Figure 5. RNA polymerase I is not associated with the newly formed nucleolar domain. Embryonic nuclei isolated 7, 9, and 11 h after fertilization were labeled with an antibody to RNA poly- merase I (a, d, and g) before performing in situ hybridization of the rRNAs (b, e, and h). When the nucleolar domain began to as- semble in nuclei 7 h after fertilization, maternally derived rRNAs were detected (b). No RNA polymerase I was seen colocalized to the FISH signal at this time (a; arrowheads). Instead, RNA poly- merase I appeared as speckles dispersed in the nucleoplasm. This distribution was maintained in nuclei 9 h after fertilization (d). RNA polymerase I was not yet colocalized with rRNA (e). Only after 11 h of development, RNA polymerase I (g) was seen par- tially colocalized with rRNAs (h). The corresponding DNA staining with DAPI is shown (c, f, and i). Bars, 10 mm. blastomeres while they remain within the vitellin membrane (Duval et al., 1990). It is of note that neither the dissociation of blastomeres nor the presence of actinomycin D appeared to block cell division. Nuclei, isolated from dissociated em- bryos that were developed in 0.2 and 10 mg/ml of actino- mycin D, were comparable in size to nuclei from control embryos. Embryos were maintained at 238C until 13 h af- ter fertilization. At this time, divisions still occurred, but gastrulation movements were abolished. It was first necessary to check the ability of actinomycin D when applied to whole embryos to selectively block RNA pol I transcription. This prompted us to use embryos 12 h after fertilization and in situ transcription assays in conditions favoring the detection of RNA pol I transcrip- tion (see Materials and Methods). Transcription, revealed by Br-UTP incorporation in isolated nuclei from control and actinomycin D–treated embryos, was characterized by its sensitivity to a-amanitin. In control embryos, the tran- scription detected in nuclei isolated 12 h after fertilization (Fig 6 a) was RNA pol I–dependent (Fig 6 c). Indeed, transcription was not sensitive to 100 mg/ml of a-amanitin and consequently was not RNA pol II– and III–depen- dent. Accordingly, with RNA pol I transcription the tran- scripts were colocalized with fibrillarin (Fig. 6 b and d). In contrast, RNA pol I transcription was not detectable in 95% of the nuclei isolated from embryos treated with 0.5 mg/ml of actinomycin D and collected 12 h after fertiliza- tion (Fig. 6 e). This demonstrates inhibition of RNA pol I in actinomycin D–treated (0.5 mg/ml) whole embryos. Inter- estingly, the recruitment of fibrillarin occurred even when onset of RNA pol I transcription was abolished (Fig. 6 f). Thus, the treatment of whole embryos by low doses of actinomycin D provides a means to follow the changes in the distribution of the rRNA processing components in em- bryos without activation of RNA pol I transcription. From the same group of actinomycin D–treated (0.5 mg/ml) em- bryos, the nuclei were isolated 9 h after fertilization. Fibril- larin was already recruited to the nucleolar domain at this time (Fig. 7 e). Maternally derived pre-rRNAs were also detected by FISH in the nucleolar domain (Fig. 7 d). In- deed, formation of the nucleolar domain was initiated nor- mally at the MBT, even if the onset of RNA pol I tran- scription was blocked. Based on these results, it appears that neither zygotic RNA pol I transcription nor the cata- lytic enzyme RNA polymerase I itself, are responsible for the recruitment of processing machinery. Recruitment of the rRNA Processing Machinery Does Not Occur in the Absence of Maternal Pre-rRNAs in the Nucleolar Domain As maternally derived pre-rRNAs were the only pre-rRNAs in embryonic nuclei at MBT, we wondered if they could play a role in recruiting the rRNA processing machinery to the nucleolar domain. A correlation could be established between the presence of pre-rRNAs in the nucleolar do- main and the ability of the rRNA processing machinery to be recruited to this domain. Two types of nuclei were ex- perimentally induced in which pre-rRNAs were not de- tected by FISH, and in both cases the rRNA processing machinery was maintained in large PNBs. The first type of nuclei was isolated from embryos treated with 2 mg/ml of actinomycin D and collected 9 h after fertilization (Fig. 7, g–i). At this time, Br-UTP was incorporated into large foci (Fig. 8 a) not colocalized with fibrillarin (Fig. 8 a, b arrows). These foci were not further detected, probably due to changes in the transcription pattern during development. Transcription observed 9 h after fertilization was inhibited by a-amanitin, indicating that it corresponds to RNA pol II and/or III activities (Fig. 8 d). RNA pol II and/or III ac- tivities were also detected in most nuclei isolated from em- bryos treated with 0.5 mg/ml of actinomycin D (Fig. 8 g). In contrast, the onset of RNA pol II and III activities was in- hibited in 86% of nuclei isolated from embryos treated with 2 mg/ml of actinomycin D (Fig. 8 j). In the latter nu- clei, fibrillarin was maintained in large PNBs (Fig. 7 h and 8 k) and no pre-rRNA was detected by in situ hybridization (Fig. 7 g). In a few nuclei only, fibrillarin and pre-rRNAs were regrouped in the nucleolar domain (Fig. 7, g and h, arrowheads). These nuclei most likely escaped inhibition of RNA pol II and III activities by actinomycin D, as indi- cated by the detection of fibrillarin clustered to nucleolar domain in the few nuclei in which RNA pol II and III ac- Verheggen et al. Assembly of Nucleolar Components in Embryos 299