Name: Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras (Video) | JoVE Journal
Uploaded: 2014-05-31T14:00:00.000+00:00
Duration: 9 min 38 s
Description: Learn to create quail-duck chimeras for studying neural crest contributions to craniofacial development. This method reveals species-specific patterns in bone and cartilage formation.

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5 minutes

This work was funded by a National Institute of Dental and Craniofacial Research (NIDCR) F32 grant (DE021929) to J.L.F. and a NIDCR R01 grant DE016402 to R.A.S.

1. Prepare Tungsten Needles
<ol>
	<li>Cut a tungsten rod in half using wire cutters so that the rod does not bend.</li>
	<li>Thread the rod through the tapered end of a 5&#160;3/4 in&#160;borosilicate glass Pasteur pipette.</li>
	<li>Leaving approximately 3/4 in&#160;of the rod sticking out of the glass, adhere the rod to the pipette tip with a small bead of &#8220;hot melt adhesive&#8221; (HMA), which is the glue stick used for a glue gun. The HMA should be quickly melted in an alcohol flame, taking care not to burn the glue and generate black smoke.</li>
	<li>Using a pair of forceps, bend the tip of the tungsten rod (about 1/4 in&#160;from the top end) until a 45&#176;&#160;angle is reached.</li>
	<li>Holding the Pasteur pipette, place about 1/8 in&#160;of the tip of the tungsten rod into the flame of a propane torch. Hold the tip steady and exactly perpendicular to the flame.</li>
	<li>Pull the needle out of the flame the moment a tiny orange speck of tungsten flies off of the needle.</li>
</ol>
2. Prepare Spemann Pipettes
<ol>
	<li>Using a Bunsen burner, heat the narrow end of a Pasteur pipette until the glass shortly beyond the taper begins to melt. Remove from flame and pull the tip until the pipette becomes sealed. Be sure that there remains a portion of the tapered part that has an O.D. of approximately 0.7-1.0 mm. Break the sealed end off about 4 cm from the taper.</li>
	<li>Attach around 2 ft&#160;(60 cm) of rubber tubing to the wide (open) end of the pipette. Blow on the other end of the rubber tubing to ensure that no air can pass through the tapered part of the Pasteur pipette.</li>
	<li>Using a propane fuel cylinder with an attached pencil flame torch, heat an oval area on one side of the pipette just below the start of the taper. Blow air very gently and constantly through the rubber tubing with a slight amount of pressure (approximately the amount of pressure needed to say the beginning of the letter &#8220;P&#8221; at conversation level). When the glass becomes soft on one side and begins to buckle outwards, quickly remove the pipette from the flame and blow hard and steady through the rubber tubing. This will cause the heated part of the glass to form a sausage-shaped bubble that may pop, which is fine. If the bubble is too small, try melting and blowing the glass again. The final open window in the glass should be around 0.25 in&#160;(6.35 mm) wide by 0.5 in&#160;(12.7 mm) long.</li>
	<li>Pointing the tapered end upwards, scrape the bubble into a glass waste container while gently blowing through the tubing to prevent glass fragments from entering the pipette.</li>
	<li>Using the propane flame, burn off the remaining edges of the bubble and fire-polish the sides of the open window. Take care not to melt the shaft of the pipette.</li>
	<li>Using a diamond point pencil, score the tapered end of the pipette approximately 1.25 in&#160;(30 mm) from the window and break off the tip with forceps so that the pipette opening is cut cleanly (discard pipettes with broken or uneven tips).</li>
	<li>Using forceps and the edge of a flame from an alcohol burner, bend the narrow portion of the pipette 0.375 in&#160;(9.5 mm) from the tip to an angle of approximately 60&#176; so that the bent portion is in a vertical plane when the window opening is at the 1:00 position for right-handed usage and 11:00 for left-handed usage. This step is best conducted in a draft-free enclosure.</li>
	<li>Fire-polish the tip using the alcohol flame under a dissecting microscope. Be sure not to close off the tip but rather keep the opening round and smooth without any constriction.</li>
	<li>Cut a 1&#160;in&#160;(25 mm) piece of rubber tubing, spray the piece of tubing with 70% ethanol, and slide the tubing over the shaft of the pipette until the window opening is completely covered. This functions as the &#8220;diaphragm&#8221; of the Spemann pipette.</li>
	<li>Place a latex rubber 2 ml bulb over the open bottom of the pipette. The bulb maintains negative pressure in the Spemann pipette.</li>
	<li>To practice using a Spemann pipette, transfer chromatography beads of around 100-200 &#181;m in diameter from a marked spot in one Petri dish to a marked spot in another under a microscope.</li>
	<li>To clean the Spemann pipette, remove the rubber bulb and place pipette, tip down, in a tall beaker lined with cotton or gauze on the bottom and filled with distilled water and glassware detergent.</li>
</ol>
3. Sterilize and Incubate Eggs
<ol>
	<li>Wipe eggs with 70% ethanol and place the eggs on plastic egg trays.</li>
	<li>Set eggs in a heated incubator at 37.5 &#176;C and 85-87%.</li>
	<li>Incubate eggs until they reach HH9.5, approximately 26 hr for quail and 48 hr for duck.</li>
</ol>
4. Window Eggs
<ol>
	<li>Remove a small piece of outer shell from the top of the egg with forceps, taking care not to puncture the inner shell membrane.</li>
	<li>Using a syringe with an 18 G by 1-inch needle, poke a hole at the narrowest tip of the egg and withdraw approximately 1-2 ml of albumen.</li>
	<li>Place transparent tape over the hole and puncture mark in the shell.</li>
	<li>Cut a &#8220;window&#8221; along the top surface of the egg (through the transparent tape) using curved scissors.</li>
</ol>
5. Visualize Embryos and Prepare for Surgery
<ol>
	<li>Using a glass rod, gently brush a small amount of Neutral Red (0.02 g/ml in Hank&#8217;s BSS) over the embryo.</li>
	<li>Cut the vitelline membrane and pull it over the embryo using a flame-sharpened tungsten needle.</li>
	<li>Re-seal the egg by placing transparent tape over the window.</li>
</ol>
6. Separate the Donor Tissue from the Donor Embryo
<ol>
	<li>Remove tape from the window on the egg of the donor embryo.</li>
	<li>To cut the neural fold from the adjacent ectoderm of the donor embryo, make slits on both sides of the neural tube, using a flame-sharpened tungsten needle (manufactured as described above).</li>
	<li>Then, cut across the neural tube at the desired anterior and posterior levels of the graft and separate the graft from the rest of the neural tube.</li>
	<li>Remove the neural fold from the donor embryo using either a Spemann pipette or other type of micropipette.</li>
	<li>Then, remove the tape from the window on the host egg and use the Spemann pipette to place the donor neural fold alongside the host embryo adjacent to the region of the neural tube that will receive the transplant.</li>
</ol>
7. Separate the Host Tissue and Transplant the Donor Tissue
<ol>
	<li>Separate the neural fold from the neural tube of the host embryo, as done with the donor. Take care to remove a graft of equal size as the donor tissue.&#160;</li>
	<li>Gently push this host tissue far away from the embryo.</li>
	<li>Then, with a blunt tungsten needle or the rounded tip of a micropipette, gently move the donor neural fold into the host neural tube, maintaining the proper antero-posterior and dorso-ventral orientations. Make sure the graft is tucked in along the sides but be sure not to poke or damage to the underlying tissues.&#160;To help keep track of the original orientation note any asymmetry in the donor tissue or differential staining from the Neutral Red. We also photo-document the donor embryo with the accompanying excised tissue, as well as the chimera immediately following surgery.</li>
	<li>Sterile saline should be added to the egg if the host embryo shows signs of desiccation.</li>
	<li>Now, carefully re-seal and label the egg, and gently return it to a high humidity incubator (70-80%) where the chimeric embryo can develop to the desired stage for analysis.</li>
</ol>
8. Collection of Chimeras
<ol>
	<li>Be sure to collect chimeric embryos in freshly made&#160;cold Serra&#8217;s fixative and immediately place them on a rocker at 4 &#176;C for an O/N fixation. This will allow for more&#160;sensitive detection of quail cells with the anti-quail antibody.</li>
	<li>For gene expression analyses via RT-qPCR, freeze embryos directly in liquid nitrogen prior to RNA extraction.</li>
</ol>

The authors have nothing to disclose.

The neural crest is a transient embryonic cell population that migrates extensively throughout the embryo and differentiates into diverse cell types, including chondrocytes and osteoblasts, that contribute to the craniofacial skeleton. Transplanting neural crest in the quail-duck chimeric system has contributed greatly to our understanding of the tissue interactions and signaling pathways that regulate development of the craniofacial skeleton. However, given the vast potential of neural crest to also generate smooth muscle cells, adipocytes, melanocytes, schwann cells, and neurons, the quail-duck chimera system has tremendous potential for future applications, particularly in conjunction with the rapid advancement of stem cell biology and regenerative medicine. Since quail and duck are both commercially bred species, a ready supply of relatively inexpensive fertilized eggs is available from a variety of farms. Thus, this technique should be accessible to researchers operating within a wide range of budgets and facility space.
Although this technique is very powerful, there remain several limitations. Like other surgical techniques, the quality and viability of quail-duck chimeras rely on the surgical skills of the researcher, and therefore, there will be more inter- and intra-individual variation between experiments as compared to other models, such as those utilizing mouse genetics. Moreover, there is also variation in the rates of development and stages of individual embryos that contributes to the reproducibility and success of each transplant. Avian embryos are also very susceptible to dehydration and therefore critical steps during surgery include keeping the light levels low, the time under the microscope to a minimum, the eggs sealed with tape as much as possible, and high humidity in the post-operative incubator to avoid desiccation.&#160;
In terms of viability of the chimeras, usually between 50-75% survive, although these percentages can decrease the older the collection stage.&#160;In a typical 4-6 hr session of surgery, an experienced surgeon can generate 10-15 chimeras.&#160;The success of the transplants also depends greatly on the quality of the tools. Good tools lead to more consistent, reproducible results. Using a propane torch to make tungsten needles allows extremely sharp needles to be made.&#160;The type of torch used makes a big difference because it controls the size of the flame. Electrolytic sharpening can also be used, but this approach does not even come close to producing needles as sharp.&#160;Use tungsten rods instead of spooled wire so that the needles can be made straight.
The Spemann micropipette, while time-consuming and difficult to make, is an ideal instrument for tissue&#160;transfer.&#160;The pipette can be customized with different-sized openings, and can be used repeatedly. A critical factor for using a Spemann&#160;micropipette is to have some fluid in the pipette before touching the tip to the surface of the embryo. Some of the fluid will always flow out when contact is made with the meniscus over the embryo.&#160;Pressing&#160;on the diaphragm allows fluid and graft tissue to be ejected very precisely, whereas slightly letting up on the diaphragm gently sucks the donor graft tissue into the pipette.&#160;Maintaining a bit of positive pressure on the diaphragm keeps the donor graft tissue at the tip of pipette during transfer, and a little additional pressure on the diaphragm allows the donor graft tissue to be deliberately placed in the host.&#160;
For protection of the Spemann pipette during storage and sterilization, remove the bulb from the wide end and carefully insert the tapered tip into the bulb.&#160;Place the Spemann pipette in a glass test tube, cover the top with aluminum foil, and autoclave prior to surgery. After multiple pipettes are ready to be sterilized again for surgery, invert the pipettes, heat them nearly to&#160;a boil in the same solution of distilled water and glassware detergent, and then rinse repeatedly with distilled water. Autoclave pipettes in their individual&#160;tubes. The rubber tubing that forms the diaphragm and the rubber bulb should be replaced after several sterilizations or when they become darkened and stiff.
Many of the components of this protocol involve dangerous equipment. For example, the procedure for making a Spemann Micropipette involves three types of flames as well as heating, pulling, blowing, bending, cutting, and polishing glass. Therefore, wearing proper personal protective equipment (PPE) that increases safety such as goggles and a lab coat is critical. Additionally, because many people suffer from, or have the potential to develop, egg allergies, always use gloves when handling eggs. With these precautions in mind, the quail-duck chimeric system is a safe, efficient, and relatively accessible method that has many future applications.

&#160;The facial skeleton develops from the growth and fusion of multiple facial processes that are composed of neural crest and mesodermal mesenchyme surrounded by ectodermal and endodermal epithelial layers1-11. Morphogenetic events within each process are governed by distinct signaling interactions between the mesenchyme and surrounding epithelia12-16. Alterations to these signaling interactions and/or their downstream effectors contribute to disease phenotypes and may also be relevant to evolution of the craniofacial skeleton17,18. Therefore, elucidating the timing and nature of tissue interactions has great potential to increase our understanding of the developmental and evolutionary biology of the facial skeleton.
The use of chimeric embryos to investigate tissue interactions has a long history in developmental biology. This approach was pioneered by Hans Spemann and his lab who discovered embryonic &#8220;organizers&#8221; by transplanting tissues between embryos of different amphibian species. Spemann was a master of micro-surgical techniques whose hand-skills were complemented by his development of specialized tools, notably the Spemann pipette. Viktor Hamburger was a graduate student in the laboratory of Hans Spemann in Freiburg during the 1920s, which is when the original transplant experiments that led to Spemann&#8217;s Nobel Prize were performed.&#160;When Hamburger moved to Washington University in St. Louis in 1935, he detailed the process of making a Spemann micropipette in his Manual of Experimental Embryology19.&#160;Drew Noden was a graduate student in Hamburger&#8217;s lab at Washington University until 1972.&#160;After moving to the University of Massachusetts, Amherst and then to Cornell University, Noden continued fabricating and using Spemann micropipettes for his surgical transplants involving quail-chick chimeras.&#160;While a graduate student, one of the authors (Rich Schneider) trained with Drew Noden at Cornell from 1995 to 1998.&#160;The following protocol for making a Spemann micropipette is based on descriptions written by Hamburger and Noden, and includes subsequent modifications made by Schneider.
The use of quail-chick chimeras for the study of craniofacial development and especially for understanding the contributions of neural crest cells was pioneered by Noden and by Le Douarin in the early 1970s, reviewed in Le Douarin et al20. This approach has been broadly adopted in many studies and by numerous other investigators1,4,5,21-38. The equivalent rates of growth and morphology of quail and chick make transplants within them ideal for the study of cell fate and lineage tracing. However, because of the similarities between quail and chick, morphological changes induced by donor cells are difficult to decipher. In contrast, other avian chimeric systems have included domestic duck as a way to study mechanisms that make embryos anatomically distinct39-50.&#160;More specifically, the quail-duck chimeric system offers multiple benefits for discerning the effects of the donor on the host, and vice versa. First, quail and duck embryos are distinct in body size and shape, which provides a direct way to explore donor- or host-specific mechanisms of pattern formation by assaying for differential domains of gene expression (Figures 1 A&#160;and B). Second, quail and duck embryos have considerably different rates of maturation, with quail hatching in 17 days and duck hatching in 28 days. Transplanted neural crest maintains its intrinsic maturation rate within the host environment, and thus, identification of temporal changes in gene expression, tissue interactions, histogenesis, and morphogenesis is possible51-57. Finally, the anti-quail nuclear antibody (Q&#162;PN) allows donor and host cellular contributions to be permanently distinguished from one another by recognizing a protein that is ubiquitously expressed in quail cells but absent from duck cells.

<table><tbody><tr><td>1x PBS</td><td>TEK</td><td>TEKZR114</td><td></td></tr><tr><td>Hank&amp;rsquo;s BSS w/o phenol red</td><td>Invitrogen</td><td>14025-092</td><td></td></tr><tr><td>Neutral Red</td><td>Sigma Aldrich</td><td>N4638-5G</td><td>0.22 &amp;micro;m filter-sterilized</td></tr><tr><td>18 G Needles</td><td>BD</td><td>305195</td><td></td></tr><tr><td>5 ml syringe</td><td>BD</td><td>309646</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>Straight Scissors</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Curved Scissors</td><td>Fine Science Tools</td><td>14029-10</td><td></td></tr><tr><td>Spemann Pipette</td><td></td><td></td><td>Hand-made in lab</td></tr><tr><td>Egg Holder</td><td></td><td></td><td>Glass ashtray and modeling clay</td></tr><tr><td>Alcohol Burner</td><td>Fisher</td><td>04-245-1</td><td></td></tr><tr><td>Transparent Tape</td><td>3M Scotch</td><td>600</td><td></td></tr><tr><td>Glass Stirring Rod</td><td>Fisher</td><td>11-380C</td><td>Tip is narrowed and rounded using a flame</td></tr><tr><td>Tungston wire (0.004 x 3 inches)</td><td>A-M Systems</td><td>7190</td><td>Tip is flame-sharpened in a propane torch</td></tr><tr><td>Bunsun Burner</td><td>Fisher Scientific</td><td>S49117</td><td></td></tr><tr><td>Pasteur Pipette</td><td>Fisherbrand</td><td>22-183-632</td><td>9-inch (229 mm)</td></tr><tr><td>Rubber Tubing</td><td>Fisher Scientific</td><td>14-178C</td><td>amber, thin wall natural rubber; wall thickness: 0.0625 in/1.6 mm; O.D.: 0.375 in/9.5 mm; I.D.: 0.25 in/6.4 mm</td></tr><tr><td>Propane Fuel Cylinder</td><td>BernzOmatic</td><td>UL2317</td><td>TX-9 with torch style &quot;A&quot; with a screw-on brass &quot;pencil flame&quot; torch</td></tr><tr><td>Diamond Point Pencil</td><td>Fisher Scientific</td><td>22-268912</td><td></td></tr><tr><td>Rubber Bulbs</td><td>Fisherbrand</td><td>S32325</td><td></td></tr></tbody></table>

assessing species-specific contributions to craniofacial development using quail-duck chimeras

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

Prior to further analysis, the efficiency of the transplant needs to be assayed. For histological, morphological, or gene expression analyses on tissue samples, quail cells should be detected by immunohistochemistry using Q¢PN antibody as described36. For RNA analyses, species-specific contributions to tissues of interest can be calculated using a PCR-based strategy58. After the efficacy of the transplant has been validated, further morphological or molecular outcome measures can be evaluated in chimeras. Interactions between the donor neural crest cells and surrounding host-derived tissues that underlie proper histogenesis and morphogenesis of the craniofacial complex have previously been extensively studied. In particular, neural crest mesenchyme directs species-specific morphology of the face7,13,51,59, feather pattern52, muscle pattern56, and cartilage53,57 through the regulation of host gene expression. For example, neural crest mesenchyme dictates when bone forms in the mandible by temporally regulating Bmp4 expression55.
Recently, investigations have centered on tissue interactions occurring very early in craniofacial development. In this regard, experiments utilizing quail-duck chimeras have shown host embryos influence neural crest migration by determining morphological boundaries. That is, in chimeric quck embryos, donor quail neural crest cells migrate into the host duck mandibular arch in a duck-like pattern (Figure 1D). Despite this host contribution to the size of the neural crest population, the donor neural crest continues to generate a mandibular skeleton that is quail-like in size and shape (Figure 1E).
<img alt="Quail-duck neural crest transplant study; adult skull comparison, embryonic development stages." fo:content-width="6in" fo:src="/files/ftp_upload/51534/51534fig1highres.jpg" src="/files/ftp_upload/51534/51534fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Quail-Duck Chimeric System. (A) Quail and (B) duck skulls display considerable differences in size and shape, and thus, are ideally suited for using a chimeric system to study craniofacial development (modified from Tokia et al.56). (C) Experimental design for generating unilateral chimeric quck embryos from stage-matched HH9.5 quail and duck embryos. The neural fold is removed from one side of quail embryos and transplanted into duck embryos after an equivalent piece of the neural fold has been removed. (D) Quail donor cells (green) can be followed in chimeras using an anti-quail antibody (Q¢PN) as shown in ventral view of HH12 chimeric embryo. (F) In quck mandibles at HH38, the quail donor-derived Meckel’s cartilage is shorter and straighter than that observed for the contralateral duck host-derived Meckel’s cartilage, which is larger and curved. Reprinted Tokita et al., Dev. Bio. 306, 377 (2007) with permission from Elsevier.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

The overall goal of this procedure is to use quail duct chimeras as a means to investigate mechanisms through which neural crust cells generate species specific patterns during cranial facial development. This is accomplished by first dissecting and removing a strip of the neural fold from the mid and anterior hind brain region of a stage 9.5 quail embryo. The second step involves transferring the quail donor neural fold to the duck host embryo.The third step involves dissecting and removing the equivalent region of a stage 9.5 duck host embryo. The final step is to place the coil neural fold into the duck, mid and anterior hindbrain region. Ultimately, the resulting chimeric embryos can pinpoint neural crest mediated changes in gene expression through in C two hybridization, and can also reveal morphological changes through histological analyses.I first had the idea for this method when I was watching Brown pelicans fly over Ocean Beach in San Francisco. I realized that neural crest cells must be the source of species specific pattern since they give rise to the bones and cartilages of their long beak skeleton. Although I was unable to track down brown pelican eggs, I was able to find white Peking duck.Given that San Francisco has the largest Chinatown outside of Asia By exploiting the differences in the rates of maturation and morphology between quails and ducks. This method can help us answer key questions in developmental biology such as understanding the origins of species specific pattern, Performing the surgery will be Dr.Jennifer Fish, who's a postdoctoral scholar In my laboratory, assisting will be Kate Ovitz, who is a graduate student Using a buns and burner heat the narrow end of a paste pipette until the glass along the taper begins to melt. Remove it from the flame and pull the tip until the pipette becomes sealed.Be sure that a portion of the tapered part has an outer diameter between 0.7 and one millimeter. Then break off the sealed end about four centimeters from the taper. Next, attach about two feet of rubber tubing to the open end of the pipette blow on the other end.There should be no air passing through the connection or other end of the pipette while providing gentle pressure by blowing on the tube. Use a propane pencil, flame torch to heat an oval area on one side of the pipette just below the start of the taper. As soon as the glass begins to buckle outwards, remove the pipette from the flame and blow gently through the rubber tubing.This will cause the heated part of the glass to form a sausage shaped bubble. Too much pressure will cause the glass bubble to pop, and this should be avoided. The resultant window in the pipette should be about one centimeter wide and one and a half to two centimeters long.Now point the tapered end upwards and scrape the bubble into a glass waste container while gently blowing through the tubing to prevent glass fragments from entering the pipette. Then using the propane flame, carefully burn off the remaining edges of the bubble and fire polish the sides of the open window. Next, hold the diamond tip pencil in a vertical position, use a diamond tip pencil to score the tapered end of the pipette one centimeter from the beginning of the taper.Using forceps, gently break the pipette at the scored line. To remove the tip, check under a dissecting microscope to make sure the break is clean and not jagged. Hold the pipette as one would hold a pencil with an index finger over the open window at approximately 9.5 millimeters from the tip.Soften the glass on the pipette by holding it adjacent to the edge of the flame of an alcohol burner. Apply pressure to the tip with forceps to bend the narrow end of the pipette down at a 60 degree angle under a dissecting scope. Quickly and carefully, fire polished the tip with the alcohol burner.The finished opening should be rounded and unobstructed. Note that the tip will become constricted and unusable if held in the flame too long. Now lubricate a 2.5 centimeter length of rubber tube with 70%ethanol.Slide it over the shaft of the pipette from the bottom up to cover the open window. Then place a two milliliter latex bulb onto the open end of the pipette. To maintain internal air pressure, first, prepare the host egg.Cut the Vitale membrane using a flame sharpened tungsten needle, and pull the membrane over the embryo. Then reseal the egg with tape as done for the donor egg. Remove the tape from the window on the donor egg to cut the neural fold from the donor embryo.Make slits on both sides of the neural tube using a flame sharpened tungsten needle. Then cut across the neural tube at the desired anterior and posterior levels of the graft and tease the graft apart from the rest of the neural tube. Pay attention to any particular anatomical details like the anterior versus posterior shape of the graft, which will the donor tissue to be oriented properly.When placed in the host, Be sure the tungsten needle is extremely sharp. Use quick and deliberate strokes in order to remove the tissue completely and in one piece, Remove the neural fold from the donor embryo using a shaman pipette. Draw up as little liquid as possible or the tissue could float away on transfer.Now remove the tape from the window on the host egg and use the shaman pipette to place the donor neural fold alongside the host embryo adjacent to the region of the neural tube that will receive the transplant. Now separate the neural fold from the neural tube of the host embryo as done with the donor. Take care to remove a graft of equal size as the donor tissue.Gently push this host tissue far away from the embryo. Then with a blunt tungsten needle or the rounded tip of a micro pipette, gently move the donor neural fold into the host neural tube, maintaining the proper anter posterior and dorsal ventral orientations. Make sure the graft is tucked in along the sides, but be sure not to poke or damage the underlying tissues.Sterile saline should be carefully and gently applied over the host embryo. If there are any signs of desiccation, now carefully reseal and label the egg and gently return it to a high humidity incubator where the chimeric embryo can develop to the desired stage. For analysis.For analysis, collect the chimeric embryos in freshly made cold Sarah's fixative. Then immediately place them on a rocker at four degrees Celsius for an overnight fixation. This will allow for more sensitive detection of quail eggs with the anti quail antibody.Another important step for gene expression analyses by real-time quantitative PCR is to freeze the embryos and liquid nitrogen prior to RNA. Extraction coils and ducks show considerable differences in size and shape, and thus their embryos are ideally suited for making a chimeric system. To study C craniofacial development.Specifically, the system is useful for investigating the role of neural crest cells. In patterning the facial skeleton in the chimeric qua embryo donor quail neural crest cells migrate into the host duck mandibular arch. In a duck like pattern, quail donor cells in green are viewed with an anti quail antibody from the ventral view.In a stage 12 chimeric embryo, the donor neural crest generates a mandibular skeleton that is quail like in size and in shape. Once mastered, each transplant can be done in approximately five minutes. If done properly any longer, puts the embryo at risk for desiccation and survival rate will decrease.While performing this procedure, it's important to be aware of your posture, relax your shoulders, and maintain good hand position. Also, develop a routine and place your surgical tools and reagents in the same place every time so that you can keep your eyes in the microscope and move your hands in an automated manner.

assessing-species-specific-contributions-to-craniofacial-development

Research

JoVE Journal

Biology

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

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

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

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

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.

Facial Transplants in Xenopus laevis Embryos

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Quantification of Orofacial Phenotypes in Xenopus

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’s cartilage.

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

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.

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

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

Creating Avian Forebrain Chimeras to Assess Facial Development

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

Learn to create quail-duck chimeras for studying neural crest contributions to craniofacial development. This method reveals species-specific patterns in bone and cartilage formation.

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

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