Worm Breeder's Gazette 13(1): 25 (October 1, 1993)


Analysis of maternal and zygotic gene expression during embryogenesis using in situ hybridization.

Geraldine Seydoux, Andy Fire.

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Carnegie Institution, Baltimore MD 21210.

We have developed an in situ hybridization technique suitable for embryos based on the protocol of Patel and Goodman(1) (many thanks to Tom Evans, David Greenstein, Mike Krause, Shohei Mitani, Neela Patel and Jorge Mancillas for sharing advice and protocols). We have used this method to analyze patterns of gene expression during embryogenesis. We first used gene-specific probes to characterize the expression pattern of 21 genes suspected of being expressed during embryogenesis. These genes were obtained from various laboratories(2) and include "housekeeping" genes as well as regulatory genes required for embryonic development. These genes fall into four classes based on the types of mRNAs produced as detected by in situs:

In this classification, maternal RNAs are defined as those RNAs that are present in the one cell-stage and zygotic RNAs are those RNAs that appear de novo during development. We plan to confirm this classification by doing in situs on embryos homozygous for deficiencies that remove specific genes. The expression patterns observed for Class 4 genes did not allow us to distinguish between maternal and zygotic RNAs.

From this survey, we draw the following general conclusions:

- Maternal mRNAs (6/6) are rapidly degraded in somatic lineages but maintained in the germline lineage. For most genes, the difference is first visible at the 8-cell stage, when P3 shows more intense staining than the other blastomeres. For some genes, the difference is seen even earlier: in the late two-cell stage, the cey-2 RNA is already present at higher levels in P1 than in AB. By the 28-cell stage, maternal RNAs appear absent from somatic lineages but detectable levels are still present in P4 .

- Zygotic mRNAs are detected as early as the four-cell stage ( pes-10 )in all somatic lineages. At least 4 genes in the set are expressed zygotically prior to the 24-cell stage ( pes-10 , crf-2 ,lin- l9 and Histone H1 ).This result agrees with the findings of Schauer and Wood(3) and Edgar, Wolf and Wood(4) who showed that zygotic transcription starts sometime prior to the 20-cell stage. So far, we have not detected any zygotic expression in the germline lineage before the onset of morphogenesis.

- The onset of morphogenesis coincides with changes in gene expression. Some genes start to be expressed just before the bean stage (HSP70A, unc-54 , RP21 C),some are turned off ( cdc2 ,Tubulin alpha), and some continue to be expressed after the bean stage but exhibit lower levels of expression ( dpy-30 and ama-1 )or become restricted to specific tissues (H1, Tubulin ß, elF4 A, cey-1 , EF1 alpha, HSP90 ).

We have also analyzed the general distribution of poly-A+ mRNAs in the embryo using an oligo-dT probe. The cytoplasm of all cells at all stages hybridizes strongly with this probe. We noticed, however, that germline precursor cells hybridize more strongly to the probe than do somatic cells. This difference is detected as early as the four-cell stage (P2) and can still be detected in Z2 and Z3 at least as late as the 2-fold stage. This phenomenon may be due to the stabilization of maternal RNAs in the germline lineage as described above. Interestingly, in the P cells, the oligo-dT staining pattern forms clusters (best seen in P2 and P3 ).We are currently testing whether these clusters are sensitive to RNase and co-localize with P granules.

1. Patel and Goodman (1992). In Non-radioactive Labeling and Detection of Biomolecules. Spring-Verlag.

2. Thank you to Ann Sluder for crf-2 ,Edward Kipreos for lin-19 ,Jim Priess for skn-1 and glp-1 ,Verena Plunger for cey-1 and cey-2 ,Dave Hsu for dpy-30 ,David Bird for ama-1 ,Deborah Roussell and Karen Bennett for eIF4 -A,and Patty Wohldmann and Bob Waterston for Actin ( act-1 coding sequence), cdc2 ,Tubulins alpha and ß, EF1 alpha, HSP70 A, HSP90 and Histone H1 .

3. Schauer and Wood (1990). Development 110,1303-1317.

4. Lois Edgar, Nurit Wolf, and W.B. Wood. Worm Meeting 1991, 92.

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