Myelinating oligodendrocytes arise from migratory and proliferative oligodendrocyte progenitor cells (OPCs). Complete myelination requires that oligodendrocytes be uniformly distributed and form numerous, periodically spaced membrane sheaths along the entire length of target axons. Mechanisms that determine spacing of oligodendrocytes and their myelinating processes are not known. Using in vivo time-lapse confocal microscopy, we show that zebrafish OPCs continuously extend and retract numerous filopodium-like processes as they migrate and settle into their final positions. Process remodeling and migration paths are highly variable and seem to be influenced by contact with neighboring OPCs. After laser ablation of oligodendrocyte-lineage cells, nearby OPCs divide more frequently, orient processes toward the ablated cells and migrate to fill the unoccupied space. Thus, process activity before axon wrapping might serve as a surveillance mechanism by which OPCs determine the presence or absence of nearby oligodendrocyte-lineage cells, facilitating uniform spacing of oligodendrocytes and complete myelination.

Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.

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SignificanceInherited pathogenic mutations in genes required for copper delivery to cytochrome c oxidase (CcO) perturb mitochondrial energy metabolism and result in fatal mitochondrial disease. A prior attempt to treat human patients with these mutations by direct copper supplementation was not successful, possibly because of inefficient copper delivery to the mitochondria. We performed a targeted search to identify compounds that can efficiently transport copper across biological membranes and identified elesclomol (ES), an investigational anticancer drug, as the most efficient copper delivery agent. ES rescues CcO function in yeast, zebrafish, and mammalian models of copper deficiency by increasing cellular and mitochondrial copper content. Thus, our study offers a possibility of repurposing this anticancer drug for the treatment of disorders of copper metabolism.

Notochord and floor plate cells are sources of molecules that pattern tissues near the midline, including the spinal cord. Hypochord cells are also found at the midline of anamniote embryos and are important for aorta development. Delta-Notch signaling regulates midline patterning in the dorsal organizer by inhibiting notochord formation and promoting hypochord and possibly floor plate development, but the precise mechanisms by which this regulation occurs are unknown. We demonstrate here that floor plate and hypochord cells arise from distinct regions of the zebrafish shield. Blocking Notch signaling during gastrulation entirely prevented hypochord specification but only reduced the number of floor plate cells that developed compared to control embryos. In contrast, elevation of Notch signaling at the beginning of gastrulation caused expansion of hypochord at the expense of notochord, but floor plate was not affected. A cell proliferation assay revealed that Notch signaling maintains dividing floor plate progenitors. Together, our results indicate that Notch signaling regulates allocation of appropriate numbers of different midline cells by different mechanisms.

Notochord, floor plate, and in anamniotes hypochord, are vertebrate embryonic midline structures that are the sources of molecules that pattern the nervous system, somites, and dorsal aorta. Midline precursor cells arise from the dorsal organizer during gastrulation, and Notch signaling is an important regulator of midline cell fate specification. To understand fully how Notch signaling regulates midline development, we investigated the role of potential Notch target genes. We show here that midline precursors express her9, a member of the hairy/Enhancer of split gene family. Although her9 inhibits notochord development and promotes floor plate specification, her9 expression in floor plate cells appears not to require Notch signaling. We show that, instead, her9 is a downstream effector of Nodal signaling for floor plate specification.

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1b. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.