Name: Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos (Video) | JoVE Journal
Uploaded: 2007-10-01T00:00:00.000+00:00
Duration: 9 min 25 s
Description: Learn to place growth factor-coated beads on E2.5 chick embryos using in ovo preparation. This method enables precise manipulation of embryonic tissues to study cell fate determination in developmental biology.

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1. Remove eggs from the incubator
At E2-2.5 (~ St. 17 HH), remove the eggs and prepare them accordingly. They can be worked on immediately or returned to the incubator for a couple hours. The eggs can be placed at room temperature safely for over an hour. Eggs can stay out for the duration of the manipulations. Sometimes these can take an hour or more.
Note: The longer they sit at room temperature, the less likely they are to survive.
2. Preparation of India ink
<ol>
<li>Prepare a fresh solution of 4% India Ink in sterile PBS.</li>
<li>Fill a 3 mL syringe with the solution.</li>
<li>Place a 27 gauge, &frac12; inch needle on the syringe.</li>
<li>Using a pair of hemostats, bend the needle to a 90&deg; angle.</li>
</ol>
3. Opening the eggs and injection of India ink
<ol>
<li>Using a pair of forceps, peel back the tape and open the window, securing it by using the reverse side of the tape on the back side of the egg.</li>
<li>Guide in the angled needle until it lies just beneath the embryo. Placing the needle under the floating disk insures that the ink, which is toxic to the embryo, will not come in direct contact.</li>
<li>You need to only inject enough ink to create a comfortable contrast. This is typically less than &frac14; mL.</li>
</ol>
4. Opening the neural tube
<ol>
<li>Take a pair of fine dissection scissors and cut the thin membrane that lies on top, or sometimes around, the embryo.</li>
<li>Take a pair of 55 forceps or a tungsten probe and open the neural tube from posterior to anterior. Make sure the opening is large enough to place a bead into it.</li>
</ol>
Note: Depending on the age, a fine tungsten probe can be used to do the same thing.
5. Retrieving a coated heparin-acrylic bead
<ol>
<li>Using a pair of 55 forceps, find an intact bead in the solution. Let the bead attach itself to the tip, slowly close the forceps.</li>
<li>Remove the bead from the solution and bring it over on top of the egg.</li>
<li>Once the bead is out of its solution, it will not likely come off until it has been placed in the albumin.</li>
</ol>
6. Placing the bead
<ol>
<li>Once the bead has been placed on top of the target area, use a tungsten probe to guide it towards the forebrain or fix it in the hindbrain.</li>
<li>Add a few drops of PBS using a 10 &micro;L pipette.</li>
</ol>
7. Closing the egg
<ol>
<li>Close the egg using the thin piece of tape to shut the window.</li>
<li>Seal the egg according to the prepping protocol, available&nbsp;at <a href="https://www.jove.com/index/Details.stp?ID=306" target="_blank">https://www.jove.com/index/Details.stp?ID=306</a>.</li>
</ol>

<ol><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Windowing+Chicken+Eggs+for+Developmental+Studies%5BTitle%5D)+AND+%22Jounal+of+Visualized+Experiments%22%5BJournal%5D)">Windowing Chicken Eggs for Developmental Studies.</a> Jounal of Visualized Experiments. 8, 

 <a target="_blank" href="http://www.jove.com/index/Details.stp?ID=306">http://www.jove.com/index/Details.stp?ID=306</a> (2007).</li>
<li>Crossley, P. H., Martinez, S., Ohkubo, Y., Rubenstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinate+expression+of+Fgf8%2C+Otx2%2C+Bmp4%2C+and+Shh+in+the+rostral+prosencephalon+during+development+of+the+telencephalic+and+optic+vesicles%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.</a> Neuroscience. 108, 183-206 (2001).</li>
<li>Alexandre, P., Bachy, I., Marcou, M., Wassef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positive+and+negative+regulations+by+FGF8+contribute+to+midbrain+roof+plate+developmental+plasticity%5BTitle%5D)+AND+%22Development+%22%5BJournal%5D)">Positive and negative regulations by FGF8 contribute to midbrain roof plate developmental plasticity.</a> Development . 133, 2905-2913 (2006).</li>
</ol>

The authors have nothing to disclose.

Depending on the protein you wish to use, there are different protocols on how to prepare the beads. In these experiments, we use heparin-acrylic beads, but affi-gel beads can also be used. We have found that the heparin-acrylic beads are easier to manipulate in the aqueous environment of the egg albumin. Beads are soaked in 5 l of protein overnight at 4&deg;C. Control beads are placed in sterile PBS until the time of placement. The advantage in using an in ovo preparation is that the embryo is still alive and free to ...

<table><tbody><tr><td>Eggs</td><td>Animal</td><td></td><td></td><td>Fertilized, E2-2.5 (~ St. 17 HH)</td></tr><tr><td>Incubator</td><td>Profi-I</td><td>Lyon</td><td></td><td></td></tr><tr><td>India Ink</td><td>Tool</td><td>Higgins</td><td></td><td>Prepare a 4% solution in sterile PBS.</td></tr><tr><td>Sterile PBS</td><td></td><td></td><td></td><td>Alternatively, an antibody cocktail can be used</td></tr><tr><td>1ml syringe</td><td></td><td></td><td></td><td></td></tr><tr><td>27G 1/2 needles</td><td>Tool</td><td>BD Biosciences</td><td>305109</td><td>Angled at 90 degrees</td></tr><tr><td>Forceps</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td></td><td>size 55</td></tr><tr><td>Hemostats</td><td></td><td></td><td></td><td></td></tr><tr><td>Dissection scissors</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>500086</td><td>vannas, 8.5cm</td></tr><tr><td>Parafilm tape</td><td></td><td></td><td></td><td>to seal eggs</td></tr><tr><td>Tungsten Probe</td><td>Tool</td><td>Electron Microscopy Sciences</td><td>62091</td><td>Tool #24. Handle optional. </td></tr></tbody></table>

placing growth factor-coated beads on early stage chicken embryos

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Welcome to the Raburn Lab. I'm Matt Korn, department of Neurobiology and Behavior at the University of California Irvine. Today we're gonna take an egg, open it and place a bead that's been soaked in growth factor.The wonderful preparation of a egg will allow us to put in a growth factor to change the fate of a number of different types of tissues in this early stage of embryonic development. Day two and a half, or stage 17. Okay, so we're here at, at the scope here.I've got my eggs and all my tools. So go, go ahead and just pull an egg here and you can see where our tape is. Window seal.I've got this nice little donut. It's just made out of artistic clay that you can get an art store. And I'm gonna take a really bad pair of five forceps.These have been around for a long time. The tip tips are pretty dull, but they work great to just lift up the tape just enough so that window comes up. I can put those down.I'm just gonna fold this over and tuck the egg, this window behind there like that. So now I can put it under the scope, make any type of manipulations. In this case, we're gonna be doing the beads, and I'm gonna go ahead and grab my India ink right now without the India ink.You

placing-growth-factor-coated-beads-on-early-stage-chicken-embryos

Research

JoVE Journal

Biology

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex.   These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants.  Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Method for Culture of Early Chick Embryos ex vivo (New Culture)

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo 

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

Learn to place growth factor-coated beads on E2.5 chick embryos using in ovo preparation. This method enables precise manipulation of embryonic tissues to study cell fate determination in developmental biology.

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

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