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Fig. 6. Transcript degradation and protection are evolutionary conserved processes. Localization of maternal Hsp83 transcripts to the pole cells of D. virilis occurs by degradation and protection as in D. melanogaster. (A) Maternal Hsp83 transcripts are initially uniformly distributed throughout a syncytial stage D. virilis embryo. (B) Subsequently, these transcripts are degraded in the somatic region but are protected from degradation in the pole cells that bud from the posterior (arrowhead). Note that Bicoid-dependent zygotic expression of Hsp83 that occurs in the anterior of D. melanogaster embryos (see Fig. 4 A and ref. 18) does not occur in D. virilis. (C) In vitro-transcribed D. melanogaster Hsp83 3′ UTR transcripts that carry the HDE(+HDE) are highly unstable when injected into X. laevis stage 6 oocytes (the time points are hours after injection). (D) In contrast, Hsp83 3′ UTR transcripts lacking the HDE (∆HDE) are stable for at least 24 h after injection. (A and B) Whole-mount RNA in situ hybridizations are shown, with anterior to the left and dorsal toward the top of the page. (C and D) Blots are shown of digoxigenin-labeled transcripts recovered the specified number of hr after injection into Xenopus oocytes. See ref. 9 for details.

Evolutionary Conservation of Degradation-Protection Mechanisms

To determine whether transcript localization by degradationprotection is conserved between distant Drosophila species, Hsp83 transcripts were examined in early embryos of Drosophila virilis, which has diverged ≈60 million years from D. melanogaster (19). Both components of the localization mechanism—degradation and protection—are conserved (Fig. 6 A and B).

When in vitro-synthesized transcripts comprising the D. melanogaster Hsp83 3′ UTR (i.e., carrying the degradation element) are injected into Xenopus laevis stage 6 oocytes or early embryos, the transcripts are unstable (Fig. 6 C and D) (9). Strikingly, deletion of the degradation element increases the half-life of injected transcripts approximately an order of magnitude (9). Thus, Drosophila cis-acting sequences can be recognized by the Xenopus trans-acting machinery, which suggests that the fundamental maternal transcript degradation machinery is conserved throughout the metazoa.

Functions of Transcript Degradation and Protection

As discussed above, maternal transcript degradation in the early embryo has been presumed to be necessary for the passage of developmental control to the zygotic genome. However, there have been few experiments that address whether this is in fact the case. Perhaps the best data derives from dosage analyses in which the concentration of maternal string transcripts was either halved or doubled (13) (string encodes the Drosophila Cdc25 cell cycle regulator). The transition from uniform maternally regulated to spatially patterned zygotically regulated cell divisions was delayed (double dose) or induced prematurely (half dose). Now that the pathways of transcript instability in the early Drosophila embryo have been defined, it should be possible to delete instability elements from string transcripts to ask whether stabilization of maternal string mRNA results in delay of the cell cycle transition at the MBT.

In contrast to the poorly defined role of maternal RNA degradation in the early embryo, the functions of posterior protection are better understood. For example, it is known that posterior protection of nanos and Pgc transcripts is crucial for the biological roles of these RNAs in germ-cell differentiation; elimination of posterior localization of these transcripts results in abnormal differentiation of the germ cells (12, 20, 21). The role of posterior protection of Hsp83 transcripts has not yet been defined.

Conclusions

The analyses summarized above have shown that transcript stability is exquisitely regulated both in time and in space in the early Drosophila embryo as well as at other developmental stages and in other cell types. Transcript localization by degradation-protection represents a distinct mechanism from that involving directed cytoplasmic transport via cytoskeletal motors, although these two mechanisms are not mutually exclusive (reviewed in refs. 10 and 22). Further analyses of degradation and protection using the combination of genetic and biochemical methods available in Drosophila are expected to lead to general insights into the mechanisms and functions of these processes during animal development.

R.L.C. has been supported in part by a Medical Research Council of Canada Graduate Scholarship and a University of Toronto Open Scholarship. Our research on RNA localization mechanisms is funded by an operating grant to H.D.L. from the Canadian Institutes of Health Research (formerly the Medical Research Council of Canada).

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