*Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neurongenerating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation 1, 2 , we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy ( Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.This assay may be modified to image a range of embryonic tissues 3,4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling 5 ) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development. Video LinkThe video component of this article can be found at https://www.jove.com/video/3920/ Protocol 1. Dish Preparation 1. Dishes used for slice culture are glass-bottomed (with a coverslip as the base) (WillCo dishes). These are placed on lens tissue in a 6 cm tissue culture dish to keep the glass bottom clean. 2. On the day before the experiment, add 2 ml 0.1% poly-L-lysine solution to the glass bottomed dish and incubate for 5min at room temperature to allow the poly-L-lysine to coat the glass bottom. 3. Remove poly-L-lysine solution and rinse three times in deionised water, and then once in 70% ethanol. 4. Leave to dry overnight. Dishes can also be dried by gently heating in a microwave at low power for 30-45 seconds. Embryo Electroporation1. Incubate eggs at 37 °C to Hamburger-Hamilton (HH) stage 10 (~36 hours) (or other desired stage). 2. Before beginning prepare glass needles (we use a Flaming/Brown model p87 microcapillary puller) and break off the tip of the needle using a fine forceps under a dissecting microscope. The end of the needle should be sharp enough to pierce the embryo while not being so narrow that it impedes injection of the DNA solution. 3. Window eggs and place electrodes (5mm apart) on either side of the embryo. 4. Inject DNA (~0.025 -0.5 μg/μl in deionised water colored with a small amount of fast green) into the neural tube. 5. Apply current -12-17 V three times, 50ms pulse length with 950 ms between pulses. 6. We use low concentrations of DNA and low electroporation voltages to achieve mosaic expression so that we can follow individual cells. 7. Cover window in the eggshell with cellotape and make sure it is sealed. 8. Allow embryos to recove...

Hypoxia and glucose deprivation, are important during many physiological and pathological processes. Cells respond to these stimuli by activating genes involved in the regulation of metabolism and angiogenesis. Platelet derived growth factor-B (PDGF-B) is involved in the regulation of angiogenesis and tumour progression and is induced by hypoxia. Most known hypoxia-induced genes are activated by the hypoxia inducible factor (HIF-1), via its binding to specific response elements. The mechanism of hypoxic induction and the effect of low glucose on PDGF-B expression have not been characterised. We show that PDGF-B exhibits a novel, biphasic regulation (induction, followed by repression below basal levels) in bladder carcinoma cells cultured under chronic hypoxia. We show that the repression observed after long-term hypoxia is due to glucose-depletion and that this can also abrogate short-term hypoxic induction. This is in contrast to the previous results showing that hypoxia/hypoglycaemia elicit the same response. We also show that a putative hypoxia response element in the PDGF-B promoter is not sufficient for hypoxic induction, although it does function as a hypoxia independent enhancer element in hepatocellular carcinoma cells.

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