198 



THE ORGANIZER 



operation and put it aside (carefully) in a separate #2 Stender with sterile medium and 

 allow it to regenerate. (See section on "Wound Healing") If there is incomplete regener- 

 ation there should be incomplete or abnormal induction of parts of the medullary plate, 

 determined within about 3 days at laboratory temperatures. (If there are abundant em- 

 bryos, remove sections of the lateral marginal zone and observe for regeneration and 

 effect on neurulation. ) 



B. IMPLANTATION OF THE DORSAL LIP MATERIAL 



Having become acquainted with the size, location, and extent of the blastocoel of the 

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

 blastocoel of an otherwise complete blastula of about stage #7 or #8. This can be accom- 

 plished best by making the transfer in a small-bore pipette (see p. 6, 8, 9, 199) and allow- 

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

 minal 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 atterr;pted with forceps or needles. 



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

 exerted on the fluid by 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 implantation to deter- 

 mine the relative "organizer" and "inductor" effects of the dorsal lip areas. 



C. EXPLANTATION OF THE DORSAL-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 3 to 4 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 

 piece including the dorsal-lip, and transfer it on the point of a needle, under 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 



