Figure 8. Fibrillarin was not recruited to the nucleolar domain when the onset of nonribosomal zygotic transcription was inhibited. The fluorescent in situ transcription assay by immunolabeling of incorporated Br-UTP coupled to immunolabeling of fibrillarin was per- formed on nuclei isolated 9 h after fertilization from control embryos (A) and embryos treated with actinomycin D (B and C). A, At this time, incorporation of Br-UTP was detected in the nucleoplasm as punctuated signals (a) not colocalized with fibrillarin to the nu- cleolar domain in control nuclei (b). This transcription signal was inhibited by incubation of nuclei in 100 mg/ml of a-amanitin before and during incorporation of Br-UTP (d). B and C, A non-RNA pol I transcription signal was also detected in nuclei isolated 9 h after fertilization when embryos were treated with 0.5 mg/ml of actinomycin D (g), but not when embryos were treated with 2 mg/ml of acti- nomycin D (j). In this latter case, fibrillarin was maintained in large PNBs dispersed in the nucleoplasm (k). Arrows indicate the posi- tion of the nucleolar domains in a and g. DNA staining with DAPI is shown in (c, f, i, and l). Bars, 10 mm. known as the PNBs, which are dispersed in the nucleo- plasm. These components are subsequently recruited to rDNA loci to form the nucleolar domain (Fig. 10). Both of these steps play an important part of the nucleolar assembly process. Interestingly, these phenomena also occur during nucleolar assembly at the end of mitosis (Hadjiolov, 1985; Scheer and Benavente, 1990; Thiry and Goessens, 1996). A comparison of these biological situations reveals the general features necessary for recruitment of the rRNA processing machinery. PNB Assembly as the First Step of Nucleologenesis In cultured cells, PNBs are formed at the end of mitosis when components of the rRNA processing machinery as- sociate (Ochs et al., 1985a; Schmidt-Zachmann et al., 1987; Jiménez-Garcia et al., 1989; Azum-Gélade et al., 1994). PNB-like structures with a similar composition also form in vitro in reconstituted interphasic nuclei (Bauer et al., 1994; Bell et al., 1992; Bell and Scheer, 1997; Bauer and Gall, 1997). During X. laevis embryogenesis, PNBs assemble in early blastula embryonic nuclei and remain as discrete struc- tures throughout interphase (Verheggen et al., 1998). In this study, we demonstrate the existence of two types of PNBs in both X. laevis embryonic nuclei and permeabilized sperm nuclei reconstituted in egg extracts. The components in PNBs I are all involved in early steps of rRNA processing, whereas the components found in PNBs II are involved in a late step of processing. Distinct PNBs for proteins involved in early and late steps of rRNA processing were also described in telophase cells (Fomproix and Hernandez-Verdun, 1999; Savino et al., 1999). To date, the mechanisms that govern PNB assembly are poorly understood. By systematic depletion of the egg ex- tract, it has been shown that fibrillarin, nucleolin, B23/NO38, and U3 are dispensable for PNB assembly in reconstituted nuclei (Bell and Scheer, 1997). At the end of mitosis, PNB formation and the onset of transcription occur simulta- neously, suggesting a common regulatory event (Fomproix et al., 1998). Nevertheless, PNB assembly occurs in the ab- sence of transcription in both cultured cells (Morcillo et al., 1976; Benavente et al., 1987) and reconstituted nuclei (Bell et al., 1992). Nucleolus-like particles with features of PNBs were assembled in a soluble extract of nucleoli (Trimbur and Walsh, 1993). PNBs were also induced in interphasic cells which had been released from hypotonic Verheggen et al. Assembly of Nucleolar Components in Embryos 301