Evolutionary development of excitatory projection neurons in tetrapod sound localization circuits

声音定位是听觉的一个基本方面。在四足动物（两栖动物、爬行动物、鸟类和哺乳动物）中，声音定位线索的处理首先发生在后脑的神经回路中。关于这些电路的一些最重要的问题与它们的发展和进化有关。真兽哺乳动物的后脑有三个声音定位回路。在这些细胞中，主要的投射神经元是VGluT2+细胞。基于共同的胚胎起源，细胞形态，以及神经支配和投射模式，我最近提出它们代表具有修饰的系列同源物。它们的分子关系和控制它们分化的遗传程序目前尚不清楚。关于四足动物的声音定位回路的进化，普遍的观点是它们是独立出现的，代表了趋同进化的例子。这是基于这些回路之间的解剖和生理差异以及脊椎动物群体中耳道的独立进化。然而，最近的细胞命运分析表明，至少一些哺乳动物和鸟类的声音定位回路神经元，包括VGluT2+细胞，来自相同的转录因子谱系。因此，关于共同祖先或趋同还不能作出简单的陈述。在这里，我们将运用比较分子发育生物学和生物信息学来评估四足动物声音定位电路的发育和进化。在第一步中，将通过RNA-seq在P0和P25位点对小鼠听觉后脑中不同的VGluT2+神经元群进行转录组分析。P0代表中间调控状态，P25代表末端分化。表达谱将与另一个独立起源的VGluT2+群体在听觉后脑进行比较。随后，生物信息学分析将应用于数据集，构建试探性基因调控网络（grn）。这些数据将i)使我们了解哺乳动物声音定位电路发展的遗传程序，ii)确定驱动序列同源物向不同细胞功能的分化程序，iii)定义这些同源物的分子特征，以及iv)定义它们的遗传关系。在最后一步，将通过广泛的RNA原位杂交对小鼠和鸡的关键GRN成分进行全面的比较表征。四足动物声音定位回路中GRN成分的表达图谱（这是第一个）将作为评估和剖析其进化的基本分子数据驱动框架。总之，我们将通过探索哺乳动物和鸟类声音定位回路中的grn来应用进化发育生物学的一个基本概念。这将提供有关中枢听觉结构进化发展的关键信息。

Sound localization is a fundamental aspect in hearing. In tetrapods (amphibians, reptiles, birds, and mammals), processing of cues for sound localization first occurs in neuronal circuits within the hindbrain. Some of the most important questions regarding these circuits relate to their development and evolution. Eutherian mammals feature three sound localization circuits in the hindbrain. In all of them, the major projection neurons are VGluT2+ cells. Based on common embryonic origin, cell morphology, as well as innervation and projection patterns, I recently proposed them to represent serial homologs with modifications. Their molecular relationship and the genetic program governing their differentiation are currently unknown. Concerning the evolution of sound localization circuits across tetrapods, the prevailing view is that they have arisen independently and represent examples of convergent evolution. This is based on anatomical and physiological differences between these circuits and the independent evolution of tympanic ears in vertebrate groups. Yet, recent cell fate analyses suggest that at least some mammalian and avian neurons of sound localization circuits, including VGluT2+ cells, are derived from the same transcription factor lineages. Thus, no simple statements concerning shared ancestry or convergence can yet be made.Here, we will deploy comparative molecular developmental biology and bioinformatics to assess development and evolution of tetrapod sound localization circuits. In a first step, transcriptome analysis of the different VGluT2+ neuronal populations in the mouse auditory hindbrain will be performed by RNA-seq at P0 and P25. P0 represents an intermediate regulatory state and P25 terminal differentiation. Expression profiles will be compared with another VGluT2+ population of independent origin in the auditory hindbrain. Subsequently, bioinformatics analyses will be applied to the data sets to construct tentative gene regulatory networks (GRNs). These data will i) inform our understanding of the genetic programs underlying development of mammalian sound localization circuits, ii) identify the differentiation programs that drive serial homologs towards distinct cell functions, iii) define the molecular signatures of these homologs, and iv) define their genetic relationship. In a final step, a comprehensive comparative characterization of key GRN components in mouse and chicken will be performed by extensive RNA in situ hybridization. The resulting expression maps (the first of this kind) of GRN components across tetrapod sound localization circuits will serve as an essential molecular data-driven framework for assessing and dissecting their evolution. Altogether, we will apply a fundamental concept in evolutionary developmental biology by probing GRNs operating in sound localization circuits of mammals and birds. This will provide key information concerning the evolutionary development of central auditory structures.