Three-Dimensional Distribution of UBF and Nopp140 in Relationship to Ribosomal DNA Transcription During Mouse Preimplantation Development1

Abstract

The nucleolus is a dynamic nuclear compartment that is mostly involved in ribosome subunit biogenesis; however, it may also play a role in many other biological processes, such as stress response and the cell cycle. Mainly using electron microscopy, several studies have tried to decipher how active nucleoli are set up during early development in mice. In this study, we analyzed nucleologenesis during mouse early embryonic development using 3D-immunofluorescent detection of UBF and Nopp140, two proteins associated with different nucleolar compartments. UBF is a transcription factor that helps maintain the euchromatic state of ribosomal genes; Nopp140 is a phosphoprotein that has been implicated in pre-rRNA processing. First, using detailed image analyses and the in situ proximity ligation assay technique, we demonstrate that UBF and Nopp140 dynamic redistribution between the two-cell and blastocyst stages (time of implantation) is correlated with morphological and structural modifications that occur in embryonic nucleolar compartments. Our results also support the hypothesis that nucleoli develop at the periphery of nucleolar precursor bodies. Finally, we show that the RNA polymerase I inhibitor CX-5461: 1) disrupts transcriptional activity, 2) alters preimplantation development, and 3) leads to a complete reorganization of UBF and Nopp140 distribution. Altogether, our results underscore that highly dynamic changes are occurring in the nucleoli of embryos and confirm a close link between ribosomal gene transcription and nucleologenesis during the early stages of development.

Nucleolus and rRNA Gene Chromatin in Early Embryo Development

“…Data also suggest that the re-establishment of nucleoli during early embryo development is tightly linked to transcription. Inactivation of Pol I with the inhibitor CX-5461 led to a reorganization of Nopp140 and UBF, which formed nucleolar caps at both the two-and four-cell stages [51]. These caps corresponded to those observed in SN oocytes and in interphase cells treated with inhibitors of Pol I transcription.…”

“…Embryos lacking RNA polymerase 1-2 (RPO1-2) or UBF arrest development before the morula stage [48,49]. Furthermore, pharmacological inhibition of rRNA gene transcription using CX-5461 [50] induced developmental delay and arrest at the four-cell stage [51].…”

“…Upon fertilization, the NLBs that disappeared during oocyte meiotic maturation reappear in both maternal and paternal pronuclei of zygotes in the form of nucleolus precursor bodies (NPBs), which persist throughout the first four cell cycles [52] (Figure 2 for RNA and detectable amounts of RNA appear on the NPB surface only after resumption of rRNA gene transcription [53]. Accordingly, during the late two-cell and four-cell stages, UBF and Nopp140, two nucleolar proteins that are associated with FC and DFC of the nucleolus, localize around the periphery of the NPBs [51]. These results also suggest that active rRNA genes, which are bound by UBF [19,20], are formed at the outer surface of the NPB.…”

“…These results also suggest that active rRNA genes, which are bound by UBF [19,20], are formed at the outer surface of the NPB. Starting at the eight-cell stage, NPBs diminish in size, disappearing by the late 16-cell stage, with the emergence of intermingled fibrillar and granular compartments and UBF positioning in the center of the former NPB [51]. Since nucleoli and activation of rRNA genes occurs at the periphery of NPBs, it has been suggested that NPBs are the building blocks of the future somatic nucleoli.…”

The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals

“…33 Indeed, treatment of embryos with CX-5461 (inhibitor of rRNA gene transcription) triggered embryonic lethality at the four-cell stage. 40 Deletion of RNA polymerase 1-2 (RPO1-2) or UBF results in developmental arrest prior to the morula stage. 29,31 In the present study, we show NOP2 is first detected in late two-cell stage and its depletion results in a significant reduction of rRNA expression, regardless of the precursor and mature rRNAs.…”