Worm Breeder's Gazette 8(1): 17

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Expression of a Gut-Specific Esterase in Early Embryos

L.G. Edgar, J.D. McGhee

We have developed a method to stain C.  elegans embryos for an 
esterase activity that appears early in embryogenesis and is 
restricted to the E cell lineage.  This provides an additional 
biochemical marker for early lineage studies.
Method:  Nuclepore filter circles (~1cm diameter polycarbonate 
filters, 8  m pore size, Nuclepore Corporation, 7035 Commerce Circle, 
Pleasanton, California   94565) are rimmed with rubber cement and 
placed on a siliconized depression slide.  Small numbers of embryos (
~100-200) are pipetted onto the filter and processed in volumes of 100 
l.  This allows quick exchange of solutions, by drawing off liquid 
from beneath the filter with a mouth-pipette under the dissecting 
scope, and minimizes loss of embryos.
Pretreatment:  Six to eight minutes 1% Triton-X100 in M9 (100  l 
volume throughout)  three washes, 0.1M KH2PO4/Na2HPO4 buffer, pH 7.0, 
Hypochlorite for 2.5 minutes (1:2 dilution of stock 4-6% NaOCl 
solution, in phosphate buffer, degassed),  three washes in buffer.
Chitinase (Serratia marcescens chitinase, US Biochemical Corp., at 
20 mg/ml in egg buffer. (Laufer, et. al., Cell 19: 569)) two to six 
minutes, observing under dissecting scope, until eggs round up and/or 
pretzels begin to unfold.  Fix three minutes at 4 C in 4% formalin in 
phosphate buffer.  Three washes, buffer (total about three minutes).  
Stain two to six hours at 4 C with freshly made stain (see below).  
Mount on slides in phosphate buffer, transferring either filter (peel 
off rubber cement) or individual embryos (better optics, but harder).  
Seal with nail polish.  Staining improves overnight in the 
Stain recipe:  (Lojda, Gossrau and Schiebler, Enzyme Histochemistry, 
Springer-Verlag, 1979)  Mix:  20  l 4% NaNO2 in H2O (make fresh weekly)
, 20  l pararosaniline solution*.  Add:  750  l 2.8% Na2HPO4 (in H2O), 
20  l 0.2M NaOH, 20  l alpha-naphthyl acetate (1% in acetone)  *(
Dissolve 400 mg pararosaniline HCl in 8 ml H2O, add 2 ml concentrated 
HCl and stir 15-30 minutes at room temperature.  Centrifuge and 
millipore.  Store refrigerated up to two months.
Esterase activity (indicated by dark red staining) appears 
consistently in four to eight contiguous cells of embryos fixed 160 to 
180 minutes after first cleavage (20 C) (i.e., with approximately 110 
to 180 total cells).  It is clearly localized to the E cells at the 
lima bean and later stages.  Eight to 12 cells of partial embryos (50 
to 100 total cells) derived from isolated P blastomeres stained for 
esterase activity, whereas no staining was found in partial embryos 
derived from isolated AB blastomeres.  Thus, expression appears 
autonomous within the P lineage.  We are currently investigating 
expression following cytochalasin and alpha-amanitin blocking of 
embryos permeabilized by cracking (Laufer, et. al.).  Long-range goals 
are to clone the gene for this esterase and investigate the regulation 
of expression of this and additional gut-specific hydrolases.