598 



EXPERIMENTAL FISH EMBRYOLOGY 



INJURY, ABLATION, AND RECOVERY OF FISH EMBRYOS 



Nicholas and Oppenheimer (19'+2) have found that up to the l6-cell stage the Pimdulus 

 hlastomerea are totipotent, and that removal of as much as 50^ of the embryonic protoplasm 

 can be survived by the embryo. On the basis of their work, the following procedures are 

 given- 



The early stages may be operated on through the chorion, (either by direct pricking 

 with a steel needle or through a window), or after the removal of the chorion. The first 

 procedure is not so easily controlled and pressure factors may be involved, and yet it has 

 the advantage of protection of the embryo against bacteria infection. The second method 

 (i.e., decapsulating the embryo) involves the possible hazard of infection but removes the 

 tension factor eind allows more accurate operational technique. (See previous section for 

 method of decapsulation.) Both should be attempted. Use any fish egg available, such as 

 Oryzias, Betta, Paradise or Hemichromus. 



Scliematic representation of suc- 

 cessive stages in the transformation 

 of the blastoderm to the embrjo. 

 Tlie blastoderm (B) slowly expands 

 over tlie yolk (Y) , as is shown In 

 Figs. A, B and C. As gastrulation 

 commences (Figs. D and E) the cells 

 are piled up at the periphery of 

 the blastoderm to form the germ ring 

 (G.R.) and the embryonic sliield (E. 

 S.); the central portion of the 

 blastoderm becomes the extra-embry- 

 onic membrane (E.M.) . During the 

 course of gastrulation the blasto- 

 derm gradually covers the yolk (dia- 

 grams F, G, H and 1); late in gas- 

 trulation a refractile streaK (N) 

 visible in tlie sliield represents the 

 keel of the central nervous system. 

 Fig. J shows the extent of embryonic 

 differentiation a few hours after 

 the yolk is completely covered; O.V., 

 optic vesicle; F.B., forebraln; M.B. , 

 mid-brain; H.B., liind-braln; N.C., 

 nerve cord; S. somite; M. unsegmented 

 mesoderm. 



The egg Is drawn In profile in 

 all figures except figure E, which 

 represents the stage shown in Fig. D 

 seen from the animal pole. 



(From Oppenheimer 1956: 

 Jour. Exp. Zool. 75 ••'+05) 



A. REMOVAL OB DESTRUCTION OF A BLASTOMERE : Two to U-cell stage embryo. 



1. With sharp-pointed steel needle invade the chorion directly over one of the 

 blastomeres of a 2 or U-cell stage, and rupture it. Avoid damage to the 

 yolk and contiguous blastomeres. 



2. Make a small window in the chorion, directly over the blastomeres. Prepare 

 a micro-pipette with terminal bore slightly smaller than the diameter of a 

 single blastomere. If the edges of the pipette opening are rough, so much 

 the better. Pass this pipette through the small aperture, and gently suck 

 up a single blastomere. This may require first the loosening of the blas- 

 tomere with a slight circular movement of the pipette. If the pipette is 

 attached by a small-bore rubber tube held in the mouth the suction can be 

 the better controlled. By this oral suction method 1 and then 2 blastomeres 

 of the U-cell stages should be removed. 



