THE ORGANIZER 213 



B. IMPLAJfTATION OF TEE DORSAL LIP MATERIAL 



Having 1)80011)6 acquainted with the size, location, and extent of the hlastocoel of 

 the Urodele, the student will now attempt to place a dorsal-lip (from stage #11) within 

 the hlastocoel of an otherwise complete blastula of ahout stage #7 or #8. This can be 

 accomplished best by making the transfer in a small-bore pipette (see figures) and allow- 

 ing gravity to carry the cells through the roof of the blastula into the blastocoel. The 

 terminal bore of the pipette should be Just large enough to hold the group of dorsal lip 

 cells to be implanted. Spemann's pipette with a side hole covered with a thin rubber 

 tubing, with pressure controlled by gentle thumb pressure over the covered hole, has proven 

 to be very satisfactory. The cells tend to fall apart and the "organizer" region becomes 

 highly disorganized when the implantation is attempted with forceps or needles. 



It must be remembered that the blastocoel is filled with a fluid and that any pres- 

 sure exerted on the fluid or contents of the pipette will tend to "blow up" the entire 

 blastula. 



When the donor cell area has been excised, suck up a small amount of medium into the 

 transfer pipette, then pick up the dorsal lip cells, and before the transfer is made 

 (under water at all times) it will be noted that the dorsal lip is pulled by gravity to 

 the tip of the pipette. It will therefore be necessary only to penetrate the roof of the 

 blastocoel and the cells to be implanted will drop in. Slowly and carefully withdraw the 

 pipette, aided (if necessary) by a hair loop. Allow the wound to heal and then do not 

 disturb for 3 days or more. 



If the student becomes proficient in the above, it is suggested that he coagulate 

 several gastrulae in hot water, excise the dorsal lip and make a similar In^jlantation to 

 determine the relative "organizer" and "inductor" e'ffects of the dorsal lip areas. 



C. KXPLANTATION OF TEE DOBSAL-LIP MATERIAL 



When the belly ectoderm of the neurula (stage #15) is peeled off as a sheet of cells, 

 it will normally round up in the form of a tube. It is possible to take advantage of this 

 fact by prior excision of the dorsal-lip material and placing it on the Inside of such a 

 sheet of cells so that the "organizer" will become wrapped up within indifferent ectoderm. 

 The whole may then be treated as the above operated gastrulae and observed for inductions 

 during 5 to 1+ days. 



D. TRANSPLANTATION OF THE DORSAL- LIP 



Select two early gastrulae (stage #10) and place in Syracuse operating dish over agar 

 and in 10^ Standard Solution. After removing the membranes, select the best specimen to 

 be the host. From the prospective host remove a small rectangular piece of ectoderm from 

 the presumptive flank or belly region. From the donor quickly excise a similarly sized 

 pi«ce including the dorsal-lip, and transfer it on the point of a needle, \mder water, to 

 the wound on the host. This is a difficult procedure because the host must be oriented 

 and kept In position within the agar depression, and also because mitosis is so rapid, the 

 cells are so large, and cell movements are so extensive that the transplants are often 

 pushed out of the wound before they have a chance of becoming adherent. It may be neces- 

 sary to use a glass bridge or Briicke to hold the transplant in position for 30 to '+5 minutes 

 during the healing process. Such a cover cannot be used longer because it interferes with 

 respirations. Observe the healing process and re-examine during 5 days. 



E. TRANSPLANTATION TO THE DORSAL- LIP REGION 



As in "D" above, select two embryos at stage #10 and remove the membranes. The 

 transplantation is to be made from the presumptive flank region of the donor to a position 

 Just anterior to the dorsal lip of the host. Since the dorsal lip cells move rapidly it 

 Is necesaaiy to: 



a. Make the excision from the donor first. 



b. Make the host wound with the donor material nearby, and complete the transfer 

 as quickly as possible. The host wound must be sufficiently anterior to the 

 forming dorsal-lip so that the transplant will "take" well before it reaches 

 the level of involution. 



