tivities were detected (data not shown). The PNBs that re- mained in the transcriptionally quiescent nuclei (Figs. 7 h and 8 k) were larger than the PNBs I observed in nuclei 6 h after fertilization (Fig. 1 b). Nucleolin, U3, and U8 were also found in these large structures (Fig. 9, d and e). B23/ NO38 was maintained in small diffuse PNBs similar to those observed in nuclei isolated 6 h after fertilization (data not shown). A second type of nuclei were in in vitro reconsti- tuted sperm nuclei. Again, the absence of nucleolar do- main formation was observed (Fig. 9, g–i). This is consis- tent with the transcriptional inactive state of these nuclei, which was previously reported (Bell et al., 1992). Discussion During early X. laevis embryogenesis, components of the rRNA processing machinery are first associated in bodies, Figure 7. Presence of maternally derived pre-rRNAs for the re- cruitment of fibrillarin to the nucleolar domain. In situ hybridiza- tion of rRNA was performed after immunolabeling of fibrillarin on nuclei isolated 9 h after fertilization from control embryos (a–c) and embryos treated with 0.5 mg/ml (inhibition of RNA pol I; d–f) or 2 mg/ml of actinomycin D (inhibition of RNA pol I, II, and III; g–i). The nucleolar domain was formed in control and 0.5 mg/ml actino- mycin D–treated nuclei. rRNAs (a and d) were colocalized with fibrillarin (b and e) in this domain. rRNA was not detected in most nuclei from embryos treated with 2 mg/ml of actinomycin D (g). In this case, fibrillarin was not recruited to the nucleolar domain, but was maintained in large PNBs in the nucleoplasm (h). Only a few The Journal of Cell Biology, Volume 149, 2000 300 Figure 6. Fibrillarin is tar- geted to the nucleolar domain even in the absence of zygotic RNA pol I transcription. The fluorescent in situ transcrip- tion assay by immunolabeling of incorporated Br-UTP cou- pled to immunolabeling of fibrillarin was performed on nuclei isolated 12 h after fertil- ization from control embryos (A) and embryos treated with 0.5 mg/ml of actinomycin D (B). A, Sites of transcription were labeled in nuclei isolated from control embryos (a) and colocalized with fibrillarin in the nucleolar domain (b). This transcription was RNA pol I transcription because it was not inhibited by incubation of nuclei in 100 mg/ml of a-amani- tin before and during incorpo- ration of Br-UTP (c). B, When whole embryos were treated with 0.5 mg/ml of actinomy- cin D during development, no RNA pol I transcription was detected (e). Although RNA pol I activity was inhibited, fibrillarin was clustered in the nucleolar domain (f) as in con- trol nuclei. Bars, 10 mm. nuclei formed a nucleolar domain containing rRNA and fibrillarin (arrowheads in g and h, respectively). The corresponding DNA staining with DAPI is shown (c, f, and i). Bars, 10 mm.