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

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.

This year-long pilot grant proposal aims to investigate whether or not the transcription factors Phox2b and Phox2a are expressed in the neurons of the lateral line system in shark embryos, in order to test our hypothesis that the lateral line system evolved as part of the afferent arm of Phox2b-dependent visceral reflex circuits that control autonomic functions. The lateral line system of fish and aquatic amphibians arises from a dorsolateral series of cranial lateral line placodes (thickened patches of head ectoderm) that migrate over the head and body to deposit lines of mechanosensory and electroreceptive lateral line hair cells. These are used in schooling, obstacle avoidance and prey detection. Lateral line placodes also form the sensory neurons in lateral line ganglia that provide afferent innervation for the hair cells. The transcription factors Phox2a and Phox2b are essential for the development of the autonomic nervous system. In particular, Phox2b is a pan-autonomic regulator essential for the formation of all neurons participating in the medullary reflex circuits that control autonomic functions. The afferent arm of these Phox2b-dependent circuits is provided by epibranchial placode-derived visceral sensory neurons in the geniculate, petrosal and nodose ganglia. Our hypothesis that the lateral line system was primitively part of the afferent arm of visceral reflex circuits is based on the expression of Phox2a in zebrafish lateral line ganglia, the dependence on Phox2b of inner ear efferent neurons (which are a subset of lateral line efferents in fish), and the fact that, in Xenopus, epibranchial placodes, lateral line (and otic) placodes arise from a common primordium. It is essential to investigate this question in a basal vertebrate taxon to confirm that expression of Phox2 genes in lateral line-associated neurons is primitive, rather than a derived character specific to euteleosts. The specific objectives and methodology of this pilot grant proposal are: (1) To isolate fragments of shark Phox2b and Phox2a cDNA using degenerate PCR on pooled cDNA from lesser-spotted dogfish (Scyliorhinus canicula) embryos or, if necessary, a low-stringency screen of a dogfish cDNA library. (2) To confirm the location in dogfish embryos of lateral line ganglia, their central targets, and lateral line efferent neurons, using immunohistochemistry and retrograde axonal labelling from lateral line neuromasts: lateral line efferents will be the only labelled cell bodies in the brain, while the medial octavolateral nucleus will appear as the central projection zone of lateral line afferents. (3) To establish whether Phox2 genes are expressed in dogfish lateral line ganglia, their central targets, neuromasts or neuromast precursors, and lateral line efferent neurons, using in situ hybridisation, and the information from Objective 2, if necessary in conjunction with retrograde axon labelling. If Phox2 genes are not expressed in lateral line-associated neurons in sharks, then our hypothesis receives no additional support (though we will have gathered valuable data on the development of lateral line-associated neurons in sharks and the comparative expression of Phox2 genes). However, if Phox2b, in particular, is expressed in all these neurons, this would provide additional support for the hypothesis, and the baseline data for a full research programme to investigate the role of Phox2 genes in the lateral line system, the mechanisms underlying their induction, and their downstream targets in the lateral line versus visceral neuronal circuits. This would shed further light on the importance of Phox2 genes, and on the relationship between lateral line and epibranchial placodes, yielding insights into cranial placode (and thus vertebrate) evolution. The experiments in this pilot grant are essential to determine whether or not the full research programme is worth pursuing.