Joint formation in chick limb bud CAM grafts

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Joint Lab Protocol

Objective: To determine whether normal joint formation occurs in chick limb buds that have already begun cartilage patterning

Materials

8 day old donor eggs

10 day old host eggs

fine forceps

Scotch tape

gentle agitating device (we used Nutator TM )

5% Trichloroacetic acid

PBS

100% ethanol

methyl salicylate

dH2O

Alcian green stain

ethanol

Procedure

Adapted from Developmental Biology website:

I. Chorioallantoic Membrane Grafting (Hamburger, 1960)

For each host embryo*, complete the following procedure:

1. Sterilize a 10-day old donor egg with 70% ethanol.

2. Poke a hole into the blunt end and peel away the shell with forceps. Open a hole in the shell approximately an inch in diameter, or large enough to insert the graft and isolate veins. There will be a whitish membrane on the surface inside the opened shell. Carefully remove this white membrane without disturbing the clear CAM membrane immediately underneath. The CAM should remain intact in order to preserve the circulatory system of the host embryo so that it can distribute nutrients and oxygen to and from the graft.

3. Look for a large Y- shaped junction of blood vessels in the clear chorioallaintoic membrane. This is where the graft will be placed once the donor limbs are prepared. Peel back the white membrane around the Y-shaped junction to prepare the area for grafting. Cover the hole with scotch tape and store the egg in the incubator at 37 degrees Celcius.

4. Sterilize an 8-day old donor egg with 70% ethanol, and then transfer to a dish with Ringer's solution.

5. Remove all membranes surrounding the embryo.

6. Excise limbs (2 wing buds and 2 hind limbs) with a tungsten knife, and include an extra flap of cells from the flank to help move the graft into the proper position.

7. Transfer one limb graft to each host egg.

8. Position transferred limb over the large Y- junction.

9. Re-tape the eggs to prevent infection and desication. Incubate for 7-10 days (but not more than 10 or the host chick will hatch! ) before removal of the graft for staining.

10. Carefully remove the tape covering the shell opening above the CAM graft (you may want to candle the embryos in a dimly lit room so as to avoid opening already-dead host chicks. The dead hosts will show little vasculature, a sallow yellow coloring on the inside of the egg, and no sign of a dense mass surrounded by extensive vasculature indicating the presence of the living host embryo).

11. Gently use forceps to excise the limb graft, which should appear as a dense mass of tissue often darker around the edges that the host tissue, with possible feather bud formation. In order to excise the limbs, you may need to clip the CAM and the surrounding vasculature as it often has a tendency to adhere to the limb bud.

*36 host embryos were used in this experiment, each containing a single limb graft. The average survival rate for host chicks was 11/16, with the successful retrieval of about 18 limbs. Therefore, have a large population of host chicks to ensure a sufficient enough sample of limbs in order to more accurately assess the occurrence of joint formation.

II. Preparing the control embryo

1. Retain one 8-day old embryo to use later as a control.

2. Excise the chick from its shell after the incubation period, and remove

Limb buds

3. Stain using Alcian green

III. Alcian green staining for cartilage formation

1. Wash 3X with PBS.

2. Fix limbs or embryos in 5% TCA for 1 hour at room temperature.

(Eviscerate embryos before fixation).

3. Immerse in Alcian green stain for 4 hours (or preferably overnight*) with gentle agitation.

*Note: If limbs are immersed in the stain overnight, the following ethanol treatments should also be conducted overnight to effectively dehydrate the limbs before the methyl salicylate wash.

4. Wash 3X with 70% ethanol. Each wash should be at least two hours long, but it can be left overnight.

5. Wash with 85%ethanol, 95% ethanol, 100% ethanol, several hours to overnight each.

6. Clear by washing 1-3X with methyl salicylate. The limb bud grafts will begin to become transparent after immersion in the methyl salicylate. The grafts can remain in this solution for an indeterminate amount of time. Photograph limb buds and record observations.

J. N. White 5/13/04

@ Cebra-Thomas, 2000
Last Modified: 13 May 2004

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		Joint Lab Protocol Objective: To determine whether normal joint formation occurs in chick limb buds that have already begun cartilage patterning Materials 8 day old donor eggs 10 day old host eggs fine forceps Scotch tape gentle agitating device (we used Nutator TM ) 5% Trichloroacetic acid PBS 100% ethanol methyl salicylate dH2O Alcian green stain ethanol Procedure Adapted from Developmental Biology website: I. Chorioallantoic Membrane Grafting (Hamburger, 1960) For each host embryo, complete the following procedure: 1. Sterilize a 10-day old donor egg with 70% ethanol. 2. Poke a hole into the blunt end and peel away the shell with forceps. Open a hole in the shell approximately an inch in diameter, or large enough to insert the graft and isolate veins. There will be a whitish membrane on the surface inside the opened shell. Carefully remove this white membrane without disturbing the clear CAM membrane immediately underneath. The CAM should remain intact in order to preserve the circulatory system of the host embryo so that it can distribute nutrients and oxygen to and from the graft. 3. Look for a large Y- shaped junction of blood vessels in the clear chorioallaintoic membrane. This is where the graft will be placed once the donor limbs are prepared. Peel back the white membrane around the Y-shaped junction to prepare the area for grafting. Cover the hole with scotch tape and store the egg in the incubator at 37 degrees Celcius. 4. Sterilize an 8-day old donor egg with 70% ethanol, and then transfer to a dish with Ringer's solution. 5. Remove all membranes surrounding the embryo. 6. Excise limbs (2 wing buds and 2 hind limbs) with a tungsten knife, and include an extra flap of cells from the flank to help move the graft into the proper position. 7. Transfer one limb graft to each host egg. 8. Position transferred limb over the large Y- junction. 9. Re-tape the eggs to prevent infection and desication. Incubate for 7-10 days (but not more than 10 or the host chick will hatch! ) before removal of the graft for staining. 10. Carefully remove the tape covering the shell opening above the CAM graft (you may want to candle the embryos in a dimly lit room so as to avoid opening already-dead host chicks. The dead hosts will show little vasculature, a sallow yellow coloring on the inside of the egg, and no sign of a dense mass surrounded by extensive vasculature indicating the presence of the living host embryo). 11. Gently use forceps to excise the limb graft, which should appear as a dense mass of tissue often darker around the edges that the host tissue, with possible feather bud formation. In order to excise the limbs, you may need to clip the CAM and the surrounding vasculature as it often has a tendency to adhere to the limb bud. 36 host embryos were used in this experiment, each containing a single limb graft. The average survival rate for host chicks was 11/16, with the successful retrieval of about 18 limbs. Therefore, have a large population of host chicks to ensure a sufficient enough sample of limbs in order to more accurately assess the occurrence of joint formation. II. Preparing the control embryo 1. Retain one 8-day old embryo to use later as a control. 2. Excise the chick from its shell after the incubation period, and remove Limb buds 3. Stain using Alcian green III. Alcian green staining for cartilage formation 1. Wash 3X with PBS. 2. Fix limbs or embryos in 5% TCA for 1 hour at room temperature. (Eviscerate embryos before fixation). 3. Immerse in Alcian green stain for 4 hours (or preferably overnight) with gentle agitation. Note: If limbs are immersed in the stain overnight, the following ethanol treatments should also be conducted overnight to effectively dehydrate the limbs before the methyl salicylate wash. 4. Wash 3X with 70% ethanol. Each wash should be at least two hours long, but it can be left overnight. 5. Wash with 85%ethanol, 95% ethanol, 100% ethanol, several hours to overnight each. 6. Clear by washing 1-3X with methyl salicylate. The limb bud grafts will begin to become transparent after immersion in the methyl salicylate. The grafts can remain in this solution for an indeterminate amount of time. Photograph limb buds and record observations.
		J. N. White 5/13/04