(Peculis and Steitz, 1993). Exoribonuclease activity is re- quired for complete processing of 5.8 S rRNAs by the exo- some complex (Mitchell et al., 1997; Briggs et al., 1998; Allmang et al., 1999). Several proteins of the rRNA processing machinery are detected in prenucleolar bodies (PNBs; Ochs et al., 1985a), which are scattered throughout the nucleus in early G1 (Benavente et al., 1987; Jiménez-Garcia et al., 1989; Ochs and Smetana, 1991; Azum-Gélade et al., 1994; Scheer and Weisenberger, 1994; Beven et al., 1996; Dundr et al., 1997; Zatsepina et al., 1997). Fibrillarin, nucleolin, Nop52, PM-Scl 100/exosome, and protein B23 are found within these PNBs, as are the U3 and U14 snoRNAs (Azum-Gélade et al., 1994; Gautier et al., 1994; Jiménez-Garcia et al., 1994; Beven et al., 1996; Mitchell et al., 1997; Fomproix and Hernandez-Verdun, 1999; Savino et al., 1999). Therefore, the PNBs appear to contain preassembled nucleolar com- plexes mainly involved in processing steps of the pre-rRNAs (Scheer and Weisenberger, 1994). Distinct PNBs are in- volved in the delivery of specific processing complexes to the nucleolar domain (Fomproix and Hernandez-Verdun, 1999; Savino et al., 1999). Since the delivery of the different PNBs follows a temporal order, it has been proposed that reassembly into nucleoli could proceed by a stepwise mech- anism that reflects the role of these complexes (Savino et al., 1999). The temporally regulated targeting of PNBs dur- ing the cell cycle thus appears dependent on the constitu- ents of these PNBs. However, the involvement of the tran- scription and/or the transcripts in this process is unknown. The PNBs fuse at the nucleolar organizer regions (NORs) at the end of mitosis, when rRNA synthesis resumes (Jiménez-Garcia et al., 1994; Fomproix et al., 1998). This fusion is inhibited by microinjection into mitotic cells of antibodies directed against RNA polymerase I (RNA pol I; Benavente et al., 1987) and against DNA topoisomerase I (Weisenberger et al., 1993). Therefore, it has been pro- posed that fusion of PNBs into nucleoli is dependent on the resumption of rDNA transcription (Scheer et al., 1993). Remarkably, during early X. laevis embryogenesis, a unique situation was revealed in which regroupment of fibrillarin and nucleolin around the rDNA occurred before the apparent activation of RNA pol I–dependent transcrip- tion (Verheggen et al., 1998). The first cell cycles of X. laevis embryogenesis provide an interesting biological situation since transcription is established de novo after 12 synchro- nized cell cycles devoid of transcription (Brown and Littna, 1964; Newport and Kirschner, 1982). At the midblastula transition (MBT), RNA pol II– and III–dependent tran- scription is activated, whereas RNA pol I transcription is initiated later (Shiokawa et al., 1981a,b; Newport and Kirschner, 1982). This biological situation makes it possible to study PNB assembly and delivery in the context of active or inactive RNA pol I transcription. Before MBT, scat- tered PNBs containing fibrillarin exhibit similar ultrastruc- tural features to postmitotic PNBs and at MBT fibrillarin regroups around the rDNA with maternal pre-rRNAs (Ver- heggen et al., 1998). At MBT, the association of rDNAs with UBF was demonstrated (Bell et al., 1997; Verheggen et al., 1998), but the presence of other partners of the transcrip- tion machinery and, in particular, the RNA pol I complex is not yet established. Indeed, at MBT it was reported that RNA pol I accumulated in nucleoplasmic structures differ- ent from PNBs (Bell and Scheer, 1999), without informa- tion on its association with rDNA. Nuclei assembled in X. laevis egg extracts contain PNB- like structures with fibrillarin, nucleolin, Nopp180, protein B23 (NO38 in X. laevis), U3, and U8 (Bell et al., 1992; Bauer et al., 1994; Bell and Scheer, 1997). Since these PNBs assembled in vitro were not observed to fuse into a nucleolus, a reasonable hypothesis was proposed that this lack of nucleolar assembly is due to the absence of tran- scription in this system (Bell et al., 1992). In the present study, we demonstrate that during both X. laevis embryogenesis and in nuclei assembled in vitro, two types of PNBs containing components of the rRNA processing machinery exist. During X. laevis embryogene- sis, the recruitment of both types of preassembled com- plexes to the nucleolar domain occurs at a time when the RNA pol I complex is not detected in the nucleolar do- main. Furthermore, this recruitment is not dependent on RNA pol I activity, but correlates precisely with the pres- ence of pre-rRNAs of maternal origin. Pre-rRNAs are ab- sent from nuclei in which RNA pol II and III transcription was inactive and, in this case, recruitment of the rRNA processing machinery does not occur. Materials and Methods Primary Antibodies and Probes Antibodies with the following specificities were used: a human auto- immune serum directed against fibrillarin (Gautier et al., 1994); a rabbit polyclonal serum directed against human nucleolin (a kind gift of C. Faucher, LBME, CNRS, Toulouse, France); a monoclonal culture supernatant recognizing the X. laevis nucleolar protein NO38, a homologue of the mammalian nucleolar protein B23 (No-63; Schmidt-Zachmann et al., 1987); and a mouse monoclonal ascites fluid recognizing the X. laevis RNA pol I complex (a kind gift of M. Schmidt-Zachmann, DKFZ, Heidel- berg, Germany). The rDNA probe for rRNA detection corresponds to the entire X. laevis ribosomal transcription unit inserted into pBR322 (clone pXcr7, kindly provided by F. Amaldi, Universito Tor Vergata, Roma, Italy). Double- stranded DNA probes were labeled with the nick-translation kit (GIBCO BRL) according to the manufacturer’s instructions using biotin-14-dCTP. Probes for the detection of U3 and U8 are oligonucleotides complemen- tary to three regions (positions 9-31; 63-85; and 101-122 for U3; and 33-60; 72-93; and 107-136 for U8). They were labeled with biotin-14-dCTP using 39 terminal transferase (Boehringer Mannheim). Assembly of Nuclei in X. laevis Egg Extract Eggs were obtained from female X. laevis and interphasic low-speed egg ex- tracts were prepared as previously described (Almouzni, 1998). This crude extract can be maintained on ice for 4 h without appreciable decay of nu- clear assembly activity. Demembraned X. laevis sperm nuclei were pre- pared and permeabilized with lysolecithin (Almouzni, 1998). For nuclear assembly, 105 sperm heads were added to 100 ml of egg extract and main- tained at 238C. After 45 min, when maximal decondensation of the nuclei occurred, nuclei were processed for photonic and electron microscopies. Preparation of Embryonic Nuclei from X. laevis Embryos were produced by in vitro fertilization (Almouzni and Wolffe, 1995) and allowed to develop at 238C in 0.13 modified Barth solution (Gurdon and Wickens, 1983) for different times after fertilization. At this temperature, embryos were collected at the following stages: early blastula, 6 h after fertilization (stage 8); midblastula, 7 h after fertilization (stage 8.5); late blastula, 9 h after fertilization (stage 9); early gastrula, 10 h after fertilization (stage 10); gastrula, 11 h after fertilization (stage 10.5); and late gastrula, 13 h after fertilization (stage 12); as specified by Nieuw- koop and Faber (1994). In some cases, actinomycin D was added to in- hibit the onset of transcription during development. As dissociation of the The Journal of Cell Biology, Volume 149, 2000 294