Figure 10. Recruitment of rRNA processing machinery related to transcription during X. laevis development. Pro- cessing machinery compo- nents were first assembled in the nucleoplasm in two types of PNBs: PNBs I (in purple) containing fibrillarin, nucleo- lin, U3, and U8; and PNBs II (in red) containing B23/NO38. PNB I and PNB II compo- nents were recruited to the nu- cleolar domain with different kinetics. Material contained in PNBs I was regrouped in the nucleolar domain before ma- terial contained in PNBs II. At the time of the recruitment process: zygotic transcription by RNA pol II and pol III was observed (1 in orange); RNA polymerase I was found in the nucleoplasm. It was relocal- ized on rDNA and activated later (1 in blue); pre-rRNAs of maternal origin were de- tected at the time of nucleolar domain formation (1 in fuch- sia). In nuclei isolated from embryos treated with 2 mg/ml of actinomycin D that inhib- ited RNA pol II and pol III transcriptions, pre-rRNAs were not detected by FISH and the rRNA processing machinery components were maintained in large PNBs in the nucleoplasm. A similar sit- uation was encountered in in vitro reconstituted nuclei. development of Ascaris lumbricoides (Sanford and Bruzik, 1999). A hypothesis could be that recruitment of the rRNA processing machinery to the nucleolar domain involves changes in phosphorylation of proteins during X. laevis embryogenesis. The major event for recruitment of the rRNA process- ing machinery is likely to be the presence of pre-rRNAs in the nucleolar domain. In cultured cells, the onset of rDNA transcription at the end of mitosis could provide pre-rRNAs required for recruitment of the rRNA processing machin- ery to the nucleolar domain (Scheer et al., 1993). This re- assembly of PNBs is prevented if activation of rDNA tran- scription is inhibited (Benavente et al., 1987; Scheer and Benavente, 1990). Remarkably, during X. laevis embryo- genesis, inhibition of rDNA transcription does not prevent nucleolar assembly. Maternally derived pre-rRNAs are maintained during X. laevis embryogenesis and targeted to the nucleolar domain at MBT (Verheggen et al., 1998). Accumulation of these pre-rRNAs in the nucleolar do- main could be a prerequisite for the recruitment of the processing machinery. The absence of nucleolar accu- mulation of maternally derived pre-rRNAs in actinomycin D–treated embryos appears to be a consequence of RNA pol II and III transcription inhibition. An event required for pre-rRNA accumulation could be promoted by zygotic transcription. Besides the predominant role of pre-rRNAs, additional regulatory mechanisms must exist to allow the delayed translocation of PNB II components relative to PNB I components. During X. laevis embryogenesis, we observed that most of B23/NO38 was recruited later than fibrillarin and nucleolin. A comparable situation was observed for nucleolus formation in telophase cell (Fomproix and Her- nandez-Verdun, 1999; Savino et al., 1999). Considering that steps for nucleolar assembly are common to both em- bryonic and cultured cells at the end of mitosis, it is tempt- ing to speculate that nucleolar assembly involves common regulatory pathways in both cases. The fact that compo- nents of rRNA processing machinery involved in distinct steps of processing are delayed in their recruitment could be in itself a regulatory mechanism for their stepwise in- volvement in rRNA processing. This is raising the ques- tion whether alteration of the timing of the recruitment process might impair processing of rRNA. Verheggen et al. Assembly of Nucleolar Components in Embryos 303