Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo.

Attractive growth cone turning requires Igf2bp1-dependent local translation of ß-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the ß-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.

Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization.

In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. We show that caspases were locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system.

Communities near toluene diisocyanate sources: an investigation of exposure and health.

Toluene diisocyanate (TDI) is a well-known cause of occupational asthma, but we know little about the potential for exposure and health effects among residents who live near facilities that release TDI. In the mid-1990's, the North Carolina Department of Health and Human Services and the Agency for Toxic Substances and Disease Registry investigated exposures to TDI and health outcomes in one community, which left some unanswered questions. This cross-sectional study evaluated the potential associations between living near a TDI source and the prevalence of three variables: asthma or asthma-like respiratory symptoms, antibodies specific to TDI, and verifiable levels of TDI in residential air. Results among North Carolina residents living near such facilities (five target communities) were compared with the results from residents living further away (five comparison communities). Overall, the prevalence of reporting either asthma or asthma-like respiratory symptoms was higher (odds ratio = 1.60; 95% confidence interval = 0.97-2.54) among residents in target communities than those in comparison communities. However, this difference was not statistically significant. Symptom prevalence varied greatly among the community populations. The prevalence of respiratory symptoms was higher near facilities with historically higher TDI emissions. Among the 351 participants who provided blood samples, only one had immunoglobulin G specific antibodies to TDI. This participant lived in a target area and may have had non-occupational exposure. TDI was detected at an extremely low level (1 ppt) in one of the 45 air samples from target communities. One ppt is one-tenth the EPA reference concentration. Overall, air sample and antibody test results are not consistent with recent or ongoing exposure to TDI.

E3 ligase Nedd4 promotes axon branching by downregulating PTEN.

Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo.

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways.

Upon arriving at their targets, developing axons cease pathfinding and begin instead to arborize and form synapses. To test whether CNS arborization and synaptogenesis are controlled by Slit-Robo signaling, we followed single retinal ganglion cell (RGC) arbors over time. ast (robo2) mutant and slit1a morphant arbors had more branch tips and greater arbor area and complexity compared to wild-type and concomitantly more presumptive presynaptic sites labeled with YFP-Rab3. Increased arborization in ast was phenocopied by dominant-negative Robo2 expressed in single RGCs and rescued by full-length Robo2, indicating that Robo2 acts cell-autonomously. Time-lapse imaging revealed that ast and slit1a morphant arbors stabilized earlier than wild-type, suggesting a role for Slit-Robo signaling in preventing arbor maturation. Genetic analysis showed that Slit1a acts both through Robo2 and Robo2-independent mechanisms. Unlike previous PNS studies showing that Slits promote branching, our results show that Slits inhibit arborization and synaptogenesis in the CNS.

Building geographic information system capacity in local health departments: lessons from a North Carolina project.

State government, university, and local health department (LHD) partners collaborated to build the geographic information system (GIS) capacity of 5 LHDs in North Carolina. Project elements included procuring hardware and software, conducting individualized and group training, developing data layers, guiding the project development process, coordinating participation in technical conferences, providing ongoing project consultation, and evaluating project milestones. The project provided health department personnel with the skills and resources required to use sophisticated information management systems, particularly those that address spatial dimensions of public health practice. This capacity-building project helped LHDs incorporate GIS technology into daily operations, resulting in improved time and cost efficiency. Keys to success included (1) methods training rooted in problems specific to the LHD, (2) required project identification by LHD staff with associated timelines for development, (3) ongoing technical support as staff returned to home offices after training, (4) subgrants to LHDs to ease hardware and software resource constraints, (5) networks of relationships among LHDs and other professional GIS users, and (6) senior LHD leadership who supported the professional development activities being undertaken by staff.

A three-dimensional atlas of pituitary gland development in the zebrafish.

The pituitary gland is unique to Chordates, with significant variation within this group, offering an excellent opportunity to increase insight into phylogenetic relationships within this phylum. The structure of the pituitary in adult Teleosts (class: Osteichthyes) is quite different from that in other chordates and is also variable among members of the class. Therefore, a complete description of the structure and development of the pituitary in members of this class is a critical component to our overall understanding of this gland. An obvious teleost model organism is the zebrafish, Danio rerio, as a significant amount of work has been done on the molecular control of pituitary development in this fish. However, very little work has been published on the morphological development of the pituitary in the zebrafish; the present study aims to fill this void. The pituitary develops from cells on the rostrodorsal portion of the head and reaches its final position, ventral to the hypothalamus, as the cephalic flexure occurs and the jaws and mouth form. The pituitary placode is juxtaposed to cells that will form the olfactory vesicles, the stomodeum, and the hatching gland. The volume of the pituitary is greatest at 24 hours post fertilization (hpf). From 24 to 120 hpf, the pituitary decreases in height and width as it undergoes convergent extension, increasing in length with the axis. The adenohypophysis is a morphologically distinct structure by 24 hpf, whereas the neurohypophysis remains indistinct until 72 hpf. The findings of this study correlate well with the available molecular data.

Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones.

Previous work has shown that guidance cues trigger rapid changes in protein dynamics in retinal growth cones: netrin-1 stimulates both protein synthesis and degradation, while Sema3A elicits synthesis, and LPA induces degradation. What signaling pathways are involved? Our studies confirm that p42/44 MAPK mediates netrin-1 responses and further show that inhibiting its activity blocks cue-induced protein synthesis. Unexpectedly, p38 MAPK is also activated by netrin-1 in retinal growth cones and is required for chemotropic responses and translation. Sema3A- and LPA-induced responses, by contrast, require a single MAPK, p42/p44 and p38, respectively. In addition, we report that caspase-3, an apoptotic protease, is rapidly activated by netrin-1 and LPA in a proteasome- and p38-dependent manner and is required for chemotropic responses. These findings suggest that the apoptotic pathway may be used locally to control protein levels in growth cones and that the differential activation of MAPK pathways may underlie cue-directed migration.