D. Experiments on the Early Embryo 



24. THE ORGANIZER AND EARLY 

 AMPHIBIAN DEVELOPMENT* 



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

 planted, or when introduced into the blastocoel of another embryo. Also, to test the 

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



MATERIALS: 



Biological: Urodele* gastrula, stage #10. 



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



METHOD: 



Precautions : 



1. Blastula and early gastrula stages 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. 



2. The membranes may be removed in 10% Standard Solution, or even easier in 

 hypotonic media, but the operation is to be performed in full-strength Stand- 

 ard Solution. After the wound has healed and the transplant has become in- 

 corporated in the host, gradually return the Urodele embryo to the Urodele 

 Growing Medium. 



3. 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 attempting these delicate operations. To do this it will be necessary to 

 dissect the various stages, to locate and identify the blastocoel, the early gas- 

 trocoel, 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 24 hours and then dis- 

 sects 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 an- 

 ticipation of one of the following operational procedures. (Remember that the 

 embryo is alive and that during delayed preparations the embryo may progress 

 from stage # 1 to stage #11.) 



A. INJURY EFFECTS AND REGENERATION OF THE DORSAL 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 excisions in different gastrulae, but sketch each embryo immediately after the 



* The Anuran embryos are not as satisfactory as the Urodele embryos for these experiments. ■ See the next exercise and page 207. 



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