Figure 4. Recruitment of PNB I and PNB II components to the nucleolar domain follows different kinetics. Double immunolabeling of B23/NO38 (FITC) and fibrillarin (Texas red) was performed on embryonic nuclei isolated 7, 9, and 11 h after fertilization. In a fraction of nu- clei 7 h after fertilization, B23/NO38 (a) was found colocalized with fibrillarin (b) in large clusters that began to form at the nucleolar do- main (c; yellow dots on the merged image). These clusters were seen by phase-contrast mi- croscopy (d; dense area). Nucleolar delivery of PNBs I (fibrillarin) and PNBs II (B23/NO38) thus appears to be initiated at the same time. In nuclei 9 h after fertilization, B23/NO38 (e) was not completely relocalized to the nucleolar do- main in contrast to fibrillarin (f). Large dots of B23/NO38 (g; green dots on the merged image) were seen outside of the nucleolar domain (g; yellow area on the merged image). In nuclei 11 h after fertilization, B23/NO38 (i) and fibrillarin (j) were regrouped and colocalized to the nucle- olar domain (k). The nucleolar domain appears dense by phase-contrast microscopy (h and l). Bars, 10 mm. quired for relocalization of the rRNA processing machin- ery to the nucleolar domain (Scheer et al., 1993). RNA pol I could play a role in this recruitment process. This prompted us to investigate the distribution of RNA pol I at the time of nucleolar assembly in X. laevis embryos. Immunolabeling of nuclei between 7 h and 11 h after fer- tilization with an mAb that immunoprecipitates the X. laevis RNA pol I complex (M. Schmidt-Zachmann, personal communication) revealed a speckled distribution (Fig. 5), as recently described (Bell and Scheer, 1999). rRNAs were previously detected in the nucleolar domain at the time of PNB gathering and were identified as 40S transcripts, probably derived from a maternal pool (Verheggen et al., 1998). We performed in situ hybridization of rRNAs on nuclei previously labeled with the antibody against RNA pol I, to simultaneously visualize the RNA pol I complex and the pre-rRNAs on the nucleolar domain in nuclei iso- lated between 7 (Fig. 5, a–c) and 9 h after fertilization (Fig. 5, d–f). Speckle-like structures containing RNA pol I were far from nucleolar domains and consequently from rDNAs. This indicated that pre-rRNAs were maternally derived. A fraction of RNA pol I was found associated with the nucleolar domain only in nuclei 11 h after fertilization (Fig. 5, g–i). This corresponded precisely with the time when RNA pol I transcription began to be observed by the in situ transcription assay (see below). Zygotic Pre-rRNA Is Not Required for the Recruitment of the Maternal Pre-rRNAs and Processing Machinery to the Nucleolar Domain To confirm that zygotic nascent pre-RNAs, even in unde- tectable amounts, were not present in the newly formed nucleolar domain between 7 h and 9 h after fertilization, embryos were treated with low doses of actinomycin D to inhibit RNA pol I transcription. To allow the drug to dif- fuse in all the embryonic cells, actinomycin D was diluted in Ca21- and Mg21-free medium, conditions that dissociate The Journal of Cell Biology, Volume 149, 2000 298