[
Worm Breeder's Gazette,
1990]
In order to follow the extension of cellular structures along the anterior-posterior axis it would be desirable to make longitudinal sections through complete worms. Sectioning a worm longitudinally is certainly more economic than cutting transverse (needs only about 10% of sections). Three dimensional reconstruction of cells and tissues is probably easier in many cases with longitudinal sections. Therefore, we have developed a method for doing this using a laser microbeam as a tool for careful fixation. Young adults are transferred from an agar plate into a drop of 2.5% Glutardialdehyde in 0.02M Phosphate buffer with 50 l/ml of a saturated solution of Trypane Blue. Worms are slightly squeezed in this solution between a microscope slide and a coverslip to make sure that they are perfectly flat. With single pulses of a laser-microbeam ( wavelength 550 nm, Rhodamin 6G) coupled to a microscope, tiny lesions are made into the cuticle. The thin layer of blue dye allows absorption of the orange laser beam. We had found earlier that other fixation methods for whole worms not involving multiple penetration sites along the body gave only satisfactory results in the anterior part of the animals. A hot aldehyde-peroxide method described for C. elegans by Byard et al., 1986 (Stain Tech. 61, 33) did not work well in our hands. Therefore we punched tiny holes with the laser into the cuticle starting from posterior to anterior. On each side 6-10 lesion are made waiting 30-60 sec after each pulse to allow penetration of the fixative. After treatment fixation in Glutaraldehyde is continued for 2 hours. Animals are then embedded individually into small agar blocks, washed in PBS (similar osmolarity as Glutaraldehyde + Trypane Blue) and postfixed in 2% OSO4 for another 2 hours. Later they are embedded in Araldite. With this method we are able to make perfect longitudinal sections through young adults from tip to tail. Fixation is of equal quality along the body. Besides holes in the cuticle the laser-induced lesions cause only tiny damages in the hypodermal and muscle layer, if at all. As the fixative does not penetrate the eggshell, so far we have chosen animals which do not carry fertilized eggs. For laser- induced fixation of eggs, see Cole & Schierenberg, 1986 (Experientia 42, 1046). With the help of longitudinal sections we can well study ultrastructure and extension of tissues like the alimentary tract, body muscles or the gonad including the distal tip cell. We imagine that this method may also be helpful for the analysis of other structures like the nervous system.
[
Worm Breeder's Gazette,
1982]
After Gunter von Ehrenstein's death of a heart attack December 26, 1980, the 6 directors of the Max-Planck-Institute for Experimental Medicine decided to close the Department of Molecular Biology as of the end of 1982. We are continuing work to finish up and publish work in progress with gradual attrition as people find new positions elsewhere and with correspondingly diminishing budget. A list of our recent papers and manuscripts is given below. Eddi Isnenghi completed his paper on the phenotypes of the Gottingen emb mutants (including TSP's and maternal tests) and is trying to microinject cloned DNA to rescue worm mutants. He is also trying to transform yeast mutants with worm DNA. Randy Cassada is still trying to map embs on LG III with deficiencies and duplications and is looking for new strains of C. elegans in the wild. Eddi has received a 6-months' extension of his DFG (= NSF) fellowship until July. Ken Denich is writing up his work on emb mutant lineages in Gottingen for a paper and as PhD Thesis for the Department of Zoology, University of Manitoba and will be returning there this summer to finish up PhD requirements. Einhard Schierenberg has continued with laser-induced cell fusion experiments. If the membrane between 2 cells is disrupted at the right time, the division of both nuclei results in 4 more or less normal cells. Development continues to a twitching lima bean monster. Fusing cells in later stages results in uncoordinated animals. All treated embryos show timing defects in the E-cell line similar to emb mutants, perhaps due to remixing of cytoplasm. A copy of Einhard's film on embryogenesis is available (with commentary) from Scott Emmons for those who want to show it to students. Einhard is going to Bill Wood's lab this fall. Khosro Radnia has left for a job in industry. Ulli Certa and Franz Scharfenberg are characterizing histone H2A variants in emb mutants. They have also identified several histone H4 DNA clones in a C. elegans genomic library using a sea urchin probe obtained from Mike Grunstein. Ulli has finished his PhD and plans to go to Grunstein's lab at UCLA this spring for a post- doc on yeast histones. Ulli and the other gene cloners have been working successfully with similar minded members of the Institute's Department of Chemistry. Franz hopes to do his PhD in that department. Shahid Siddiqui has just joined Joe Culotti at Northwestern. Tom Cole is very happy with his new Zeiss EM 109 electron microscope. Our computer-aided reconstruction program with color display now works for LM and EM input. We are trying to transfer the programs and data to the MRC, so they will still be available after our department has closed. Chris Carlson has gone to the EMBL in Heidelberg as of Jan 1 to work on computer graphics of molecules. Ursula Reuter also has a position at EMBL as of Jan 1 as an EM technician. Ed Hedgecock visited for 3 weeks this fall to show us microinjection, antibody staining (with anti-actin from Mary Osborne and Klaus Weber), post- embryonic lineage techniques and the like. It was a very useful and pleasant visit. We hope to have some of you as visitors in 1982!