blastomeres was required for drug accessibility, embryos 3 h 30 min after fertilization were transferred to Ca21- and Mg21-free medium containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, and 7.5 mM Tris HCl, pH 7.6, as previously described (Duval et al., 1990). Actinomycin D (Sigma Chemical Co.) was added to the incubation medium at the desired con- centration from a stock solution at 5 mg/ml. As a control, embryos were incubated in Ca21- and Mg21-free medium without actinomycin D. Other drugs, such as a-amanitin and 5,6-dichloro-b-D-ribofuranosylbenzimida- zole (DRB; Sigma Chemical Co.) could not be used as inhibitors of tran- scription in X. laevis embryos. When a-amanitin was added to the incuba- tion medium, it did not penetrate into the dissociated blastomeres and DRB had a deleterious effect on the division of the embryos. Suspensions of embryonic nuclei were prepared from embryos taken at precise times after fertilization as described (Verheggen et al., 1998). Immunofluorescence and In Situ Hybridization In vitro reconstituted nuclei and suspensions of embryonic nuclei were fixed with 1 vol 4% paraformaldehyde in PBS. The suspensions of fixed nuclei could be stored several weeks at 48C. For immunofluorescence and in situ hybridization studies, nuclei were centrifuged onto a coverslip. Af- ter washing, the different coverslips were postfixed in methanol, perme- abilized in 0.1% Triton X-100 (IBI) in PBS, and rinsed. For immunofluorescence labeling, the coverslips were incubated with primary antibodies, followed by FITC- or TRITC-conjugated secondary antibodies (anti-human, anti-mouse, or anti-rabbit IgG; Jackson Immuno- Research Laboratories) or cy3-conjugated anti-mouse antibodies (Sigma Chemical Co.), rinsed, counterstained with DAPI (49-6-diamidino-2-phe- nylindole dihydrochloride; Polysciences, Inc.) and mounted with an anti- fading solution (Citifluor). In situ hybridization of rRNAs was performed after immunolabeling as previously described (Verheggen et al., 1998). The rRNA hybridization mixture contained 40% formamide (GIBCO BRL), 10% (wt/vol) dextran sulfate (Sigma Chemical Co.), 50 ng/ml sonicated salmon testes DNA (Sigma Chemical Co.), and the biotinylated rDNA probe diluted to a final concentration of 1 ng/ml in 23 SSC. For the detection of U3 and U8, the hy- bridization mixture contained 30% formamide, 10% (wt/vol) dextran sul- fate, 50 ng/ml sonicated salmon testes DNA, and the 39 end biotinylated oligo- nucleotides complementary to U3 and U8 diluted to a final concentration of 2 ng/ml in 23 SSC. As a control for the detection of RNA, hybridization was preceded by RNase digestion as described (Highett et al., 1993). Electron Microscopy In vitro reconstituted nuclei were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, at 48C. They were washed in cacodylate buffer, postfixed in 1% OsO4 for 1 h at 48C, dehydrated in alcohol, and embedded in Epon 812. Ultrathin sections were contrasted with uranyl ac- etate for 1 h and lead citrate for 2 min, and examined in a Philips EM412 electron microscope. In Situ Transcription Assay To localize transcription sites, 5-bromouridine-59-triphosphate (Br-UTP) incorporation into embryonic nuclei was performed as previously de- scribed (Verheggen et al., 1998). In brief, nonfixed nuclei were permeabi- lized and incubated in transcription buffer in the presence of Br-UTP for 20 min at room temperature (Masson et al., 1996). Before immunolabel- ing, the nuclei were fixed with 2% paraformaldehyde in PBS and perme- abilized with 0.1% Triton X-100. A monoclonal anti–Br-deoxyuridine an- tibody which also recognizes Br-UTP (Boehringer Mannheim) was used. Human antifibrillarin antibodies were used as a nucleolar marker. Tran- scription and fibrillarin signals were obtained using FITC-conjugated goat anti–mouse and TRITC-conjugated goat anti–human antibodies (Jackson ImmunoResearch Laboratories), respectively. Optical Microscopy Images were taken with a Leica epifluorescence microscope equipped with a thermoelectronically cooled charge-coupled device (CCD) camera (Leica). Grayscale images were collected separately with filter sets for FITC and rhodamine/TRITC using an oil immersion lens (633, I.4 plan Apochromat). Grayscale images were pseudocolored and merged us- ing the Adobe Photoshop 5.0 software. Results Components of the rRNA Processing Machinery Are Located in Two Types of PNBs in Early Embryonic and Reconstituted Nuclei During the building process of the nucleolus in X. laevis embryonic nuclei, components of rRNA processing ma- chinery are associated in bodies called PNBs dispersed in the nucleoplasm. A first class of PNBs (PNBs I) containing fibrillarin and nucleolin, is observed 6 h after fertilization. A maternal pool of U3 is maintained during early develop- ment of X. laevis (Xia et al., 1995) and we compared its distribution to fibrillarin in embryonic nuclei 6 h after fer- tilization. Fluorescent in situ hybridization (FISH) of U3 snoRNAs was performed after immunolabeling of fibril- larin. U3 snoRNAs were colocalized with fibrillarin in all PNBs I and were also present on some other dots in the nucleoplasm (Fig. 1, a–d). Moreover, U8 snoRNAs, impli- cated both in early and late steps of rRNA processing in X. laevis, were also detected in PNBs I by FISH (see Fig. 3). On the contrary, B23/NO38, a nucleolar protein involved in a late step of pre-rRNA processing, did not colocalize with fibrillarin when both proteins were revealed on embryonic nuclei isolated 6 h after fertilization (Fig. 1, e–h). Instead, B23/NO38 appeared diffuse in the nucleoplasm and some proteins were distributed in numerous small dots through- out the nucleoplasm, and were termed PNBs II. This could be an intermediate step in the recruitment of B23/NO38 to PNBs II since later, B23/NO38 becomes distributed in PNBs II of large size (see Fig. 4). Consequently, the for- mation of PNBs II appears to be delayed, compared with PNBs I. The recruitment of nucleolar proteins and snoRNAs of the rRNA processing machinery to fibrillar PNB-like structures has been described using in vitro reconstituted nuclei (Bell et al., 1992; Bauer et al., 1994). We performed double immunolabeling experiments on reconstituted nu- clei to see whether the components of the processing ma- chinery were in the same structures or in different classes of PNBs as in embryonic nuclei. Permeabilized sperm nuclei incubated in a low-speed X. laevis egg extract formed largely decondensed nuclei. After immunolabeling of fibril- larin, U3 and U8 snoRNAs were detected by FISH in these reconstituted nuclei. U3 was colocalized with fibrillarin in large round-shaped structures (Fig. 1, i–l). U8 displayed a similar distribution (not shown). B23/NO38 did not colo- calize with fibrillarin (Fig. 1, m–p), but showed a distribu- tion similar to that of the embryonic nuclei 6 h after fertili- zation. In particular, B23/NO38 accumulated in small and numerous dots. In EM, two types of nucleolar bodies were observed (Fig. 2). In reconstituted nuclei, the general view of sections makes it possible to discriminate few dense struc- tures and numerous small nuclear bodies (Fig. 2 a). The dense bodies (mean diameter, 0.5 mm) exhibited a fibrillar matrix containing densely packed granules of 10–15 nm (Fig. 2 b), structures also observed at preMBT in embryonic PNBs (data not shown). Their size, distribution, and mor- phological features are compatible with the identification of these bodies as PNBs I. On the contrary, the small nuclear bodies (mean diameter, 0.1 mm) were gray without visible dense granules (Fig. 2 c). Based on their size, distribution Verheggen et al. Assembly of Nucleolar Components in Embryos 295