THE ORGANIZER AND EARLY AMPHIBIAN DEVELOPMENT 



FUBPOSE : To test the organizing potencies of the dorsal lip of the blastopore when trans- 

 planted, or when Introduced into the hlastocoel of another embryo. Also, to teat the 

 Inductive capacities of other regions, and other (non-living) substances. 



MATEBIALS : 



Biological : Urodele* gaatrula, stage #10. 



Technical : Syracuse dishes with agar bases and standard operating equipment. 



METHOD : 



Precautions : 



a. Blastula and early gaatrula stagea are very delicate and must be handled with 

 extreme caution. A soft agar base is best. Avoid shaking or Jarring after the 

 membranes have been removed. 



b. The membranes may be removed in lOjd Standard Solution, or even more hypotonic 

 media, but the operation is to be performed in full-strength Standard Solution. 

 After the wound has healed and the transplant haa become incorporated in the host, 

 gradually return the Urodele embryo to the Urodele Growing Medium. 



c. Use sterile instruments throughout. 



Control : The control for dorsal-lip transplantation or implantation is the use of a 

 comparable sized piece of tissue from the same donor, but from a region other than 

 that of the dorsal lip. 



Procedure : The student should be thoroughly acquainted with the morphology and with the 

 morphogenetic movements of the late blastula and the early gastrula before attempt- 

 ing these delicate operations. To do this it will be necessary to dissect the vari- 

 ous stages, to locate and identify the blastocoel, the early gastrocoel, and the 

 surrounding yolk and cell layers. It will further familiarize the student with the 

 conditions of the early gastrula if he removes the membranes and hardens some of the 

 specimens in 10^ formalin for 2l4- hours and then dissects some and cuts others in 

 sagittal and other planes. (Avoid contamination of usual dissecting instruments 

 with fixative.) (See the section on "Morphogenetic Movements and Vital Staining.") 



The Jelly and vitelline membranes are to be removed from the early gastrula 

 while it is in 10^ Standard Solution over the agar base in a Syracuse dish. Do not 

 use as hosts any embryos which have been injured unintentionally. Place the denuded 

 embryo in a depression in the agar, and orient it with a hair loop in anticipation 

 of one of the following operational procedures. (Remember that the embryo is alive 

 and that during delayed preparations the embryo may progress from stage #10 to stage 

 #11.) 



A. INJURY EFFECTS AND REGENERATION OF THE DOBSAL LIP 



With sharp pointed glass needles and a hair loop, remove a rectangular group of cells 

 from the mid-dorsal region. Just anterior to the dorsal lip. Vary the size and the shape 

 of the exciaions in different gastrulae, but sketch each embryo Immediately after the 

 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 regenera- 

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

 determined within about 5 days at laboratory temperatures. (If there are abundant embryos, 

 remove sections of the lateral marginal zone and observe for regeneration and effect on 

 nexurulation. ) 



The Anuran embryos are not as satisfactory as the Urodele embryos for these experiments. 



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