Fertilised GFP chicken eggs were supplied by Prof. Helen Sang, The Roslin Institute, and University of Edinburgh, UK. The Roslin Transgenic Chicken Facility is funded by the Wellcome Trust and by the Biotechnology and Biological Sciences Research Council (BBSRC). The work was partly funded, and NT supported, by Great Ormond Street Hospital Children&#8217;s Charity, London, UK.&#160;The authors thank Ben Jevans, UCL Institute of Child Health, for help with preparing embryos for grafting.

1. Preparation of Micro-scalpel for Neural Tube Ablations
<ol>
	<li>Shape a micro-scalpel from a commercially available steel sewing needle.
	<ol>
		<li>First flatten the needle on both sides using a grinding wheel mounted on a powered bench grinder.</li>
		<li>Start shaping the scalpel, first on a coarse grade Arkansas stone using a controlled circular motion, in alternate directions, on both sides of the needle.</li>
		<li>Carry on the same sharpening movements on an extra fine grade Arkansas stone to shape an ultra fine micro-scalpel, with a well defined cutting edge (Figure 1A, B). 
		NOTE: Alternatives to the micro-scalpel could be electrolitically sharpened needles, commercially available tungsten needles, or pulled glass needles.</li>
	</ol>
	</li>
</ol>
2. Incubate Wild Type and GFP Eggs to the Desired Stage
<ol>
	<li>Store fertilized chicken eggs and transgenic GFP chicken eggs in a cooled incubator at 14 - 15 &#176;C prior to incubation as development is halted at this temperature. Store eggs for a few days, up to one week.</li>
	<li>To commence development, place wild type and GFP eggs on a tray horizontally and incubate simultaneously in a humidified (58 - 60%) incubator at 37.5 &#186;C, so that embryos are at matching stages for neural tube grafting.</li>
	<li>To obtain embryos at the 10 - 12 somite stage of development for carrying out vagal neural tube grafting, incubate eggs for 1.5 days (33 - 38 hrs) and stage embryos according to the development tables of Hamburger and Hamilton21.</li>
</ol>
3. Prepare Eggs for Windowing and Grafting
<ol>
	<li>Move one egg at a time to a custom-made egg holder for windowing. Make a small hole in the egg shell by repeatedly tapping, with straight scissors, on the upper surface of the pointed end of the egg.</li>
	<li>Remove 2 - 3 ml of albumin from the egg with an 18&#189; G hypodermic needle and 5 ml syringe. Removing the albumin lowers the yolk within the egg and facilitates subsequent windowing without causing any damage to the embryo.
	<ol>
		<li>Discard the albumin. Seal the hole with a small strip of clear tape cut to size with fine scissors.</li>
	</ol>
	</li>
	<li>Using curved scissors, tap another hole in the upper surface of the egg shell. Insert the tip of the scissors into the hole and, keeping the scissors parallel to the bench, work in a circular motion to cut a ~2 cm diameter window on top of the shell.
	<ol>
		<li>Keep the scissors in a stationary position and rotate the egg. Discard the removed disk of egg shell. At E1.5, the embryo is recognizable as a darker yellow disk on top of the yolk.</li>
		<li>Remove any shell debris that has fallen within the egg using tweezers. Discard any unfertilized eggs (identified by a small white spot on top on the otherwise light yellow yolk).</li>
	</ol>
	</li>
</ol>
4. Prepare the Host Embryo to Receive Grafted Tissue
<ol>
	<li>Adjust the stereo-microscope to eye level and optimize the orientation of gooseneck light source to adequately illuminate the embryo without causing reflections.</li>
	<li>To visualize the embryo proper, inject a small amount of Indian ink under the center of the darker yellow disk, using a mouth tube and a pulled glass micro-pipette (Figure 1C, Oii).
	<ol>
		<li>Prepare the ink 50:50 with PBS containing Penicillin/Streptomycin at 100 &#956;g/ml final concentration. Insert the micro-pipette through the yolk membrane outside the perimeter of the blastoderm then carefully angle its tip directly beneath the embryo.</li>
		<li>Deliver ink underneath the embryo by blowing on the mouth tube. If mouth pipetting is not permitted, use a 1 ml syringe instead. Be careful not to introduce any air bubbles underneath the embryo, which can lead to contamination, then carefully remove the glass micro-pipette. This is a delicate step that can lead to the death of the embryo if not done with precision.</li>
	</ol>
	</li>
	<li>Stage the embryo by reference to Hamburger and Hamilton21 and record the stage in a lab book.</li>
	<li>Using a custom made micro-scalpel (or a fine tungsten needle) mounted on a needle holder, make a very small gash in the vitelline membrane, next to the area where the micro-surgery will be performed.
	<ol>
		<li>Carefully apply 2 - 3 drops of PBS over the membrane tear (using a glass micro-pipette and the mouth tube) to create space between the embryo and the membrane. Cut a larger window in the membrane to expose the entire region where the micro-surgery will take place.</li>
	</ol>
	</li>
	<li>Remove the neural tube region of interest using the micro-scalpel, starting with rostral and caudal transverse incisions across the entire dorsal neural tube (at the level of somite 1 to 7 in the video).
	<ol>
		<li>Cut bilaterally between the neural tube and the somites to separate the neural tube from surrounding tissues, without damaging the somites.</li>
		<li>Very gently separate the neural tube from the underlying notochord, which should remain intact. Note that successful neural tube excision will leave all surrounding tissues perfectly intact (Figure 2).</li>
		<li>Remove the excised neural tube by aspirating it into a glass micro-pipette, then discard.</li>
	</ol>
	</li>
	<li>Record the level of neural tube ablation in a lab book. The host embryo is now ready to receive the donor neural tube.</li>
</ol>
5. Prepare the Donor Graft Tissue
<ol>
	<li>Select a windowed, stage-matched GFP embryo by viewing under a fluorescent stereo-microscope with FITC filter. The GFP fluorescence makes it very easy to visualize the somites and stage the embryo.</li>
	<li>Once a stage-matched embryo has been identified, remove the embryo from the egg by making 4 incisions, with Pascheff-Wolff spring scissors (Figure 1C, l) in a rectangle shape around the embryo then gently pick it up with an embryo spoon.</li>
	<li>Place the embryo in a square watch glass with a sylgard polymer base. Gently shake the embryo with Dumont #5 tweezers to remove any attached yolk. Remove the vitelline membrane and pin the embryo onto the polymer base using stainless minutien pins (Figure 1C).</li>
	<li>Using the Spring Scissors, make 4 incisions in a rectangular shape around the neural tube and surrounding somites, in the same region that has been removed from the host embryo.</li>
	<li>Using a plastic transfer pipette, transfer the neural tube and somite tissues from the donor GFP embryo into a watch glass containing 0.2% pancreatin in Pen/Strep PBS.</li>
	<li>Allow enzymatic digestion to proceed for 10 min at RT to help separate the tissues. After incubation in enzyme, use stainless minutien pins mounted on a handle to manually separate the neural tube from all adjacent tissues.</li>
	<li>Using a glass micro-pipette, transfer the dissociated neural tube to another watch glass containing DMEM + 10% serum (e.g., goat, horse or fetal calf) on ice, to rinse the excess pancreatin and stop enzymatic digestion. After 5 min, the dissected neural tube is ready to be grafted orthotopically into the chick host (Figure 2 and S1).</li>
</ol>
6. Graft the Tissue
<ol>
	<li>Using a glass micro-pipette, carefully transfer the dissected neural tube from the watch glass to the host embryo. Position the neural tube in the correct anterior-posterior orientation and gently push the explant adjacent to the excised region of the chick host using the micro-scalpel. Leave a small fragment of ectoderm attached to, or by cutting a small nick in, the dorsal surface to identify the orientation of the neural tube.
	<ol>
		<li>If necessary, use the micro-scalpel to trim the explant to the exact size of the excised region.</li>
	</ol>
	</li>
	<li>Gently guide the neural tube into the ablated region and position it such that the dorsal side is correctly orientated. Use a glass micro-pipette, mounted to a mouth tube, to remove PBS and/or fluid surrounding the graft. This helps the donor and host tissues to adhere and the graft to become established.</li>
	<li>Seal the entire window with 24 mm wide clear tape to prevent dehydration and contamination.</li>
	<li>Label the chimeric embryo by marking with a pencil on the eggshell and record its number in the lab book. Return the egg to the incubator for further development.</li>
</ol>
7. Inject DiI into Blood Vessels of Host Embryo
<ol>
	<li>At the desired experimental time point (here, 3 - 10 days later), retrieve the chimeric embryo from the incubator and remove the clear tape using straight scissors to gain access to the embryo within the egg.</li>
	<li>If necessary, enlarge the window in the shell using the scissors. Be careful not to damage the chorioallantoic membrane if it is attached to the shell, which would result in hemorrhage and jeopardize the blood vessel labeling.</li>
	<li>Choose an accessible vein on the yolk making sure the blood flow is directed towards the embryo. Choose a branching point of one of the vitelline veins (Figure 3B, C). 
	NOTE: At E6.5 - E7.5, the chorioallantoic membrane may need to be gently moved aside with tweezers to access the yolk veins. After E8.5, the only option is to inject into one of the chorioallantoic membrane veins since, by this stage, the chorioallantoic membrane fully covers the embryo.</li>
	<li>Remove the vitelline membrane above the chosen injection point using two Dumont #5 tweezers by tearing in opposite directions.</li>
	<li>Break a pulled glass needle using a Dumont #5 and adjust its diameter to the approximate size of the vein prior to loading with CellTracker CM-DiI. Make the DiI stock solution at 40 &#181;g/&#181;l in DMSO and store at -20 &#176;C. Prepare the working solution in 0.3 M sucrose/PBS at a concentration of 4 &#181;g/&#181;l.
	<ol>
		<li>Aspirate between 5 - 10 &#181;l of DiI in 0.3 M sucrose/PBS into the needle using suction with a mouth tube. Older embryos might require up to 25 &#181;l or more. From E8.5, embryos have larger, more muscular veins, which may need to be held in position with a Dumont #5 before stabbing with the DiI-loaded glass needle.</li>
	</ol>
	</li>
	<li>Swiftly insert the needle into the vein and blow steadily with the mouth tube to allow the DiI to join the blood flow slowly without forming a clot. Alternatively, use a pressure injector for DiI delivery.</li>
</ol>
8. Harvest Embryos for Sectioning or Wholemount Examination
<ol>
	<li>To retain as much DiI within the embryo as possible, harvest the embryo immediately after the injection by scooping it onto a perforated spoon and cutting the blood vessels and connective tissues with a pair of straight scissors, to free the embryo from the yolk.</li>
	<li>Remove any loose membranes and dissect out the organs of interest (i.e., the lungs and digestive tract in this tutorial), taking great care not to compress the tissue, which creates diffusion of the DiI. Immediately fix the tissues by immersion in 4% PFA for 1 - 2 hrs at RT.</li>
	<li>Rinse the tissue for 5 min in PBS, then 15 min in PBS containing 5 &#956;g/ml DAPI. Mount the samples on a bridged microscope slide for whole mount examination or embed them for cryo-sectioning.</li>
</ol>

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The authors declare that they have no competing financial interests.

The method of intraspecies neural tube grafting, combined with blood vessel labeling described here, takes full advantage of the ease of access of the avian embryo within the egg (compared with other vertebrate embryos) to study the co-development of an element of the autonomic nervous system (the ENS) and the vascular system.
For labeling NCC derivatives, the chickGFP-chick intraspecies grafting method we describe has a number of advantages over the classical quail-chick chimera method that was established over 40 years ago1-3. First, under FITC light, GFP fluorescence is extremely bright, to the extent that GFP+ cells are easily discernable in live chimeric embryos. This allows the success of the graft to be checked in ovo, whereas quail-chick grafting requires the embryo to be killed, processed and immunostained using QCPN, before the success of the graft can be ascertained2. Second, GFP expression in the transgenic chickGFP is cytoplasmic, therefore it not only labels cell bodies, but also allows the projections of the transplanted cells to be visualized22. This permits intricate neuronal networks to be observed at high resolution (note that fine projections are best visualized when the sample is immunostained with anti-GFP antibody). As QCPN labeling is restricted to the quail cell nucleus, such networks are not revealed using quail-chick chimeras. Third, intraspecies grafting eliminates any potential species differences between cells within the chimeric embryo. Since quail embryos have a shorter incubation period than chick (19 days versus 21 days) it has been suggested that quail cells have a higher proliferation rate than chick cells, which could potentially affect the development of the chimeric tissues23. Interestingly also, it has been shown in plants that interspecies grafting could produce extensive alterations in DNA methylation patterns in the host 24. Fourth, chickGFP facilitates back-transplant experiments to address topics such as NCC fate and cell commitment25. Fifth, the transgenic chickGFP is also useful for many other techniques including FACS sorting of GFP+ cell subpopulations, organotypic culture of organs containing GFP+ cells, genetic manipulation of GFP+ grafted tissue via electroporation of expression plasmids26, and other imaging technologies such as optical projection tomography27.
The neural tube transplantation approach can be modified by microsurgically replacing shorter amounts of neural tube. By using smaller segments of neural tube the microsurgery is potentially less damaging to the embryo and survival can be improved. However, the downside of transplanting less neural tube is that the numbers of GFP+ NCC in the host will be reduced. Users could try and achieve a balance between the amount of neural tube transplanted to give optimum survival of embryos, and the numbers of GFP+ NCC within host intestine sufficient to give informative results.
For vessel painting, DiI has the advantage that its fluorescence is very bright and robust. Also, it has the capacity to diffuse during fixation assuring staining of the finest opened capillaries. Since it is a vital dye, embryos can survive the injection procedure and carry on developing with a stained vascular system (up to 24 hr in our hands, although the staining becomes more punctate over time, see Figure 3E). The combination of chickGFP grafting with DiI vascular painting is therefore compatible with live imaging. Besides all these advantages, it is important to note that vascular injection only labels luminised vessels and therefore does not identify unopened capillaries, endothelial tip cells or isolated endothelial cells. However, further progress in avian transgenesis could provide new ways to circumvent such issues, as exemplified by experiments using Tg(tie1:H2B-eYFP) quail embryos to study vascular morphogenesis28. Another limitation of this technique is that, for effective vessel labeling in embryos at E7.5 and beyond, larger amounts of dye need to be injected, which can make experiments expensive. However, a modification of the technique could include low cost blood vessel labelingusing highlighter ink14, although this approach has not been tried in our hands.
Critical steps of the procedures include the process of visualizing the embryo by injecting ink underneath the blastodisc. If the membrane covering the yolk is torn by the ink-filled needle at this stage then embryo survival is severely compromised. Also, it is important, when preparing a donor neural tube, that the tissue is not left for an excessively long time in pancreatin (consider approximately 10 min&#160;as a maximum). Prolonged exposure to pancreatin damages the tissue and the neural tube is then difficult to handle and it will not incorporate well into the host.&#160;Gaining experience of the DiI injection technique on wild type embryos is essential before injecting chimeric embryos, as only one attempt at injecting is generally possible for each embryo. DiI volume and needle diameter are critical parameters for each embryo and should be assessed on wild type, stage matched controls. &#160;
In conclusion, our dual labeling method of neural tube transplantation and DiI vessel painting in live chick embryos can be used to investigate the inter-relationships between NCC and blood vessel networks during organogenesis. Considering the mechanisms responsible for establishing correct target innervation and vascularization during organ development are still largely unknown, this methodology holds potential for future discoveries in this field.

The chicken embryo is an invaluable model organism in vertebrate developmental biology, not least because its development in ovo permits experimental manipulations that are otherwise impossible to perform in vertebrates that develop in utero. This accessibility and ease of manipulations has led to the chick embryo playing key roles in many seminal discoveries in the field of developmental biology. Amongst the most powerful techniques has been the use of quail-chick chimeric embryos to study cell fate, a method pioneered by Professor Nicole Le Douarin in the 1970&#8217;s1-3. In particular, quail-chick chimeras have been especially useful to genetically mark and follow highly migratory neural crest cell (NCC) populations during early development. NCC are a multipotent population of migratory cells, arising in the dorsal ectoderm at the margins of the neural tube, that give rise to a wide range of cell types throughout the vertebrate embryo. These include craniofacial structures (cartilage, bone, muscles), neurons and glia (in the sensory and autonomic nervous systems), melanocytes, and a subpopulation of cells of the endocrine system2,4,5. One of the most important factors influencing NCC fate is their initial location along the anterior-posterior axis of the neural tube. For example, enteric NCC, which give rise to the neurons and glia of the enteric nervous system (ENS), arise from two discrete sub-populations: the first located in the vagal (caudal hindbrain) region, and the second in the sacral region of the neural tube6-13. Inter or intra-species grafting of the corresponding regions of the neural tube have been the techniques of choice to permanently label these cells and subsequently allow tracking, from their birth on the margins of the neural tube, to their final destinations within the digestive tract6,7,10.
Another embryonic manipulation easier to perform in chick, compared to other animal models, is the vital labeling of the vascular system. Indeed, as the chick embryo develops, it lays on top of an extra-embryonic vascular network that circulates oxygen and nutrients from the yolk. This accessible vascular network, located on the surface of the yolk, can be used as a gateway to label the embryo&#8217;s developing vascular system during organogenesis12,14-17. Intravascular injection of various dyes, such as the lipophilic dye DiI, makes it possible to delineate/stain all the luminised vessels of the nascent vascular network.
Because 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), the two networks develop alongside each other and share striking similarities in their branching architecture18-20. Here we report embryonic manipulations that allow us to study the simultaneous development of the NCC-derived ENS, along with the vascular system, during organogenesis. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, including the neural crest, combined with vascular DiI injection. As an advance from quail-chicken chimeras, our method uses transgenic GFP chick embryos for intraspecies grafting, making the transplant technique more powerful, in terms of imaging cells and their projections, and eliminating any potential bias linked to species differences.

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			<td height="21" style="height:21px;width:528px;">Fertilised chick eggs</td>
			<td style="width:312px;">Henry Stewart and Co, Louth, UK</td>
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			<td height="40" style="height:40px;width:528px;">Fertilised GFP chick eggs</td>
			<td style="width:312px;">The Transgenic Chicken Facility, The Roslin Institute, The University of Edinburgh</td>
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			<td height="21" style="height:21px;width:528px;">Egg incubator (Profi-H Hatcher)</td>
			<td>Lyon Technologies, CA, USA</td>
			<td style="width:320px;">910-033</td>
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			<td style="width:312px;">Precision Cooled Incubator, Leec Ltd., Nottingham, UK</td>
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			<td height="21" style="height:21px;width:528px;">Goose neck halogen cold light source</td>
			<td style="width:312px;">Advanced Imaging Concepts, Inc</td>
			<td>KL 1500 LCD&#160;</td>
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			<td height="21" style="height:21px;width:528px;">181&#8260;2 G hypodermic needle&#160;</td>
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			<td style="width:320px;">HSWNH181</td>
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			<td style="width:312px;">SIGMA - ALDRICH</td>
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			<td style="width:312px;">SIGMA - ALDRICH</td>
			<td style="width:320px;">Z350796&#160;</td>
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			<td height="21" style="height:21px;">Borosillicate glass capillaries, thin wall without filament</td>
			<td>Harvard apparatus</td>
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			<td>Fine Science Tools</td>
			<td>14058-09</td>
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			<td>Fine Science Tools</td>
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			<td height="21" style="height:21px;">Needle holders (Nickel-plated pin holder)&#160;</td>
			<td>Fine Science Tools</td>
			<td>26018-17&#160;</td>
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			<td height="21" style="height:21px;">Pascheff-Wolff Spring Scissors&#160;</td>
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			<td style="width:320px;">15371-92</td>
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			<td height="21" style="height:21px;">Dumont #5 forceps&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>11251-30&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Minutien pins&#160;</td>
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			<td style="width:320px;">26002-15</td>
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			<td height="21" style="height:21px;">Dumont AA forceps, Inox Epoxy- coated&#160;</td>
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			<td>11210-10&#160;</td>
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			<td height="21" style="height:21px;">Perforated spoon</td>
			<td>Fine Science Tools</td>
			<td style="width:320px;">10370-18</td>
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			<td>Fine Science Tools</td>
			<td style="width:320px;">10130-05</td>
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			<td height="21" style="height:21px;width:528px;">Sellotape (clear, 24mm width)</td>
			<td style="width:312px;">Any Supplier</td>
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			<td height="21" style="height:21px;width:528px;">Pen/Strep (Penicillin, Streptomycin) Solution</td>
			<td style="width:312px;">VWR international</td>
			<td>101447-068&#160;</td>
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			<td height="21" style="height:21px;width:528px;">Sylgard 184 silicone elastomer kit</td>
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			<td>S09 512 516</td>
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			<td height="21" style="height:21px;">Pelikan black ink</td>
			<td style="width:312px;">Pelikan</td>
			<td>211-169</td>
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			<td height="21" style="height:21px;">CellTracker CM-DiI&#160;</td>
			<td style="width:312px;">Molecular Probes</td>
			<td style="width:320px;">C-7001</td>
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			<td height="21" style="height:21px;width:528px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
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			<td style="width:320px;">D1306</td>
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			<td height="21" style="height:21px;">Settings for glass needle puller&#160;</td>
			<td style="width:312px;">Sutter Instruments</td>
			<td>Flaming/Brown micropipette puller model P-86</td>
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			<td height="21" style="height:21px;">Heat 950; Pull 150; Velocity 100; Time 200; Pressure 500</td>
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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.

Figure 1 shows typical instruments required to carry out microsurgical isolation and transplantation of the neural tube. Figure 2 shows the transplant procedure. Following transplantation embryos are screened for transplant success. This involves examining the embryo under a stereo fluorescence microscope, typically the morning after microsurgery, for the presence of graft-derived (GFP+) NCC. If transplantation has been a success, then GFP+ NCC can be observed in the vicinity of the neural tube and in early migration pathways leading toward the foregut. If the procedure has not been successful, GFP+ NCC will not be observed outside the neural tube, or if they are present in the host they may be in smaller numbers. These unsuccessful embryos are discarded. Typically, 5-8 neural tube transplants are performed in one day, and of these 80% are successful. Reasons for unsuccessful neural tube transplantation include death of the embryo due to tissue damage incurred during microsurgery, or failure of the neural tube to integrate into the host embryo. The latter can result from poor placement of the neural tube within the host or from a poor quality neural tube due to poor dissection technique or from excessive exposure to dissociation enzyme. The initial screening step, as well as similar later examinations for GFP+ cells, is useful as it means that time and resources are not wasted by performing experiments on embryos that do not have GFP-labelled NCC within the gut.
Figure 3 shows the procedure for DiI injection of the blood vessels.&#160;The efficiency/success of the DiI injection technique depends on: first, cutting the injection needle to the optimum diameter for the targeted vein, second a precise gesture when inserting the needle in the vein (so not to pierce through the other side), and third avoiding the needle getting plugged during the injection by blowing at a constant rate. If any of these three parameters is done incorrectly, the embryo will bleed out or will need several hours to recover before a second attempt is made as the hemorrhage makes it almost impossible to re-inject immediately. Successful embryos should be selected immediately by viewing under a stereofluorescence microscope and must be dissected rapidly. In successful embryos, DiI labeled blood vessels are present throughout the embryo (Figure 3C,D) including capillary beds (Figure 3D).
Upon harvesting of embryos and examination of tissue sections or wholemount gastrointestinal tracts, typical results reveal GFP+ NCC within the primitive ENS and the fine structure of the DiI-labeled gut blood vessel networks (Figure 4) Wholemount preparations can be examined using confocal microscopy whereby image stacks produce three dimensional (3D) reconstructions showing the interrelationships between the fine projections of GFP+ ENS cells and the DiI stained vascular system (Figure 4 A-C; G-I; Videos 1 and 2).
<img alt="Figure 1" src="/files/ftp_upload/52514/52514fig1.jpg" /> 
Figure 1. Recommended microsurgery instruments.&#160;(A) micro-scalpel shaped from a sewing needle. (B) fine Arkansas stone for shaping a micro-scalpel. (C) a) straight scissors, b) curved scissors, c) 5 ml syringe with 181/2 G hypodermic needle, d) plastic pipette, e) custom made egg holder, f) black ink, g) square watch glass, h) square watch glass with black sylgard base, i) micro-scalpel on needle holder, j) minutien pins, k) minutien or tungsten needle on needle holder, l) Pascheff-Wolff spring scissors, m) Dumont #5 tweezers, n) perforated spoon, oi) short fire-pulled transfer needle, oii) long fire-pulled inking needle, p) mouth tube. <a href="https://www.jove.com/files/ftp_upload/52514/52514fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52514/52514fig2.jpg" /> 
Figure 2. Intraspecies neural tube transplant. Chick embryo/GFP neural tube images have been modified from Delalande et al.12. Vascularization is not necessary for gut colonization by enteric neural crest cells. <a href="https://www.jove.com/files/ftp_upload/52514/52514fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52514/52514fig3.jpg" /> 
Figure 3. Intravenous DiI injection. (A)&#160;Recommended instruments: a) CellTracker CM-DiI drop on parafilm, b) pulled glass injection needle, c) mouth tube. (B)&#160;Schematic diagram of intravenous DiI injection into E4 chimeric chick embryo. (C)&#160;in ovo DiI intravenous injection showing fine glass needle containing DiI inserted into vein (arrow). (D) E4 chimeric&#160;embryo post DiI injection (red) with GFP+ neural tube (arrow).&#160;(E) DiI stained fine blood vessel network in a live embryo, 24 hr post-injection. Br: brain; H: heart; LB: limb bud; A: allantois. Images in (C) and (D) have been modified from Delalande et al.12 Vascularization is not necessary for gut colonization by enteric neural crest cells. <a href="https://www.jove.com/files/ftp_upload/52514/52514fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52514/52514fig4.jpg" /> 
Figure 4: Representative results in the stomach and caecum of an E5.5 chick embryo. (A-C)&#160;3 dimensional (3D) reconstruction of a confocal image stack in the region of the stomach showing (D) the GFP+ enteric neural crest cells (ENCC) (E) the DiI stained vascular system and (F) a merged image of both networks D-F Histological sections at the level of the stomach showing (G) the GFP+ ENCC (H) the DiI stained vascular system and (I) a merged image of both networks. Nuclei are stained with DAPI (cyan).(G-H) 3D reconstruction of a confocal image stack in the caecum region showing (A) the GFP+ ENCC migration front in green, (B) the DiI stained vascular system in red, and (C) a merged image of both networks. Images (A-F) have been modified from Delalande et al.12 Vascularization is not necessary for gut colonization by enteric neural crest cells. <a href="https://www.jove.com/files/ftp_upload/52514/52514fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure S1" src="/files/ftp_upload/52514/52514figS1.jpg" /> Figure S1.&#160;Isolation of a donor GFP+ neural tube from the surrounding tissues by enzymatic digestion and micro-dissection. (A)&#160;GFP+ neural tube and adjacent somites dissected from the donor embryo. (B) Isolated neural tube after pancreatin digestion and micro-dissection using stainless minutien pins. So: somites; NT: neural tube; Nc: Notochord.
<img alt="video 1" src="/files/ftp_upload/52514/52514video1.jpg" /> Video 1.&#160;3-dimensional 360&#176; rotation of the image in Figure 4C, showing the vascular system and the ENCC in the stomach at E5.5 (HH27-28). <a href="https://www.jove.com/files/ftp_upload/52514/JoVE_Video1.avi" target="_blank">Please click here to view this video.</a>
<img alt="Video 2" src="/files/ftp_upload/52514/52514video2.jpg" /> Video 2.&#160;3-dimensional 360&#176; rotation of the image in Figure 4I, showing the vascular system and the ENCC migration front in the region of the caecum at E5.5 (HH27-28). <a href="https://www.jove.com/files/ftp_upload/52514/JoVE_Video2.avi" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection at JoVE.co

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.

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, ...

dual-labeling-neural-crest-cells-blood-vessels-within-chicken-embryos

Research

JoVE Journal

Developmental Biology

8.0K Views.  UCL Institute of Child Health. 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. 

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...