Axial patterning in the vertebrate inner ear: the role of Hedgehog signalling

内耳对于平衡感和听觉非常重要。它由一个复杂的充满液体的迷宫组成，里面有各种非常敏感的感觉结构，可以对声音，运动和重力做出反应。为了正确的内耳功能，这些组件中的每一个都必须在正确的位置形成，因为任何干扰都可能导致耳聋或平衡障碍。事实上，先天性耳聋是一个重要的临床问题，大约每千名出生儿童中就有一名受到影响。我们的目标是了解内耳在胚胎中是如何发育的，以及确保耳朵中不同类型的细胞出现在正确位置的机制，以便它们能够准确地发挥作用。我们在研究中使用了一种小型热带鱼--斑马鱼的胚胎，因为它是研究脊椎动物内耳发育的极好模型。重要的是，斑马鱼拥有一个内耳，在大多数方面与其他脊椎动物（如我们）非常相似，胚胎是透明的，这意味着内耳-即使是一个内部器官-可以在活的生物体中可视化。此外，斑马鱼是一种强大的遗传有机体，这意味着我们可以专门破坏单个基因以发现它们的功能。存在许多不同的斑马鱼品系，其中耳朵发育有特定的缺陷，或者其中不同的细胞类型用荧光染料标记，这些可以用来识别正确形成耳朵所需的关键基因。此外，胚胎丰富，容易获得，易于操作。在这个项目中，我们将专注于了解刺猬（Hh）蛋白对耳朵发育的影响。Hh蛋白质是一种信号分子，它可以给胚胎细胞发出指令，告诉它们如何发育以及在哪里发育。已知Hh在区分耳朵的一个区域与另一个区域方面具有关键和早期作用，但其确切功能尚未完全了解。特别是，它似乎在斑马鱼和哺乳动物中起着不同的作用：在鱼类中，它主要调节耳中前后（头到尾）轴的发育，而在哺乳动物中，它的主要作用是调节背腹（背到腹部）轴，该轴垂直于前后轴。这是令人惊讶的，因为成年动物头部的内耳方向和大多数内耳结构在两组之间非常相似。哺乳动物和鱼类的耳朵发育机制真的如此不同吗？回答这个问题很重要，因为斑马鱼被广泛用作人类听力和耳聋的模型。我们的初步研究表明，Hh在小鼠和斑马鱼之间的作用比最初出现的更相似。特别是，我们的研究表明，Hh信号也参与区分背侧和腹侧区域的斑马鱼内耳。我们的目标是确认这一点，使用一个新的和独特的面板斑马鱼突变体。在这些鱼中，编码Hh信号抑制剂的基因的功能被破坏，这意味着所有细胞现在都经历高水平的这种信号分子。我们将利用这些突变体来探索斑马鱼耳背腹图案形成的机制。特别是，我们的目标是确定Hh在耳朵发育过程中的确切作用时间，以及它是否与第二组信号分子（Wnt家族的信号分子）一起作用于耳朵。我们还将测试Hh是否在小鼠胚胎发育中的内耳中具有额外的AP模式作用。这项工作将导致更好地了解内耳如何在脊椎动物胚胎中发育，提供重要的基础知识，有助于为临床研究提供信息，以了解导致人类耳聋的许多遗传条件。

The inner ear is enormously important for the senses of balance and hearing. It consists of an intricate fluid-filled labyrinth housing a variety of extraordinarily sensitive sensory structures that respond to sound, movement and gravity. For correct inner ear function it is essential that each of these components form in exactly the right place, as any disturbance can lead to deafness or balance disorders. Indeed, congenital deafness is an important clinical problem, affecting approximately one in every thousand children at birth. Our aim is to understand how the inner ear develops in the embryo, and the mechanisms that ensure that the different cell types in the ear arise in the correct positions so that they can function accurately. We use embryos of a small tropical fish, the zebrafish, in our research, as it is a superb model for the study of vertebrate inner ear development. Importantly, zebrafish possess an inner ear that is very similar in most respects to those of other vertebrates like ourselves, and the embryo is transparent, meaning that the inner ear - even though an internal organ - can be visualised in the live organism. Moreover, the zebrafish is a powerful genetic organism, meaning that we can disrupt individual genes specifically to discover their function. Many different lines of zebrafish exist in which the ear develops with specific defects, or in which different cell types are marked with fluorescent dyes, and these can be used to identify key genes that are required for correct formation of the ear. In addition, embryos are abundant, easily available and amenable to manipulation. In this project, we will focus on understanding the effects of Hedgehog (Hh) proteins on ear development. Hh proteins are signalling molecules that give instructions to cells in the embryo, telling them how and where to develop. It is known that Hh has a crucial and early role in distinguishing one region of the ear from another, but its exact function is not fully understood. In particular, it does not seem to play the same role in zebrafish and mammals: in fish, it primarily regulates development of the anterior-posterior (head to tail) axis in the ear, whereas in mammals, its primary role is in regulation of the dorsal-ventral (back to belly) axis, which is perpendicular to the anterior-posterior axis. This is surprising, as the orientation of the inner ear in the head of the adult animal, and the majority of inner ear structures, are very similar between the two groups. Are mechanisms of ear development really so different in mammals and fish? It is important to answer this question, as the zebrafish is widely used as a model for human hearing and deafness. Our preliminary investigations now suggest that the role of Hh is more similar between mouse and zebrafish than it first appears. In particular, our studies indicate that Hh signalling is also involved in distinguishing between dorsal and ventral regions of the zebrafish inner ear. We aim to confirm this using a novel and unique panel of zebrafish mutants. In these fish, the function of genes that code for inhibitors of Hh signalling is disrupted, meaning that all cells now experience high levels of this signalling molecule. We will use these mutants to explore the mechanisms involved in formation of dorsal-ventral patterning in the zebrafish ear. In particular, we aim to establish exactly when Hh is acting during ear development, and whether it acts together with a second group of signalling molecules, those of the Wnt family, in patterning the ear. We will also test whether Hh has additional AP patterning roles in the developing inner ear of the mouse embryo. This work will lead to a greater understanding of how the inner ear develops in the vertebrate embryo, providing important basic knowledge that will help to inform clinical research into the many genetic conditions that lead to deafness in humans.