Chorioallantoic membrane grafting with chick embryo limb buds
Objective
This experiment explores the ability of the chick chorioallantoic membrane (CAM) to support an excised limb bud from a donor embryo. The chick system will allow observation of general cartilage formation in limb grafts on the chorioallantoic membrane and to study the role of the CAM in calcium transport in ovo.
Introduction
Extraembryonic membranes regulate crucial functions in the chick egg, including water retention and gas and ion exchange. Membranes such as the chorioallontoic membrane (CAM) provide a vascular system of blood vessels that facilitate oxygen, calcium, and nutrient transport to the embryo (Tuan, 1987). The chorioallantoic membrane results from the fusing of the mesodermal layer of the allantoic membrane with the mesodermal layer of the chorion which completely surrounds the embryo after 10 days of incubation (Gilbert, 2003). The CAM is attached to the internal surface of the shell membrane and provides a barrier between the watery environment of the embryo and the air space. The structure allows the embryo to harvest the calcium from the shell for bone development. The importance of the shell for supplying calcium is shown in shell-less cultures of chick embryos where the embryos exhibit retarded growth and calcium deficiency (Dunn, 1987). It is estimated that 80% of the 140 mg of calcium found in a hatched chick is derived from the shell (Dunn, 1987). Since the CAM is the primary means of calcium transport between the shell and the developing embryo, it is thus critical for bone formation in normal chick development.
Previous experiments have also shown that the CAM can support the development of limb grafts from donor embryos. The vascular CAM transports essential nutrients and gases to the graft, thereby facilitating differentiation and cartilage formation in the limb. This experiment will allow us to study the ability of the chorioallantoic membrane to support development and cartilage formation in limb grafts.
Materials and Methods
Prep list for week 1 (12 - 15) 10- or 11-day old chick embryos (6) 5-day old chick embryos (3) 7-day old chick embryos 70% ethanol Forceps, scissors, plastic spoon 60 mm petri dishes Scotch tape Plastic tubes Gentle agitator Incubator at 37 degrees Celcius | Prep list for week 2 5% trichloroacetic acid (TCA) 70%, 85%, 95%, and 100% ethanol Phosphate buffered saline (PBS) Methyl salicylate Dissecting tools Glass vials Gentle agitator Dissecting microscope/ LCD digital camera |
Procedure (adapted from Hamburger, 1960) Week 1 1) Sterilize host embryo shell (10-day old embryo) with 70% ethanol. Candle the egg to locate blood vessels near the blunt end of egg by holding egg against light (fig. 1). At the blunt end of the egg, create a hole approximately 1.5 cm in diameter with forceps. Remove shell and shell membrane, keeping the CAM (membrane that lies beneath) intact. After locating a large Y-junction of blood vessels (fig. 2), seal the hole with scotch tape (fig. 3) and store egg in 37 degree Celcius incubator. Figure 1. Candling procedure to locate blood vessels in egg.Figure 2. Y-junction on chorioallantoic membrane.Figure 3. Hole punctured on blunt end of egg covered with Scotch tape. 2) Sterilize donor embryo (5 day-old) shell with 70% ethanol. Open blunt end of shell with forceps and transfer the embryo from egg into dish of Ringer's solution. Remove and peel away surrounding membranes from the embryo and photograph the embryo using digital microscopy. Excise limbs (2 forelimbs and 2 hindlimbs) from embryo using fine forceps. Include extra flap of cells from flank if possible to help position graft onto CAM. 3) Transfer one limb graft into each host embryo. Gently position graft over Y-junction of blood vessels and reseal egg with scotch tape to prevent infection. Incubate at 37 degrees Celcius for 7 days. 4) Isolate 5-day old donor embryo as a control for cartilage development at the 5-day stage. Fix in 4% PFA in plastic tube and gently agitate overnight. 5) Set aside untampered 5-day old donor embryo (shell intact) in 37 degree Celcius incubator for 7 days to serve as a control for normal cartilage development in intact embryo. 6) Repeat entire procedure using 7-day old donor embryo.
Week 2 7) Candle hosts to determine which are viable. Remove tape and dissect graft from CAM. Excise limbs from intact donor controls from step 5. Place limbs in individual glass vials. Immerse in 5% trichloroacetic acid (TCA) for 1 hour at room temperature to fix sample. 8) Wash 3 times with enough phosphate buffered saline (PBS) to cover the limb or embryo in order to remove TCA and return to proper pH and salt concentration. 9) Immerse in Alcian green stain for cartilage overnight. 10) Wash 3 times with 70% ethanol to dehydrate sample. Repeat with one wash each in 85%, 95%, and 100% ethanol to remove remaining water from sample. 11) Transfer to methyl salicylate to clear limbs and further dehydrate the sample for better visualization. Allow sample to equilibrate overnight. 12) Photograph limbs under microscope linked to LCD digital camera. Compare development of limb structures and formation of cartilage/bone in the graft limb and in the controls. Try to identify major bones in graft and control limbs and observe any differences in appearance. |
Results
At 5 days of development, the forelimbs and hindlimbs of the donor embryo appear as elongated protrusions from the embryo body (fig. 4). Staining with Alcian green allows visualization of cartilage formation up to this stage in development. The 5-day old and 7-day old embryos exhibited faint cartilage at the location of the future forelimb and hindlimbs (fig. 5a-b).
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Figure 4. 5-day old donor embryo before graft excision. | Figure 5a. 5-day old embryo stained with Alcian green for cartilage development. |
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Figure 5b. 7-day old embryo stained with Alcian green for cartilage development. | Figure 6. Hindlimb graft derived from 7-day old donor embryo incubated on chorioallantoic membrane for 7 days. |
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Figure 7a. Control forelimb of 12-day old embryo. Note the upper part of the limb is missing. | Figure 7b. Control hindlimb of 12-day old embryo. |
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Figure 7c. Control forelimb of 14-day old embryo. | Figure 7d. Control hindlimb of 14-day old embryo. |
Of the 12 grafts attempted from 5-day old donor embryos in one experiment, none were successful. Of the 3 grafts derived from the 7-day old donor embryos, one was successful. Many of the embryos died from infection during the 7-day incubation period, as evidenced by the yellow-green appearance and odor of the egg contents. Of the embryos that survived, the grafts were difficult or impossible to locate, possibly because the grafts were concealed by membrane or lost in the liquid environment below the membrane.
The only successful graft was a hindlimb derived from a 7-day old embryo. After the 7-day incubation period on the chorioallantoic membrane, the graft showed considerable cartilage development (compare fig. 5b, 6). However, only the most distal structures developed, compaared with the intact 7-day control embryos incubated for 7 days (fig. 7d).
Though no grafts derived from 5-day old embryos or forelimbs from 7-day old embryos were successful, it is worthwhile to compare limb development in the controls that were prepared. After 7 days of incubation, the embryos were 12 and 14 days old, respectively. There is extensive cartilage and bone formation in both groups. Distinct joints can be discerned, as well as clearly distinguished digits in the forelimbs and hindlimbs (fig. 7a-d).
In a second experiment, both hindlimb and forelimb grafts were able to form cartilage, but again developed primarily distal structures compared to control embryos (fig. 8) Figure 8. Cartilage development in 5-day chick limbs grafted on the chorioallantoic membrane. Discussion The results of this experiment suggest that cartilage formation is possible in limb grafts, but that cartilage formation is maximal when the limbs and chorioallantoic membrane are left completely intact in embryos.. Since limbs are capable of moderate cartilage formation when grafted onto the chorioallantoic membrane, the CAM is able to supply calcium to extraneous limb grafts through mere contact. These results suggest that the chorioallantoic membrane plays an active role in facilitating calcium transport for bone formation in chick embryos.
