Pilot investigation: expression of Phox2 genes in neurons associated with the lateral line system in shark embryos

脊椎动物是如何进化的？我们与脊椎动物联系在一起的许多特征，比如有头骨的头和特殊的感觉器官（一对有晶状体的眼睛，有毛细胞的耳朵，用于听力和平衡，鼻子用于嗅觉），都来自两个胚胎细胞群。神经嵴构成了大部分的头骨，以及大部分的外周神经元（大脑/脊髓外）。头盖骨是胚胎头部的增厚皮肤斑块，形成了成对的特殊感觉器官和头部的大多数外周感觉神经元。在鱼类和两栖动物中，另一组感觉毛细胞，就像内耳中的毛细胞一样，在头部和身体表面呈直线排列：它们检测水的运动和电磁场。这种侧线系统起源于侧线基序；它对探测猎物和躲避障碍物很重要。神经嵴和颅基构成了脊椎动物身体结构的许多独特特征，因此它们的进化对脊椎动物的进化至关重要。它们被认为是随着脊椎动物祖先从相当被动的滤食性生活方式进化到主动的掠食性生活方式而进化的。这是一个试点项目，旨在测试关于侧线系统最早进化的假设：它的进化是否不是为了远距离接收（它今天的功能），而是作为控制无意识身体（内脏）功能的神经元反射回路的一部分。来源于鳃外PLACODES的神经元将味蕾和其他器官（如心脏和肺）内的感觉受体的信息传递给大脑，告诉大脑需要什么样的反射动作，例如呼吸频率是否需要加快。值得注意的是，这种“内脏反射回路”中的所有神经元都需要Phox2b转录因子（一种开启或关闭特定基因的蛋白质）才能正常形成。不仅鳃外基板衍生的感觉神经元需要Phox2b（以及它的近亲Phox2a），它们在大脑中与之交流的神经元以及与腺体和平滑肌交流的神经元也需要Phox2b。有趣的是，Phox2a在斑马鱼的侧线感觉神经元中也有表达，而Phox2b是小鼠大脑中神经元的形成所需要的，这些神经元向内耳毛细胞发送信息。在鱼类中，这些神经元也与侧线毛细胞对话。依赖phox2的内脏反射系统和侧线系统之间是否存在胚胎/进化上的联系？前者的感觉神经元来源于鳃外基板，后者的毛细胞和感觉神经元来源于侧线基板。如果侧线神经元是依赖于phox2b的，就像鳃外基质来源的神经元一样，那么也许它们最初是作为依赖phox2的内脏反射回路的感觉臂的一部分进化而来的？在这个试点项目中，我们的目标是了解Phox2基因是否在侧线感觉神经元中表达，它们在大脑中与神经元对话，以及大脑中与侧线毛细胞对话的神经元。我们将使用鲨鱼胚胎进行这些实验，看看Phox2在侧线相关神经元中的表达是一种古老的脊椎动物特征，还是在斑马鱼等现代硬骨鱼中进化出来的新东西。如果Phox2基因在侧线相关神经元中不表达，那么我们就放弃了我们的假设。然而，如果它们是，我们将建议研究Phox2基因在侧线系统中的作用，它们是如何在侧线与内脏反射相关的细胞中打开的，以及它们自己在侧线与内脏反射神经元中打开/关闭的基因。这将增加我们对这些显著的转录因子的理解，阐明侧线和鳃外基板之间的关系，并提供对颅基板（从而脊椎动物）进化的见解。然而，首先我们需要发现Phox2基因是否在侧线相关神经元中表达。

How did vertebrates evolve? Many of the features we associate with vertebrates, such as a head with a skull and special sense organs (paired eyes with lenses for acute vision, ears with hair cells for hearing and balance, nose for smell), arise from two embryonic cell populations. The NEURAL CREST makes much of the skull, and most peripheral neurons (outside the brain/spinal cord). CRANIAL PLACODES are patches of thickened skin on the embryonic head that make paired special sense organs and most peripheral sensory neurons in the head. In fish and amphibia, another set of sensory hair cells, like those in the inner ear, are arranged in lines over the surface of the head and body: these detect water movements and electromagnetic fields. This LATERAL LINE SYSTEM arises from LATERAL LINE PLACODES; it is important for detecting prey and avoiding obstacles. Neural crest and cranial placodes make many of the unique features of vertebrate body plans, so their evolution was crucial for vertebrate evolution. They are thought to have evolved as the vertebrate ancestor moved from a fairly passive, filter-feeding existence to an active predatory lifestyle. This is a pilot project to test a hypothesis about the earliest evolution of the lateral line system: whether it might have evolved not for distance reception (its function today), but instead as part of the neuronal reflex circuits that control unconscious bodily (visceral) functions. Neurons derived from the EPIBRANCHIAL PLACODES send information to the brain from taste buds, and other sensory receptors inside organs such as the heart and lungs, telling the brain what reflex action is needed, e.g. whether the breathing rate needs to go up. Remarkably, all neurons in such 'visceral reflex circuits' need the Phox2b transcription factor (a protein that turns specific genes on or off) to form properly. Not only do the epibranchial placode-derived sensory neurons need Phox2b (and its close relative Phox2a): so do the neurons they talk to in the brain, and the neurons that talk back to glands and smooth muscle. Interestingly, Phox2a is also expressed in lateral line sensory neurons in zebrafish, while Phox2b is needed for the formation of neurons in the mouse brain that send information to inner ear hair cells. In fish, these neurons also talk to lateral line hair cells. Is there an embryonic/evolutionary link between the Phox2-dependent visceral reflex system, whose sensory neurons arise from epibranchial placodes, and the lateral line system, whose hair cells and sensory neurons arise from lateral line placodes? If lateral line neurons are Phox2b-dependent, like epibranchial placode-derived neurons, then perhaps they first evolved as part of the sensory arm of Phox2-dependent visceral reflex circuits? In this pilot project, we aim to learn whether Phox2 genes are expressed in lateral line sensory neurons, the neurons they talk to in the brain, and the neurons in the brain that talk back to the lateral line hair cells. We will use shark embryos for these experiments, to see if Phox2 expression in lateral line-associated neurons is an ancient vertebrate trait, or something new that evolved in modern bony fish like zebrafish. If Phox2 genes are not expressed in lateral line-associated neurons, then we discard our hypothesis. However, if they are, we would then propose to investigate the role of Phox2 genes in the lateral line system, how they are switched on in lateral line vs visceral reflex-associated cells, and what genes they themselves turn on/off in lateral line vs visceral reflex neurons. This would increase our understanding of these remarkable transcription factors, illuminate the relationship between lateral line and epibranchial placodes, and provide insight into cranial placode (and thus vertebrate) evolution. However, first we need to find out if Phox2 genes are expressed in lateral line-associated neurons.