High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Abstract

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...

Integrin signalling regulates the expansion of neuroepithelial progenitors and neurogenesis via Wnt7a and Decorin

“…Culture conditions were adapted from Das et al . 56 ; neurobasal media were supplemented with 1% penicillin and streptomycin, 1% Glutamax and 2% B-27, explants were cultured at 37 °C at 5% CO 2 .…”

Chicken Embryo Spinal Cord Slice Culture Protocol

“…Culture conditions that facilitate neuron survival and cell body migration in embryo slice cultures of higher vertebrates are thus required. Currently, spinal cord slice cultures of higher vertebrates have been used to seed dissociated neurons on top of the slices [12][13][14] , investigate fluorescently labelled axons in the periphery of the slice 18 , or of progenitor cell behavior in early spinal cord development 19 . Slice culture conditions for later spinal cords, particularly those that maintain motor neuron development are currently lacking.…”

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

“…Here, we established a method combining in ovo electroporation and the culture of organotypic tissue slices to study exogenous gene functions in the CNS during chicken embryo development. Some of these methods have been established for the CNS tissue slice culture during chicken embryo development . Compared with the established methods, we have combined in ovo electroporation with tissue slice culture to study the function of exogenous genes in the CNS during chicken embryo development.…”

“…Today, organotypic brain slice culture is mainly applied to mouse and rat tissue, as well as for disease modelling, such as with transgenic mice . However, there are few reports related to the study of organotypic slice culture using chicken embryos . The chicken embryo is a good animal model, used for both developmental biology and neurobiology studies.…”

“…[10][11][12][13][14][15][16][17] However, there are few reports related to the study of organotypic slice culture using chicken embryos. 18,19 The chicken embryo is a good animal model, used for both developmental biology and neurobiology studies. Particularly for the study of embryonic CNS development, the chicken embryo has the advantages of being abundant, easy to manipulate and easy to access to collect material from.…”