A transcription factor network controls cell migration and fate decisions in the developing zebrafish pineal complex.

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.

Fgf signaling governs cell fate in the zebrafish pineal complex.

Left-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.

Light and melatonin schedule neuronal differentiation in the habenular nuclei.

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.

A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites.

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.

Lineage labeling of zebrafish cells with laser uncagable fluorescein dextran.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling and pressure injection of traceable enzymes to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularity attractive tools for lineage labeling and to observe the migration of cells in vivo. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequently, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.