57. INTRA-EMBRYONIC 

 TRANSPLANTATIONS 



The most productive and successful investigators in this field are Willier and Hamburger, 

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

 eye, or neural crest) is merely inserted into the coelom through a slit in the somato- 

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

 it may be incorporated in an otherwise normal organ. These latter transplantations are 

 the more difficult, but possibly the more significant. 



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

 allantois, 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, coelomic epi- 

 thelium, or the surface of the gonad and mesonephric primordia. Such grafts cannot be 

 properly innervated, and limbs may develop morphologically but without movement. 

 Hamburger and Waugh (1940) 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 transplants are 

 to be introduced directly into the tissues of the (3 day) host. 



Limb primordia grafted into the coelom: 



1. Under aseptic conditions, expose a 3-day embryo through a shell aperture, and 

 apply a small strip of sterile Neutral Red 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 another 

 embryo, clean it of all excess yolk and membranes, and place the watchglass 

 containing the donor tissue on a warming plate at 38 C. 



3. Prepare the host by opening the shell, adding a drop of sterile chick Ringer'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 attachment. 



4. 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 (cutting) 

 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 po- 

 sterior 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 in- 

 sertion 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 trans- 

 plant 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 24 hours. 



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