Figure 1. Components of rRNA processing machinery were regrouped in two types of PNBs both in embryonic and reconstituted nuclei. FISH of U3 (a; FITC) was performed after immunolabeling of fibrillarin (b; Texas red) on embryonic nuclei isolated 6 h after fertiliza- tion. U3 was distributed in numerous dots dispersed in the nucleoplasm. A large number of these dots colocalized with fibrillarin (c; yel- low dots on the merged image). They were called PNBs I. Some other dots of U3 were also seen in the nucleoplasm (c; green dots on the merged image). In contrast, B23/NO38 (e; cy3) was not colocalized with fibrillarin (f, FITC) in a double immunolabeling experiment on embryonic nuclei isolated 6 h after fertilization. Labeling of B23/NO38 was diffuse and partially clustered in small dotted structures. These structures were called PNBs II. Distribution of the rRNA processing machineries was similar in in vitro reconstituted nuclei. Per- meabilized sperm nuclei were incubated in low-speed X. laevis egg extract and further treated for in situ hybridization of U3 (i; FITC) af- ter immunolabeling of fibrillarin (j; Texas red). U3 was completely colocalized with fibrillarin (k; yellow dots on the merged image). PNBs I formed in these nuclei were larger and easily distinguished by phase-contrast microscopy (l) in contrast to PNBs I in embryonic nuclei (d and h). Immunolabeling of B23/NO38 on reconstituted nuclei (m) showed the same distribution as in embryonic nuclei 6 h after fertil- ization. B23/NO38 (m; cy3) was not accumulated on the large PNBs I containing fibrillarin (n; FITC). Bars, 10 mm. (Fig. 2 a), and number, we postulated that these nuclear bod- ies might correspond to PNBs II. Small nuclear bodies with low contrast were also observed in embryonic nuclei (data not shown). Immunolabeling using B23/NO38 antibody was attempted, but without success, most probably because for EM, the antibody has too low a titer. Thus, morphologically different nuclear bodies were formed in reconstituted nuclei. We propose that they correspond to PNBs I and PNBs II. PNBs I and PNBs II Are Targeted to the Nucleolar Domain with Different Kinetics during Development We next defined the kinetics of recruitment of PNBs I and PNBs II to the nucleolar domain. Embryonic nuclei were isolated at various times during development, between 7 and 9 h after fertilization. Labeling of PNB I components (fibrillarin, nucleolin, U3, and U8) revealed a progression towards regrouping in nucleolar domain. PNBs I were first seen gathered in two restricted areas of the nucleoplasm in some nuclei 7 h after fertilization (Fig. 3, a–c). By 9 h after fertilization, they had fused in two large nucleolar domains in most nuclei (Fig. 3, d–f). Intermediate structures larger than PNBs I and smaller than the nucleolar domain were formed (Fig. 4 b) and were visible by phase-contrast mi- croscopy (Fig. 4 d) in nuclei between 7 and 9 h after fertili- zation. B23/NO38 colocalized with fibrillarin in these structures (Fig. 4 c). Thus, fusion of the PNB I and PNB II components to the nucleolar domain was initiated nearly The Journal of Cell Biology, Volume 149, 2000 296