Figure 2. In vitro reconstituted nuclei observed by EM. a, General view of the nucleus surrounded by the nuclear envelope. The chro- matin is decondensed and two types of nuclear bodies are visible: a dense body (arrow) and several gray bodies (arrow heads). Bar, 1 mm. b, High magnification of a dense nuclear body. This body corresponds to PNB I. Granules are densely packed inside. Bar, 0.1 mm. c, High magnification of two gray bodies exhibiting a fibril- lar feature. These bodies correspond to PNB II. Bar, 0.1 mm. at the same time. In contrast, large amounts of B23/NO38 remained in PNBs II in the nucleoplasm, even when all PNBs I had completely fused (Fig. 4 g). PNBs II in nuclei 7 (Fig. 4 a) and 9 h (Fig. 4 e) after fertilization were larger than in nuclei 6 h after fertilization (Fig. 1). Complete tar- geting of B23/NO38 to the nucleolar domain was only ob- Figure 3. Targeting of PNB I components to the nucleolar do- main. Embryonic nuclei isolated 7 and 9 h after fertilization were labeled with antibodies to fibrillarin (b and e) before performing in situ hybridization of U8 (a and d). In nuclei 7 h after fertiliza- tion, U8 (a) and fibrillarin (b) were colocalized on PNBs I and gathered in two areas of the nucleoplasm as a first step through the recruitment process. In most nuclei isolated 9 h after fertiliza- tion, fibrillarin (e) and U8 (d) were colocalized in two nucleolar domains. The corresponding DNA staining with DAPI is shown (c and f). Bars, 10 mm. served in nuclei 11 h after fertilization (Fig 4 i). Targeting of B23/NO38 to the nucleolar domain was thus achieved at a later time, compared with the other rRNA processing com- ponents found in PNBs I. Similar delay between B23 and fibrillarin targeting was also observed in early G1 X. laevis A6 cells (data not shown). Fusion of PNB components to the nucleolar domain does not occur in in vitro reconstituted nuclei after several hours of incubation in the egg extract. B23/NO38 distribu- tion remained diffuse and in small PNBs, as in embryonic nuclei 6 h after fertilization. Therefore, we attempted to identify the events that occur in embryonic nuclei and not in reconstituted nuclei that could be responsible for target- ing of the rRNA processing machinery to the nucleolar domain. RNA Polymerase I Was Excluded from the Nucleolar Domain during the Recruitment of Maternally Derived Pre-rRNAs and their Processing Machinery It is generally accepted that the onset of rDNA transcrip- tion in cultured cells at the end of mitosis is an event re- Verheggen et al. Assembly of Nucleolar Components in Embryos 297