SummaryWe demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo’s enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.

Background As they migrate through the developing gut, a sub-population of enteric neural crest-derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs. Methods The appearance of neuronal sub-types in the gut of embryonic mice was examined using immunohistochemistry. The effects of blocking various forms of neural activity on ENCC migration and neuronal differentiation were examined using explants of cultured embryonic gut. Key Results Nerve fibers were present in close apposition to many ENCCs. Commencing at E11.5, neuronal nitric oxide synthase (nNOS), calbindin and IK Ca channel immunoreactivities were shown by sub-populations of enteric neurons. In cultured explants of embryonic gut, tetrodotoxin (TTX, an inhibitor of action potential generation), nitro-L-arginine (NOLA, an inhibitor of nitric oxide synthesis) and clotrimazole (an IK Ca channel blocker) did not affect the rate of ENCC migration, but tetanus toxin (an inhibitor of SNAREmediated vesicle fusion) significantly impaired ENCC migration as previously reported. In explants of E11.5 and E12.5 hindgut grown in the presence of TTX or tetanus toxin there was a decrease in the number nNOS+ neurons close to the migratory wavefront, but no significant difference in the proportion of all ENCC that expressed the pan-neuronal marker, Hu. Conclusions & Inferences (i) Some enteric neuron sub-types are present very early during the development of the enteric nervous system. (ii) The rate of differentiation of some sub-types of enteric neurons appears to be influenced by TTX-and tetanus toxin-sensitive mechanisms.

Summary During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging in zebrafish reveals that differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord before axon initiation. These protrusions express Delta protein, consistent with the hypothesis they influence Notch signaling at a distance of several cell diameters. Experimental reduction of Laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that neuronal differentiation pattern is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers a stereotyped protrusive activity of newborn neurons that organize long-distance spatiotemporal patterning of differentiation.

During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging of zebrafish reveals differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord prior to axon initiation. These protrusions express Delta protein consistent with the possibility they influence Notch signalling at a distance of several cell diameters. Experimental reduction of laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that the pattern of neuronal differentiation is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers novel, stereotyped protrusive activity of new-born neurons that organizes long distance spatiotemporal patterning of differentiation.

Maintenance of the neural progenitor pool during embryonic development is essential to promote growth of the central nervous system (CNS). The CNS is initially formed by tightly compacted proliferative neuroepithelial cells that later acquire radial glial characteristics and continue to divide at the ventricular (apical) and pial (basal) surface of the neuroepithelium to generate neurons. While neural progenitors such as neuroepithelial cells and apical radial glia form strong connections with their neighbours at the apical and basal surfaces of the neuroepithelium, neurons usually form the mantle layer at the basal surface. This review will discuss the existing evidence that supports a role for neurons, from early stages of differentiation, in promoting progenitor cell fates in the vertebrates CNS, maintaining tissue homeostasis and regulating spatiotemporal patterning of neuronal differentiation through Delta-Notch signalling.

BACKGROUND: Nebulizer therapy is an important treatment component for patients with cystic fibrosis (CF). Nebulizer manufacturers' guidelines advocate thorough nebulizer drying after washing. The aim of this study, therefore, was to examine the microbiology associated with nebulizer drying, particularly related to Pseudomonas control, and to examine microbiologically non-adherence to the recommended drying procedures. METHODS: Four aspects of nebulizer drying were examined in 3 common nebulizers, including examination of the drying profile, improvement to the drying profile of assembled nebulizers, survival of Pseudomonas aeruginosa in tap water and in tap water plus 0.5% (v/v) dishwashing detergent, and the effect of drying of P. aeruginosa in tap water and tap water plus residual sputum (1%v/v, 10%v/v). Microbiologic examination was performed by using P. aeruginosa (5 clinical CF strains plus 1 National Collection of Type Cultures Reference strain). RESULTS: There were differences in the time to complete dryness between disassembled and fully assembled nebulizers. Vigorous repeated shaking was unable to drive off all residual water on assembled nebulizers. P. aeruginosa counts did not decrease significantly in either tap water or in tap water plus detergent after 24 h storage at ambient temperature. In contrast, all Pseudomonas organisms were killed when nebulizers were dried for 24 h, even when contaminated with 1% and 10% sputum. Dishwashing detergent did not demonstrate any antibacterial activity. CONCLUSIONS: This study demonstrated that nebulizer drying, if applied properly, had the ability to reduce counts of P. aeruginosa to non-detectable levels. Equally, this study showed that, if the device was not dried thoroughly and moisture remained, then the device was able to support the survival of P. aeruginosa at high numbers, which constituted an infection risk to the patient with CF. This information may help educate and inform the patient with CF about the importance of proper nebulizer drying for Pseudomonas control to improve patient awareness and safety.

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During brain development, the tissue pattern and specification are the foundation of neuronal circuit formation. Contact-mediated lateral inhibition is well known to play an important role in determining cell fate decisions in the nervous system by either regulating tissue boundary formation or the classical salt-and-pepper pattern of differentiation that results from direct neighboring cell contacts. In many systems, however, such as the Drosophila notum, Drosophila wing, zebrafish pigmented cells, and zebrafish spinal cord, the differentiation pattern occurs at multiple-cell diameter distances. In this review, we discuss the evidence and characteristics of long-distance patterning mechanisms mediated by cellular protrusions. In the nervous system, cellular protrusions deliver the Notch ligand Delta at long range to prevent cells from differentiating in their vicinity. By temporal control of protrusive activity, this mechanism can pattern differentiation in both space and time.