Worm Breeder's Gazette 13(2): 22 (February 1, 1994)
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.
Previously, we have developed a method to amplify the 3'-end region (0.5 -1 kb) of all mRNA (cDNA) species from a single embryo or a single blastmere quantitatively (Y. Kohara, WBG 11#1, p.67, H. Tabara & Y. Kohara '93 Worm Meeting). The amplified cDNA have been used for two types of differential screening which have given several candidate clones for differentially expressed genes; (l) The cDNA were used as probes in conventional screening of cDNA libraries, (2) aliquots of the amplified cDNA from various stages of single embryos were dot-blotted on strips of nylon membrane and then probed by individual cDNA clones. The validity of the amplification was confirmed by the fact that consistent hybridization signals were obtained in model experiments in which the strips of the dot-blot were probed with stage-specific gene probes; glp-l for very early stage, unc-54 and myo-2 for mid-late stage and vit-2 for no expression in embryogenesis. However, PCR amplification involved in the method has potential uncertainty with respect to quantitativeness, and information about the distribution of mRNA within an embryo is not available from these analyses. Thus, we have developed a method for in situ hybridization on embryos.
The in situ method is based on our protocol for separating blastmeres, in situ protocol for Drosophila embryos and G. Seydoux's one. The point is that egg shell is removed by chitinase treatment and then the vitelline membrane is partially broken by shearing force before fixation, which takes a bit time but allow us to regulate the extent of fixation and proteinase digestion finely, leading to high signal-to-noise ratio for early embryos. The results of a model experiment using the stage-specific gene probes ( unc-54 , myo-2 and glp-l) verified the method. As to glp-l, both AB and P1 blastmeres were positive, which was consistent with the observation by Evans and Kimble ('93 Worm Meeting).
The results of in situ analysis on 4 cDNA clones are as follows;
(l) clone 4-1: This clone was obtained by differential screening between the amplified cDNA from single embryos at 1.5hr and 9.5 hr after 1st cleavage, which were positive around 9.5hr embryo in the dot-blot analysis. In situ analysis showed that this gene gave signals on the embryos from comma to 2-fold stages which exactly coincided with the period expected from the dot-blot analysis, and more precisely, the expression was limited in the cells placed along the worm body.
(2) clone 4-3: This was obtained from the same screening with clone 4-1, but specific for 1.5hr embryo. The results of in situ analysis was very interesting. No expression was observed in embryos earlier than 4-cell stage and later than the end of gastrulation. The expression was first detectable at 8-cell stage in all blastmeres except for P3 ,and culminated at the beginning of gastrulation when one cell, probably P4 ,was still free from the expression.
(3) ant-l: This clone was obtained from differential screening using cDNA amplified from AB and P1 blastmeres. However, in situ analysis showed no clear localization of the mRNA at 2-cell stage and showed rather ubiquitous expression during early embryogenesis. Since this gene has similarity to a cell cycle related gene, CKS1 ( cdc28 kinase subunit), the differential screening might have detected the difference between the stages of cell cycle in the AB and P1 cells from which the cDNA were amplified. We are going to continue the differential screening. (4) skn-1 :Although skn-1 gene product was reported to be localized in P1 and therefore a good example of maternal genes which showed localized expression in early embryos, skn-1 probes hybridized equally to dot-blots of the cDNA amplified from AB and P1 blastmeres, suggesting no localization of the mRNA. In situ analysis also showed the existence of the mRNA in both AB and P1 cells. These data suggest that the expression of skn-1 gene is regulated post-transcriptionally.
* We thank G. Seydoux for giving us her protocol.
** The protocol of our in situ method is available.