Worm Breeder's Gazette 6(1): 32
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.
In situ hybridization should prove useful for analyzing changes in RNA populations during C. elegans development without isolating biochemical quantities of similarly staged embryos or specific tissues. RNA can be detected in various fixed preparations with well- preserved morphology. Embryos, squashed according to the procedure of Gossettand Hecht (1979 C. elegans meeting), maintain their cell boundaries so that the number of cells, and thus the developmental age, is easily determined. Larvae and adults, fixed in ethanol/acetic acid (3:1), frozen in O.C.T. compound from Tissue-Tek, and sectioned at -20 C, retain good tissue morphology. With Nomarski optics, well- preserved muscle, gonad and intestinal tissues can be recognized, and with polarization optics, muscle birefringence and gut granules are visible. Alternatively, adults can be cut open in M9 such that the gonad and intestine extrude as separate tissues from the body of the worm. When squashed in 45% acetic acid, frozen,and then fixed in ethanol/acetic acid, these tissues retain good Nomarski morphology. After hybridization of a radioactive DNA probe to these preparations, followed by autoradiography, grain densities can be determined for particular embryonic cells or adult tissues. I can currently detect abundant RNAs using recombinant DNA probes. In situ hybridizations with cloned nick-translated DNAs containing ribosomal, histone, actin (from Jim Files), and myosin (from John Karn) genes produce strong signals (10 to 200 fold over background after 10- day exposure) under conditions in which non-homologous DNA (Charon 3) gives no signal over background. In addition, three cloned DNAs from our Charon 3 library that plaque-hybridize with C. elegans cDNA also hybridize to RNAs in situ. These three probes show differential hybridization between young embryos (1-50 cells) and old embryos ( comma to hatching) and between two adult tissues (gonad and intestine). Present work involves more precisely characterizing the amount of hybridization as functions of developmental time and tissue specificity for these and other clones.