EXPERIMENTAL CHICK EMBRYOLOGY khj 



INTRA-EMBRYONIC TRANSPLANTATIONS 



The most productive and successful Investigators in this field are Wlllier and Ham- 

 hurger, and the student is directed to their papers. In some instances the graft (whether 

 limt, eye, or neural crest) is merely Inserted into the coelom through a slit in the soma- 

 topleure and in other instances the transplant is placed in a more solid (flank) region 

 where it may he incorporated in an otherwise normal orgeua. llieae latter transplantations 

 are the more difficult, but poasihly the more significant. 



The coelom of the 3-day embryo allows freer expansive growth than does the chorio- 

 allantols, and it is an ideal nutritive environment. However, the graft may attach itself 

 to any of a number of surfaces within the coelom, such as mesenteries, coelomlc epithelium, 

 or the surface of the gonad and mesonephrlc primordia. Such grafts cannot be properly in- 

 nervated, and limbs may develop morphologically but without movement. Hamburger and Waugh 

 (19'<-0) found excellent histological differentiation which rapidly regressed without such 

 Innervation. 



The method of candling, excising the blastoderm, trimming away the yolk and non- 

 essential parts, and the final dissecting out of the organ anlage are all described 

 (above) and are so well known that they will not be repeated here. The student must be 

 reminded, however, of the increased necessity of absolute aseptic conditions, since trans- 

 plants are to be Introduced directly into the tissues of the (5 day) host. 



Limb primordia grafted into the coelom : 



1. Under aseptic conditions, expose a 5-day embryo through a shell aperture, and 

 apply a small strip of sterile Neutral Bed stained Agar to the flank region, 

 cover the opening of the shell, and return the egg to the Incubator for 10 

 minutes. It is best to rupture the vitelline membrane with #5 watchmaker's 

 forceps so that the dye can penetrate the faster. 



2. Under aseptic conditions excise the wing or leg bud (72 hours or older) of an- 

 other embryo, clean it of all excess yolk and membranes, and place the watch- 

 glass containing the donor tissue on a wanning plate at 58°C. 



5. Prepare the host by opening the shell, adding a drop of sterile chick Blnger's 

 solution, and removing the dyed agar with forceps. Should the blastoderm ad- 

 here to the shell or window, shake it gently to separate it from the attach- 

 ment. 



h. If the amino and chorion have grown over the flank region. Insert a (sterile) 

 glass needle into the amnion, parallel with the body, and with an upward (cut- 

 ting) movement, make a slit in the extra-embryonic membrane directly above the 

 flank region where the graft Is to be Inserted. The membranes will heal, par- 

 ticularly if there has been no hemorrhage. 



5. Make a longitudinal slit posterior to the forelimb bud. Just above the entrance 

 of the vitelline veins into the body. Avoid injury to any blood vessels. The 

 posterior cardinal veins lie ventral to the lateral edges of the somites, and 

 the lower splanchnopleure is highly vascular. Make the slit long enough for 

 the insertion of the transplant. 



6. Suck the transplant Into the end of a micro-pipette and, under good lighting 

 and a dissecting (binocular) microscope with high power objectives, drop the 

 transplant onto the embryo as near the slit as possible. With (sterile) glass 

 needles orient the transplant and insert It into the coelom. It may be further 

 oriented after the insertion, by using a blunter needle. Add a few drops of 

 sterile chick Ringer's. 



7. Replace the shell and seal the window with melted paraffin. Return the egg to 

 the Incubator in the same position for 2*1 hours. 



8. Allow the host to continue development for about 9 days, and recover the trans- 

 plant at about 12 days of incubation of the host. The recovery may be diffi- 

 cult since the transplant may be hidden by some of the host tissues, and it may 

 require an exploratory dissection of the host. 



9. Make drawings, gross and histological analysis of the development of the trans- 

 plant. 



