前庭系统中仍有一些毛细胞。为了确定这些细胞与野生型细胞的不同之处，我们使用单细胞RNAseq来比较野生型毛细胞和Pou4f3阴性毛细胞。结果表明，Pou4f3阴性细胞停滞在未成熟状态。这为利用基因疗法恢复前庭毛细胞的正常功能提供了潜在的机会。我们正在使用病毒载体在Pou4f3突变小鼠中重新表达Pou4f3，以验证这一假设。 转录抑制因子SALL1的变异会导致人类的汤斯-布罗克斯综合征，包括听力损失。SALL1是SALL1-4基因家族中的一员，其中SALL1、2和3在耳蜗组织中表达。为了更好地了解SAL基因在内耳功能中的作用，我们正在为不同的SAL基因产生单一和复合突变体。 转录因子Prox1只在耳蜗区表达，该区域将产生外毛细胞。为了确定Prox1的作用，我们在耳蜗内对该基因进行了有条件的缺失。对听觉功能的评估表明，这些小鼠有明显的听力损失，而表型分析表明成年小鼠毛细胞丢失。我们将使用单细胞RNAseq来探索这些缺陷背后的转录变化。 在与外部研究人员的合作中，我们对发育中的耳蜗核进行了转录图谱分析，并检查了内耳器官中产生的神经元和内源性螺旋神经节神经元之间的转录相似性。最后，与Michael Burger博士合作研究外周输入对听觉中枢神经系统结构的影响，最终在2022年将R01授予Burger博士。由于Burgers博士实验室的员工流动率，我们现在才刚刚开始推进这些合作实验。

Auditory and vestibular function are dependent of the formation of a functional inner ear. While there are multiple components for both of these systems, this laboratory focuses on the development of the sensory epithelia, which contain mechanosensory hair cells and associated cells called supporting cells and on the innervation of those hair cells by neurons from the VIIIth (acousticovestibular) cranial nerve. All three of these cell types are derived from the otocyst, a placodal structure that forms adjacent to the hindbrain early in development. Identifying the factors that specify each of these cell types and then direct their assembly into functional units is a key goal of the Section on Developmental Neuroscience. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. With the lifting of all remaining COVID restrictions, productivity in the laboratory has largely returned to pre-pandemic levels. A project using single cell RNA sequencing to profile the development of spiral ganglion neurons was published in P.N.A.S. in November of 2023. This study used single cell RNAsequencing to explore the transcriptional changes that occur as neurons develop within the spiral ganglion. By collecting developing neurons at different time points and then using single cell RNA-Seq to generate transcriptional profiles, we were able to assemble a developmental trajectory for each of four neuronal subtypes that are present in the functioning spiral ganglion. In addition, we were able to identify candidate transcription factors that may influence the formation of different subtypes. As a next step, we are now developing/generating conditional mouse mutants for several of these factors. Preliminary data suggest that deletion of Tle4, a gene that was identified in our single cell study, leads to a decrease the percentage of neurons that develop with several of the known phenotypes, suggesting that this factor plays a role in specific of those specific cellular phenotypes. In July, 2023, we published the results of a study examining the role of Leucine Rich Repeat Neuronal 1 (Lrrn1) in patterning of the cochlear sensory epithelium. Analysis of cochlear development using single cell RNAseq had identified Lrrn1 as being expressed at one edge of the inner ear sensory epithelium. To examine the role of Lrrn1, we generated Lrrn1 mutant mice. Analysis of their ears indicated a disruption in cellular patterning within the ear. In subsequent studies we demonstrated that Lrrn1 acts to enhance activation of Notch1 in cells located adjacent to the sensory epithelium, preventing them from becoming hair cells. POU4F3 is a human deafness gene that has been reported to be required for hair cell survival. However, one of the Research Fellows in the laboratory discovered that some hair cells persist in the vestibular system of Pou4f3 mutant mice. To determine how these cells differ from wild type cells, we are using single cell RNAseq to compare wild type and Pou4f3 negative hair cells. Results indicate that Pou4f3 negative cells are arrested in an immature state. This provides a potential opportunity to use gene therapy to restore normal function to vestibular hair cells. We are in the process of testing this hypothesis using viral vectors to re-express Pou4f3 in Pou4f3 mutant mice. Variants in Sall1, a transcriptional repressor, cause Townes-Brocks syndrome in humans which includes hearing loss. Sall1 is a member of a family of genes, Sall1-4 of which Sall1,2 and 3 are expressed in the cochlea. To better understand the role of the Sall genes in inner ear function, we are generating single and compound mutants for the different Sall genes. The transcription factor Prox1 is expressed only in the region of the cochlea that will give rise to outer hair cells. To determine the role of Prox1 we generated a conditional deletion in this gene within the cochlea. Assessment of auditory function indicates significant hearing loss in these mice while phenotypic analysis indicates a loss of hair cells in adult mice. We will use single cell RNAseq to explore transcriptional changes underlying these defects. In collaborations with external researchers, we have performed transcriptional profiling of the developing cochlear nucleus and examined the transcriptional similarities between neurons generated in inner ear organoids and endogenous spiral ganglion neurons. Finally, an existing collaboration with Dr. Michael Burger to examine effects of peripheral inputs on the structure of the auditory CNS resulted in the awarding of an R01 to Dr. Burger in 2022. Because of employee turnover in Dr. Burgers lab, we are just now beginning to move forward with these collaborative experiments.