Bruce Appel

In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development

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.

Delta-Notch signaling regulates oligodendrocyte specification

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analysing mutant zebrafish embryos, we found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, we also learned that constitutive Notch activity could promote formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. Our data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate.

Neuronal activity biases axon selection for myelination in vivo

An essential feature of vertebrate neural development is ensheathment of axons with myelin, an insulating membrane formed by oligodendrocytes. Not all axons are myelinated, but mechanisms directing myelination of specific axons are unknown. Using zebrafish, we found that activity-dependent secretion stabilized myelin sheath formation on select axons. When VAMP2-dependent exocytosis was silenced in single axons, oligodendrocytes preferentially ensheathed neighboring axons. Nascent sheaths formed on silenced axons were shorter in length, but when activity of neighboring axons was also suppressed, inhibition of sheath growth was relieved. Using in vivo time-lapse microscopy, we found that only 25% of oligodendrocyte processes that initiated axon wrapping were stabilized during normal development and that initiation did not require activity. Instead, oligodendrocyte processes wrapping silenced axons retracted more frequently. We propose that axon selection for myelination results from excessive and indiscriminate initiation of wrapping followed by refinement that is biased by activity-dependent secretion from axons.

Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling

Graded Hedgehog (Hh) signaling patterns the spinal cord dorsoventral axis by inducing and positioning distinct precursor domains, each of which gives rise to a different type of neuron. These domains also generate glial cells, but the full range of cell types that any one precursor population produces and the mechanisms that diversify cell fate are unknown. By fate mapping and clonal analysis in zebrafish, we show that individual ventral precursor cells that express olig2 can form motoneurons, interneurons and oligodendrocytes. However, olig2+ precursors are not developmentally equivalent, but instead produce subsets of progeny cells in a spatially and temporally biased manner. Using genetic and pharmacological manipulations, we provide evidence that these biases emerge from Hh acting over time to set, maintain, subdivide and enlarge the olig2+ precursor domain and subsequently specify oligodendrocyte development. Our studies show that spatial and temporal differences in Hh signaling within a common population of neural precursors can contribute to cell fate diversification.

CNS-derived glia ensheath peripheral nerves and mediate motor root development

Motor function requires that motor axons extend from the spinal cord at regular intervals and that they are myelinated by Schwann cells. Little attention has been given to another cellular structure, the perineurium, which ensheaths the motor nerve, forming a flexible, protective barrier. Consequently, the origin of perineurial cells and their roles in motor nerve formation are poorly understood. Using time-lapse imaging in zebrafish, we show that perineurial cells are born in the CNS, arising as ventral spinal-cord glia before migrating into the periphery. In embryos lacking perineurial glia, motor neurons inappropriately migrated outside of the spinal cord and had aberrant axonal projections, indicating that perineurial glia carry out barrier and guidance functions at motor axon exit points. Additionally, reciprocal signaling between perineurial glia and Schwann cells was necessary for motor nerve ensheathment by both cell types. These insights reveal a new class of CNS-born glia that critically contributes to motor nerve development.

Oligodendrocyte Specification in Zebrafish Requires Notch-Regulated Cyclin-Dependent Kinase Inhibitor Function

Cyclin-dependent kinase inhibitors (Cdkis) influence both cell-cycle progression and differentiation of neural cells. However, the precise roles of Cdkis in coordinating formation of neurons and glia and the mechanisms that regulate expression of genes that encode Cdkis in the vertebrate CNS remain unknown. Here, we report that, in zebrafish, expression of the Cdki gene cyclin-dependent kinase inhibitor 1c (cdkn1c), a p57 homolog, is negatively regulated by Delta-Notch signaling and that Cdkn1c function is required for neural plate cells to stop dividing and differentiate as neurons on schedule, even in the absence of Notch signaling activity. Furthermore, Cdkn1c function is required for specification of oligodendrocytes from ventral spinal cord precursors. We propose that levels of cdkn1c expression are an important factor in regulating neural development: high levels of Cdkn1c promote cell-cycle exit and neuronal development, whereas, during late embryogenesis, neural cells that have low but functional levels of Cdkn1c, regulated by Notch activity, are specified for oligodendrocyte fate.

Retinoids Run Rampant: Multiple Roles during Spinal Cord and Motor Neuron Development

Learning how the incredible diversity of neurons in the vertebrate central nervous system (CNS) is generated is a central focus of developmental neuroscience. Three studies in the September 25, 2003, issue of Neuron bring us closer to this goal by revealing how the interplay between Fibroblast Growth Factor (FGF), retinoic acid (RA), and Sonic hedgehog (Shh) signaling regulate progression of spinal cord progenitor cells through various phases of development and specify particular types of spinal motor neurons (MNs).

An olig2 Reporter Gene Marks Oligodendrocyte Precursors in the Postembryonic Spinal Cord of Zebrafish

Continuous production of new neurons and glia in adult mammals occurs within specialized proliferation zones of the forebrain. Neural cell proliferation and neurogenesis is more widespread in adult amphibians, reptiles, and fish but the identity of neural stem cell populations in these organisms has not been fully described. We investigated expression of a reporter gene driven by olig2 regulatory DNA at postembryonic stages in zebrafish. We show that olig2 expression marks a discrete population of spinal cord radial glia in larvae and adults that divide continuously. olig2+ radial glia have hallmarks of stem cells and their divisions appear to be asymmetric, producing new oligodendrocytes but not neurons or astrocytes. Developmental Dynamics 236:3402–3407, 2007.

Notch-Regulated Oligodendrocyte Specification From Radial Glia in the Spinal Cord of Zebrafish Embryos

During vertebrate neural development, many dividing neuroepithelial precursors adopt features of radial glia, which are now known to also serve as neural precursors. In mammals, most radial glia do not persist past early postnatal stages, whereas zebrafish maintain large numbers of radial glia into adulthood. The mechanisms that maintain and specify radial glia for different fates are still poorly understood. We investigated formation of radial glia in the spinal cord of zebrafish and the role of Notch signaling in their maintenance and specification. We found that spinal cord precursors begin to express gfap+, a marker of radial glia, during neurogenesis and that gfap cells give rise to both neurons and oligodendrocytes. We also determined that Notch signaling is continuously required during embryogenesis to maintain radial glia, limit motor neuron formation and permit oligodendrocyte development, but that radial glia seem to be refractory to changes in Notch activity in postembryonic animals. Developmental Dynamics 237:2081–2089, 2008.

Notch signaling regulates neural precursor allocation and binary neuronal fate decisions in zebrafish

Notch signaling plays a well-described role in regulating the formation of neurons from proliferative neural precursors in vertebrates but whether, as in flies, it also specifies sibling cells for different neuronal fates is not known. Ventral spinal cord precursors called pMN cells produce mostly motoneurons and oligodendrocytes, but recent lineage-marking experiments reveal that they also make astrocytes, ependymal cells and interneurons. Our own clonal analysis of pMN cells in zebrafish showed that some produce a primary motoneuron and KA′ interneuron at their final division. We investigated the possibility that Notch signaling regulates a motoneuron-interneuron fate decision using a combination of mutant, transgenic and pharmacological manipulations of Notch activity. We show that continuous absence of Notch activity produces excess primary motoneurons and a deficit of KA′ interneurons, whereas transient inactivation preceding neurogenesis results in an excess of both cell types. By contrast, activation of Notch signaling at the neural plate stage produces excess KA′ interneurons and a deficit of primary motoneurons. Furthermore, individual pMN cells produce similar kinds of neurons at their final division in mib mutant embryos, which lack Notch signaling. These data provide evidence that, among some postmitotic daughters of pMN cells, Notch promotes KA′ interneuron identity and inhibits primary motoneuron fate, raising the possibility that Notch signaling diversifies vertebrate neuron type by mediating similar binary fate decisions.