Worm Breeder's Gazette 10(2): 115

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More on Phorbol Ester TPA and C. elegans Embryogenesis

Ken-ichiro Higashi, Yo Tabuse and Johji Miwa

The tumor promoting phorbol ester TPA causes severe perturbations in 
the growth and reproduction of larval and adult C.  elegans.  One  
g/ml of TPA arrests the growth of L1 larvae.  We reported previously 
that the partial embryo that comprised an AB or a P1 cell underwent 
many rounds of cell division to form cell clusters and that E-cell 
specific autofluorescence developed normally in a P1-derived partial 
embryo in 1 g/ml of TPA.  Thus, we concluded that C.  elegans early 
embryogenesis was unaffected by TPA.  The observations have been 
confirmed in the presence of 5  g/ml TPA using laser microsurgery.  We 
have, however, remained unable to tell whether the late embryogenesis 
is also unaffected by TPA, because gastrulation and morphogenesis do 
not proceed normally in the partial embryo.  In an attempt to solve 
this problem, we used laser microsurgery to puncture an eggshell 
without causing apparent damages in individual cells.  We punctured 
the eggshell of two-cell stage embryos with laser microbeam (wave 
length 560nm, about 1 m diameter at the focal plane, pulsed beam of 
6ns duration) in the embryonic culture medium and followed the 
embryonic development.  To insure that an effective hole was opened at 
the egg shell, a nontoxic fluorescent dye, 0.1% lucifer yellow, was 
added to the culture medium.  All laser-treated embryos underwent many 
rounds of cell division in the absence of TPA.  Although many stopped 
developing probably at the beginning of morphogenesis, about 10% of 
the treated embryos completed morphogenesis and hatched.  Hatchees 
stained with lucifer yellow assured that an effective hole had been 
made in the eggshell.  Some hatchees stained with lucifer yellow were 
observed even in the presence of 10  g/ml TPA.  Muscle differentiation 
was also investigated in both the presence and the absence of TPA.  
Laser-treated embryos were cultured and stained with anti-myosin 
monoclonal antibody.  As expected from the above experiment, myosin 
was stained in the presence of 10  g/ml TPA just as in its absence.  
These results suggest that TPA has little or no effect on C.  elegans