Name: creating avian forebrain chimeras to assess facial development
Uploaded: 2021-02-18T13:00:00
Description: Watch this Scientific Journal Video about Creating Avian Forebrain Chimeras to Assess Facial Development at JoVE.com

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R01DE019648, R01DE018234, and R01DE019638.

White Pekin duck (Anas platyrhynchos), white Leghorn chicken (Gallus gallus) and Japanese quail (Cortunix coturnix japonica) are incubated at 37 &#176;C in a humidified chamber until stage-matched at HH7/817.
1. Preparing the donor tissue
NOTE: Preparation of reagents and tools and how to open eggs for experimental manipulation has been described6.
<ol>
	<li>Prepare DMEM media with neutral ...

<ol>
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The method described allows examination of the tissue interactions between the basal forebrain and the adjacent ectoderm. This approach differs from previous forebrain transplant methods, because the donor tissue was restricted to the ventral forebrain. This eliminates transplantation of the neural crest cells, which have been shown to participate in patterning facial morphology9,10. Hence, restricting the graft to the basal forebrain was essential to evaluate th...

Much contemporary research in developmental biology focuses on the role of genes in shaping embryos. There are good tools to examine developmental mechanisms from a genetic perspective. However, embryos are assembled and undergo morphogenesis in response to tissue interactions. The avian system is a classic tool used to assess the variety of tissue interactions that regulate development for the following reasons: the embryology is well-understood, the embryos are easily accessible, tools for analysis of avian systems are well-developed, and the embryos are inexpensive.
The avian transplantation system has been widely employed for lineage tracing and to assess tissue interactions during development for almost a century1,2,3,4. This system was used to investigate a signaling center, the Frontonasal Ectodermal Zone (FEZ), that regulates morphogenesis of the upper jaw5, and a video was published describing that technique previously6. In addition to quail-chick, other species have also been used to produce chimeras for analysis of tissue interactions. For example, the mouse FEZ was transplanted from wild type7 and mutant mice8, and others have used a duck, quail and chick systems to assess the role of neural crest in patterning the facial skeleton9,10,11,12.
In this work, the role of the forebrain in regulating the pattern of gene expression in the FEZ by transplanting the ventral forebrain reciprocally among quail, duck, and chick embryos was assessed, because a signal from the forebrain is required to induce Sonic hedgehog expression in the FEZ. Forebrain transplants are not unique in the field. These transplants were used to assess development of motility in quail and duck embryos13, although in these experiments tissues that contributed to non-neural derivatives were also transplanted. In other work, auditory circuits in birds have been assessed by forebrain transplantation14, but these transplants contained presumptive neural crest cells, which contribute to facial shape9,10 and participate in regulating SHH expression in the FEZ15. Hence, a system to transplant just the ventral forebrain from one species of bird to another prior to closure of the neural tube was devised to assess the role of the brain in facial shape16 (Figure 1A,B). This method was devoid of neural crest contamination of the graft. In this article, the method is illustrated and the expected results are described, and the challenges faced are discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>TEK</td>
			<td>TEKZR114</td>
			<td></td>
		</tr>
		<tr>
			<td>35x10 mm Petri dish</td>
			<td>Falcon</td>
			<td>1008</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermofisher</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red</td>
			<td>Sigma</td>
			<td>553-24-2</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 5 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipets</td>
			<td>Thermofisher</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>QCPN antibody</td>
			<td>Developmental Studies Hybridoma bank, Iowa University, Iowa, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten Needle</td>
			<td>Fine Science Tools</td>
			<td>26000</td>
			<td></td>
		</tr>
	</tbody>
</table>

creating avian forebrain chimeras to assess facial development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

Assessment of Chimerism and Transplant Contamination 
In order to assess the chimeras, the extent of chimerism and contamination of the graft with other cell types should be addressed. Creating chimeras by transplanting quail tissues into chick embryos allows for this type of analysis. Using the QCPN antibody quail cells can be visualized and distinguished from the host tissues (Figure 1 C,D). In this case, only tissues derived from the ventral forebrai...

Watch this Scientific Journal Video about Creating Avian Forebrain Chimeras to Assess Facial Development at JoVE.com

Creating Avian Forebrain Chimeras to Assess Facial Development

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

This method allows investigators to assess tissue interactions between the basal forebrain and the face. The main advantage of this technique is that it isolates the basal forebrain for assessment and prevents contamination with neural crest cells, which would confound the analysis. The difficult part of this technique is the removal of the forebrain from the host and replacement with the donor tissue.Demonstrating the procedure will be Diane Hu, a research scientist from Dr.Ralph Marcucio's laboratory. To begin, prepare DMEM media with neutral red, a glass transfer pipette, and sharpened tungsten. Using a 10 milliliter syringe and an 18 gauge needle, remove 0.5 milliliters of albumin from the pointed end of the eggshell.Make a small hole on top of the shell using the point of scissors. Then place a piece of tape over the hole and cut a circular opening to expose the embryo. Stain the embryo with neutral red.Harvest tissue grafts from the left side of the basal forebrain of stage seven or eight embryos. Use curved sharpened tungsten needles to gently incise a piece of the forebrain. In order to not include the underlying endoderm, slide the needle beneath the forebrain and parallel to the axis of the neural tube.Pick the graft up from the donor embryo using the glass transfer pipette, and transfer it into DMEM containing neutral red for two minutes to stain it. Then place the stained graft into DMEM that does not contain neutral red until ready for engraftment. Incubate fertilized eggs from white Leghorn chicken at 37 degrees Celsius in a humidified chamber until Hamburger-Hamilton stage seven to eight.Expose embryos as previously demonstrated. Using sharpened tungsten needles, prepare the graft site by cutting and then removing a 0.3 by 0.2 millimeter piece of basal forebrain to accommodate the graft. Avoid excessive disruption of the underlying endoderm, which will be evident as yolk granules will begin to leak through any tear that is made.After transferring the graft to the host, position the graft to replace the extirpated basal forebrain of the host. Place tape tightly over the hole, and return the embryos to the 37 degree Celsius incubator until they are ready for analysis. Initially, chimeras were created by transplanting quail tissues into chick embryos.Using the QCPN antibody, quail cells were visualized and distinguished from the host tissues. The quail-chick system confirmed that all of the graft was comprised only of neural tissue and was not contaminated with other cell types. Transplantation of quail tissue into duck embryos led to severely deformed chimeras due to a faster developing quail brain.Therefore, transplantation of duck tissues into chicken embryos was performed. The resulting duck-chick chimeras suggest that the brain participates in regulating morphology. Whole mountain C2 hybridization was used to assess Sonic hedgehog expression in chimeras.Similar to morphology, Sonic hedgehog expression on the duck side of the chimera appeared more duck-like. Preparing the host site takes practice. A large number of embryos do not survive, so creating a large number of chimeras can help ensure proper samples for analysis.This method made it possible to assess how signals from the brain shape the Sonic hedgehog expression domain in the frontonasal ectodermal zone.

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