Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

Abstract

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Fig. 1.

Cellular localization of LIN28 protein in fully grown mouse CD1 oocytes. Confocal immunofluorescence analysis of endogenous LIN28 in whole-mount oocytes. B23 (NPM) was used as a nucleolar marker. DNA was counterstained with DAPI. In NSN oocytes (A-D) LIN28 and NPM localize at the periphery of the nucleolus. During transition from NSN to SN configuration, both LIN28 and NPM gradually disappear from surface of the nucleolus and they are not detected there in SN oocytes (E-H). Insets show higher magnification of nucleus. Scale bar: 20 μm.

Fig. 2.

Cellular localization of LIN28 protein during mouse preimplantation development. Confocal immunofluorescence analysis of endogenous LIN28 in whole-mount embryos. B23 (NPM) was used as a nucleolar marker. DNA was counterstained with DAPI. An enlarged view of the boxed regions show that LIN28 (green) colocalizes with NPM (red). (A-D) In zygotes, homogenous nuclear signals of both LIN28 and NPM were observed. No enrichment at the periphery of nucleolar precursor bodies (NPBs) was observed for either marker. (E-P) At the early and mid 2-cell stage, LIN28 localizes at the periphery of nucleolar precursor bodies (NPBs) (arrow), where it colocalizes with NPM (E-H). LIN28 remains colocalized with NPM at NPBs (arrow) at the 4-cell (I-L) and 8-cell (M-P) stages. (Q-T) As NPBs have transformed to mature nucleoli in morula embryos, LIN28 remains nucleolar, as confirmed by the colocalization with NPM. (U-X) At the blastocyst stage, the localization of LIN28 in the nucleolus is concordant with NPM. LIN28 was present in both TE and ICM cells. Scale bar: 20 μm.

Fig. 3.

Cellular localization of a LIN28-Tomato fusion protein in early mouse embryos. Confocal immunofluorescence analysis of LIN28-Tomato fusion protein in whole-mount embryos following microinjection of mRNA, in vitro culture and fixation. B23 (NPM) was used as a nucleolar marker. DNA was counterstained with DAPI. As control, zygotes were microinjected with mRNA coding only for tdTomato protein. (A-H) Tomato expression is throughout the cytoplasm and nuclei in 4-cell embryos (A-D) and in 8/16 embryos (E-H). (I-L) At the 2-cell stage, the LIN28-Tomato fusion protein signal appears at distinct cytoplasmic foci that are formed around the nucleus (arrows). NPM staining is restricted to the nucleoplasm. (M-P) Focal plane of one blastomere of a late 2-cell embryo, where LIN28-Tomato localizes to one NPB, which is stained by NPM at the periphery (arrows). (M′-P′) Focal plane of second blastomere of the same late 2-cell embryo, where LIN28-Tomato signal is absent at presumptive NPBs, which lack NPM staining at periphery. (Q-T) At the 4-cell stage, LIN28-Tomato colocalizes with NPM at NPBs (an enlarged view of the LIN28-Tomato signal is shown in the boxed region). (U-X) In 8/16 embryos, LIN28-Tomato associates with mature, differentiated nucleoli. Scale bars: 20 μm in A-X; 10 μm in inset in U.

Fig. 4.

Expression of Lin28 mRNA during mouse preimplantation development. High levels of Lin28 are detectable in MII-arrested oocytes. As development proceeds, Lin28 mRNA levels start to decrease in zygotes reaching low levels at late G2 of the 2-cell stage. An increase of Lin28 expression is detected again at the late 4-cell stage and subsequent cleavage stages.

Fig. 5.

Cellular localization of LIN28 protein in early embryos and ESCs of the common marmoset monkey. Confocal immunofluorescence analysis of endogenous LIN28. B23 (NPM) was used as a nucleolar marker. DNA was counterstained with DAPI. (A-D) In 11-cell embryos, LIN28 is enriched in the nucleoplasm and not at the periphery of NPBs, whereas NPM is already present at the periphery of NPBs. An enlarged view of the boxed region shows that LIN28 (green) does not colocalize with NPM (red). (E-H) In blastocyst embryos, LIN28 colocalizes with NPM at the nucleolus, but only in trophoblast (TE) and not ICM cells. ICM cells only show a NPM signal (red). Boxed region in H shows full projection of ICM and TE cells, which are displayed as z-sections in supplementary material Fig. S5. (I-L) In ESCs, LIN28 is predominantly cytoplasmic and absent from the nucleolus. Scale bars: 20 μm.

Fig. 6.

LIN28 depletion after fertilization results in a developmental arrest. (A,B) A significant number of Lin28A-MO-injected embryos arrest at the 2- or 4-cell stage after 48 hours of in vitro culture in comparison with controls (P<0.001). Lin28A-MO-injected embryos never develop to morula or blastocyst stage after 96 hours of in vitro culture, unlike the majority of control embryos. Error bars represent mean ± s.e.m. from at least three independent sets of experiments. (C) Immunostaining confirmed that nucleolar LIN28 expression is absent in Lin28A-MO-injected embryos, arrested at the 4-cell stage, but present in Control-MO-injected and uninjected embryos. DNA was counterstained with propidium iodide. Scale bar: 20 μm.

Fig. 7.

NPM expression and chromocenter formation following LIN28 depletion. Confocal immunofluorescence analysis of NPM (green) and HP1 (red) in whole-mount embryos. DNA was counterstained with DAPI. (Ad-f) Different optical slices illustrate that NPM is distributed throughout the nucleoplasm and is not enriched at the periphery of presumptive NPBs in 2-cell arrested embryos. (Aj-l) In control late 2-cell embryos, NPM is enriched at the periphery of NPBs. (Aa-c,g-i) Heterochromatin and chromocenter clustering is brightly stained by DAPI (arrow). Heterochromatic foci are outside (Aa,b,g) or in association (Ac,h) with NPBs (arrow). (Ba-c′) Chromocenters were strongly enriched in HP1. An enlarged view of a single optical slice shows that HP1 (red) colocalizes with condensed DNA (arrow) in 2-cell arrested (Ba-c) and control (Ba′-c′) embryos. Scale bar: 20 μm.