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 Howard's Ringers solution Forceps, scissors, plastic spoon 60 mm petri dishes Scotch tape 4% paraformaldehyde (PFA) Plastic tubes Gentle agitator Incubator at 37 degrees Celcius

Prep list for week 2 Alcian green stain 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

 

 

 

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).

 

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)

Discussion