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.
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 refrigerator. 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.