Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.

SUMMARYLeft-right (L-R) asymmetries in neuroanatomy exist throughout the animal kingdom, with implications for function and behavior. The molecular mechanisms that control formation of such asymmetries are beginning to be understood. Significant progress has been made by studying the zebrafish parapineal organ, a group of neurons on the left side of the epithalamus. Parapineal cells arise from the medially located pineal complex anlage and migrate to the left side of the brain. We have found that Fgf8a regulates a fate decision among anterior pineal complex progenitors that occurs just prior to the initiation of leftward migration. Cell fate analysis shows that in the absence of Fgf8a a subset of cells in the anterior pineal complex anlage differentiate as cone photoreceptors rather than parapineal neurons. Fgf8a acts permissively to promote parapineal fate in conjunction with the transcription factor Tbx2b, but might also block cone photoreceptor fate. We conclude that this subset of anterior pineal complex precursors, which normally become parapineal cells, are bipotential and require Fgf8a to maintain parapineal identity and/or prevent cone identity.

23Interactions between epithelial cells and neurons influence a range of sensory 24 modalities including taste, touch, and smell. Vertebrate and invertebrate 25 keratinocytes/keratinocyte-like epidermal cells ensheath peripheral arbors of 26 somatosensory neurons, including nociceptors, yet the developmental origins and 27 functional roles of this ensheathment are largely unknown. Here, we describe an 28 evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of 29 somatosensory neurites. We found that somatosensory neurons in Drosophila and 30 zebrafish induce formation of epidermal sheaths, which wrap neurites of different types 31 of neurons to different extents. Neurites induce formation of plasma membrane 32 phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a 33 filamentous actin network, and recruitment of junctional proteins that likely form 34 autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation 35 destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. 36Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular 37 process that contributes to the morphogenesis and function of nociceptive sensory 38 neurons. 39Recent findings that keratinocytes express sensory channels (Peier et al. 2002; Bidaux 53 et al. 2015; Y. Chen et al. 2016), respond to sensory stimuli (Koizumi et al. 2004; Xu et 54 al. 2006; Moehring et al. 2018), release compounds that modulate sensory neuron 55 function (Woolf et al. 1997; Koizumi et al. 2004; Moehring et al. 2018), and can drive 56 sensory neuron firing (Baumbauer et al. 2015; Pang et al. 2015), underscore the 57 importance of understanding the coupling of keratinocytes to sensory neurons. 58 Anatomical studies have demonstrated that peripheral arbors of some 59 mammalian somatosensory neurons insert into keratinocytes, not just intercalate 60 between them (Munger 1965; Cauna 1973). Several factors have hindered 61 characterization of sensory neuron-keratinocyte interactions in mammalian systems, 62 4 including region-specific differences in sensory neuron-epidermis interactions 63 (Kawakami, Ishihara, and Mihara 2001; Liu et al. 2014), a still-growing repertoire of 64 neuronal cell types that innervate the epidermis (Usoskin et al. 2015; Nguyen et al. 65 2017), and a shortage of markers that label discrete populations of sensory neurons. 66 Peripheral arbors of somatosensory neurons are likewise inserted into keratinocytes or 67 keratinocyte-like epidermal cells in invertebrate and non-mammalian vertebrate model 68 systems, making these promising settings for characterizing epithelial cell-neurite 69 interactions. Notably, portions of Drosophila melanogaster larval nociceptive class IV 70 dendrite arborization (da) neuron dendrites and Danio rerio (zebrafish) larval trigeminal 71 and Rohon-Beard (RB) sensory axons become ensheathed by epidermal cells (Han et 72 al. 2012; Kim et al. 2012; O'Brien et al. 2012), and studies in these systems have ...

The formation of the embryonic brain requires the production, migration, and differentiation of neurons to be timely and coordinated. Coupling to the photoperiod could synchronize the development of neurons in the embryo. Here, we consider the effect of light and melatonin on the differentiation of embryonic neurons in zebrafish. We examine the formation of neurons in the habenular nuclei, a paired structure found near the dorsal surface of the brain adjacent to the pineal organ. Keeping embryos in constant darkness causes a temporary accumulation of habenular precursor cells, resulting in late differentiation and a long-lasting reduction in neuronal processes (neuropil). Because constant darkness delays the accumulation of the neurendocrine hormone melatonin in embryos, we looked for a link between melatonin signaling and habenular neurogenesis. A pharmacological block of melatonin receptors delays neurogenesis and reduces neuropil similarly to constant darkness, while addition of melatonin to embryos in constant darkness restores timely neurogenesis and neuropil. We conclude that light and melatonin schedule the differentiation of neurons and the formation of neural processes in the habenular nuclei.

The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.