Worm Breeder's Gazette 12(5): 84 (February 1, 1993)

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.

Progress with in situ Hybridization to Nematode Embryos

Karen L. Bennett

Department of Molecular Microbiology and Immunology
University of Missouri-Columbia

We plan to use frozen sections of synchronous Ascaris embryos to test whether cyclin B RNA is localized to the nematode P4 cell as it is to the germline precursor pole cells in Drosophila (see Kreutzer, et al., this volume). To establish whether we could detect a hybridization signal that is localized to a single cell, or a group of cells, we have used various 35S labeled riboprobes. Our first positive results were to the eliminated Ascaris satellite DNA. This DNA is lost from the somatic genome by the 28 cell stage, but retained in the germline. After hybridizing early Ascaris embryos with the satellite probe, only the P4 cell is positive; while at the two fold-stage, the Z2 and Z3 ,cells are detected. To detect RNAs, we first used an antisense probe transcribed from a small subclone of the Ascaris 26S ribosomal RNA gene cluster (Back, Mčller, and Tobler 1984. NAR 12,no.3:1333-1346). This RNA probe gave an extremely strong signal in all cells, as expected. At the same time we tested the sense strand of this ribosomal probe, as a negative control. Upon long exposure (~ 1 4 days) this sense strand probe. which we call E3 ,is not negative, but surprisingly, hybridizes specifically to a single cell in the anterior of the 28 cell embryo. Following the localized hybridization through older embryonic stages, this cell appears to be following the lineage pattern of the hyp-2 cells of C. elegans; therefore we have tentatively identified the one cell detected in the 28 cell embryo as the Ascaris equivalent of the Caenorhabditis ABalpha cell. (Based on the ~100 year old diagrams of the lineage of Ascaris embryogenesis of Boveri, zur Strassen and Mčller, the early lineages look similar, if not identical, to those of Caenorhabditis). The signal is very specific and the result is reproducible (3 different experiments). Sequence gazing at the 26S C.elegans ribosomal gene (Ellis et al. 1986. NAR 14no.5:2345-2364) reveals at least three small ORFs of 60-137aa on the opposite strand, which start with a methionine. We will sequence our Ascaris subclone, which is 770bp, and will use it on Northerns of early embryonic RNA, looking for recognition of a small RNA. However because the signal is cell specific, even in young embryos, detecting an RNA that is rare may be difficult. We have yet to show the signal goes away with RNAsing, but the only way we detect DNA hybridization with the satellite probe is after denaturing the chromosomal DNA, which we don't do before hybridizing with E3 .And we haven't tried E3 on mixed stage, sectioned C. elegans embryos yet.

The Kodacolor "baby pictures" of these embryos (combined DAPI stained and Nomarski) have been fun to look at, and have caused a stir at the local Photomat. I'd be glad to send copies, and share protocols with others doing in situs. The bottom line is we're now fairly convinced that we'll be able to detect the cyclins, which aren't rare messages, whether or not the RNA is localized.