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 red, a glass transfer pipette, and sharpened tungsten needles.</li>
	<li>Expose embryos (as shown in6).</li>
	<li>Harvest tissue grafts from the left side of basal forebrain of stage 7/8 embryos. Use curved sharpened tungsten needles6 to gently incise a piece of the forebrain measuring ~0.3 mm in length by 0.2 mm in width, making sure to not include the underlying endoderm by sliding the needle beneath the forebrain so that the needle is parallel to the axis of the neural tube.</li>
	<li>Using the glass transfer pipette, pick the graft up from the donor embryo.</li>
	<li>Transfer the graft into DMEM containing Neutral Red (0.01% in PBS, 23 &#176;C) for 2 minutes to stain it, and then place the stained graft into DMEM that does not contain Neutral Red until ready for engraftment.</li>
</ol>
2. Preparing the host
<ol>
	<li>Incubate fertilized eggs from white Leghorn chicken (Gallus gallus) at 37 &#176;C in a humidified chamber until HH7/8 17.</li>
	<li>Expose embryos (as shown in6).</li>
	<li>Using sharpened tungsten needles, prepare the graft site by gently cutting, then removing a 0.3 mm by 0.2 mm piece of basal forebrain from the left side to accommodate the graft as was done to isolate the donor tissue.</li>
	<li>Take care to avoid excessive disruption of the underlying endoderm, which will be evident as yolk granules will begin to leak through any tear that is made. This takes practice and not all attempts will be successful.</li>
	<li>After transferring to the host6, position the graft to replace the extirpated basal forebrain of the host.</li>
	<li>Place tape tightly over the hole and return the embryo to the 37 &#176;C incubator for the appropriate length of time for analysis.</li>
</ol>

<ol><li>Waddington, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+Mechanics+of+Chicken+and+Duck+Embryos%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Developmental Mechanics of Chicken and Duck Embryos.</a> Nature. 125, 924-925 (1930).</li>
<li>Noden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+neural+crest+in+patterning+of+avian+cranial+skeletal%2C+connective%2C+and+muscle+tissues%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues.</a> Developmental Biology. 96 (1), 144-165 (1983).</li>
<li>Borue, X., Noden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+and+aberrant+craniofacial+myogenesis+by+grafted+trunk+somitic+and+segmental+plate+mesoderm%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm.</a> Development. 131 (16), 3967-3980 (2004).</li>
<li>Teillet, M. A., Ziller, C., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quail-chick+chimeras%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Quail-chick chimeras.</a> Methods in Molecular Biology. 461, 337-350 (2008).</li>
<li>Hu, D., Marcucio, R. S., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+zone+of+frontonasal+ectoderm+regulates+patterning+and+growth+in+the+face%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A zone of frontonasal ectoderm regulates patterning and growth in the face.</a> Development. 130 (9), 1749-1758 (2003).</li>
<li>Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+signaling+properties+of+ectodermal+epithelia+during+craniofacial+development%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Assessing signaling properties of ectodermal epithelia during craniofacial development.</a> Journal of Visualized Experiments. (49), (2011).</li>
<li>Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+organization+of+the+frontonasal+ectodermal+zone+in+birds+and+mammals%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Unique organization of the frontonasal ectodermal zone in birds and mammals.</a> Developmental Biology. 325 (1), 200-210 (2009).</li>
<li>Griffin, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fgf8+dosage+determines+midfacial+integration+and+polarity+within+the+nasal+and+optic+capsules%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Fgf8 dosage determines midfacial integration and polarity within the nasal and optic capsules.</a> Developmental Biology. 374 (1), 185-197 (2013).</li>
<li>Schneider, R. A., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cellular+and+molecular+origins+of+beak+morphology%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The cellular and molecular origins of beak morphology.</a> Science. 299 (5606), 565-568 (2003).</li>
<li>Tucker, A. S., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+cells+provide+species-specific+patterning+information+in+the+developing+branchial+skeleton%5BTitle%5D)+AND+%22Evolution+%26+Development%22%5BJournal%5D)">Neural crest cells provide species-specific patterning information in the developing branchial skeleton.</a> Evolution & Development. 6 (1), 32-40 (2004).</li>
<li>Fish, J. L., Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+species-specific+contributions+to+craniofacial+development+using+quail-duck+chimeras%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Assessing species-specific contributions to craniofacial development using quail-duck chimeras.</a> Journal of Visualized Experiments. (87), (2014).</li>
<li>Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+and+the+origin+of+species-specific+pattern%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">Neural crest and the origin of species-specific pattern.</a> Genesis. 56 (6-7), 23219(2018).</li>
<li>Sohal, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+reciprocal+forebrain+transplantation+on+motility+and+hatching+in+chick+and+duck+embryos%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Effects of reciprocal forebrain transplantation on motility and hatching in chick and duck embryos.</a> Brain Research. 113 (1), 35-43 (1976).</li>
<li>Chen, C. C., Balaban, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interspecies+avian+brain+chimeras+reveal+that+large+brain+size+differences+are+influenced+by+cell-interdependent+processes%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Interspecies avian brain chimeras reveal that large brain size differences are influenced by cell-interdependent processes.</a> PLoS One. 7 (7), 42477(2012).</li>
<li>Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+cells+pattern+the+surface+cephalic+ectoderm+during+FEZ+formation%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Neural crest cells pattern the surface cephalic ectoderm during FEZ formation.</a> Developmental Dynamics. 241 (4), 732-740 (2012).</li>
<li>Hu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signals+from+the+brain+induce+variation+in+avian+facial+shape%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Signals from the brain induce variation in avian facial shape.</a> Developmental Dynamics. 244 (9), 1133-1143 (2015).</li>
<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li>
<li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlations+Between+the+Morphology+of+Sonic+Hedgehog+Expression+Domains+and+Embryonic+Craniofacial+Shape%5BTitle%5D)+AND+%22Evolutionary+Biology%22%5BJournal%5D)">Correlations Between the Morphology of Sonic Hedgehog Expression Domains and Embryonic Craniofacial Shape.</a> Evolutionary Biology. 42 (3), 379-386 (2015).</li>
<li>Eames, B. F., Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genesis+of+cartilage+size+and+shape+during+development+and+evolution%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The genesis of cartilage size and shape during development and evolution.</a> Development. 135 (23), 3947-3958 (2008).</li>
<li>Merrill, A. E., Eames, B. F., Weston, S. J., Heath, T., Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchyme-dependent+BMP+signaling+directs+the+timing+of+mandibular+osteogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mesenchyme-dependent BMP signaling directs the timing of mandibular osteogenesis.</a> Development. 135 (7), 1223-1234 (2008).</li>
</ol>

All authors have nothing to disclose.

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><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 ...

creating-avian-forebrain-chimeras-to-assess-facial-development

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Developmental Biology

1.1K Views.  University of California. This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain during craniofacial development.

Video: Creating Avian Forebrain Chimeras to Assess Facial Development

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

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

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

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.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

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

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

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.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Biology

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Creating Avian Forebrain Chimeras to Assess Facial Development

Summary

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