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Review
. 2017 Jun 3;14(6):752-760.
doi: 10.1080/15476286.2016.1255397. Epub 2016 Nov 18.

Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos

Patricia Heyn  1 , Hanna Salmonowicz  1 , Jonathan Rodenfels  2 , Karla M Neugebauer  2
Affiliations
Review

Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos

Patricia Heyn et al. RNA Biol. .

Abstract

Nuclear bodies are cellular compartments that lack lipid bilayers and harbor specific RNAs and proteins. Recent proposals that nuclear bodies form through liquid-liquid phase separation leave the question of how different nuclear bodies maintain their distinct identities unanswered. Here we investigate Cajal bodies (CBs), histone locus bodies (HLBs) and nucleoli - involved in assembly of the splicing machinery, histone mRNA 3' end processing, and rRNA processing, respectively - in the embryos of the zebrafish, Danio rerio. We take advantage of the transcriptional silence of the 1-cell embryo and follow nuclear body appearance as zygotic transcription becomes activated. CBs are present from fertilization onwards, while HLB and nucleolar components formed foci several hours later when histone genes and rDNA became active. HLB formation was blocked by transcription inhibition, suggesting nascent histone transcripts recruit HLB components like U7 snRNP. Surprisingly, we found that U7 base-pairing with nascent histone transcripts was not required for localization to HLBs. Rather, the type of Sm ring assembled on U7 determined its targeting to HLBs or CBs; the spliceosomal Sm ring targeted snRNAs to CBs while the specialized U7 Sm-ring localized to HLBs, demonstrating the contribution of protein constituents to the distinction among nuclear bodies. Thus, nucleolar, HLB, and CB components can mix in early embryogenesis when transcription is naturally or artificially silenced. These data support a model in which transcription of specific gene loci nucleates nuclear body components with high specificity and fidelity to perform distinct regulatory functions.

Keywords: Cajal body; coilin; fibrillarin; histone locus body; liquid phase separation; zebrafish; zygotic genome activation.

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Figures

Figure 1.
Figure 1.
Activation of rDNA transcription recruits fibrillarin from the CB. (A) Immunostaining for the CB marker coilin (green) and the snoRNP component fibrillarin (magenta) over a developmental time course from the 64-cell stage to 8 hours post-fertilization (hpf). Insets magnify (2x) 2 representative bodies in the same nucleus (indicated by gray and yellow arrowheads). All scale bars, 10 μm. A Gaussian blur filter was applied for display reasons. (B) Detection of the onset of rDNA transcription through RT-qPCR amplification of pre-rRNA. Primers complementary to the 5′ external transcribed spacer (ETS) of 18S RNA were used to amplify pre-rRNA; signal (y-axis) was normalized to RT-qPCR values for pre-mitochondrial RNA, which was previously shown to be constant over these time points.
Figure 2.
Figure 2.
HLB assembly coincides with the onset of zygotic transcription. (A) Embryonic nuclei imaged at the indicated stages after injection of fluorescent U2 snRNA (red) to mark CBs and U7 snRNA (green) to mark HLBs. Upper panel: DIC image from in vivo imaging of whole zebrafish embryos with 10x magnification. Scale bar equals 100μm. Middle panel: Single confocal sections of in vivo imaging of zebrafish embryos labeled with microinjected U7 and U2 snRNA. Scale bar equals 10μm. Arrowheads point to the nuclear body shown 2x magnified in bottom panel. A Gaussian blur filter was applied for display reasons. (B) Immunostaining for SLBP (red in the merged panel) shows the concentration of SLBP in the same foci as those marked by injected U7 snRNA (green). Scale bar equals 10 μm (C) Scatterplot showing the levels in FPKM of individual transcripts metabolically labeled by 4s-UTP injection into 1-cell embryos and purified from 128-cell (x-axis) and 512-cell (y-axis) embryos; histone genes with a fold change in transcript level of log2 ≥ 2 are displayed in red, showing that transcription of histone genes begins between the 128-cell and 512-cell stage. Data taken from ref. .
Figure 3.
Figure 3.
Separation of CBs and HLBs is transcription-dependent. Zygotic transcription was blocked by injection of α-amanitin into 1-cell embryos. Single Z-stacks of confocal series from fixed blastula stage embryos. Untreated (- amanitin) and treated (+ amanitin) embryos are labeled with antibodies specific for SLBP (A) or coilin (C); co-injection of U4 (red) and U7 snRNAs (green) provides an independent view of CBs and HLBs, respectively (B). White square indicates area shown magnified in each inset (4x magnification). Scale bars equal 10 μm. (D) Quantification of CBs per nucleus (coilin immunostaining, as in C; data from n=18 embryos from 3 independent experiments at 4hpf), HLBs per nucleus (SLBP, as in A; data from n = 18 untreated and n = 17 α-amanitin injected embryos from 3 independent experiments at 4hpf), and of the overlapping signals between HLBs and CBs (U4 and U7 snRNAs, as in B; n = 31 nuclei from untreated embryos and n = 16 nuclei from α-amanitin injected embryos at 3hpf). Distribution, mean number (dashed line) and median number (bold line) are shown. Asterisks indicate statistical significance (Welch 2-sample t-test, P = 0.004702 for CBs, P = 1.043e-10 for HLBs and P = 7.208e-05 for U4/U7 snRNA).
Figure 4.
Figure 4.
Sm ring composition specifies snRNA localization to CBs and HLBs. (A) Co-injection of U2 (magenta) and U7 (green) snRNAs shows independence of CBs and HLBs in 3 dimensions. Projection of the fluorescent data collected by confocal imaging is shown in the left panel; rendering of CB and HLB centers of mass, obtained through the use of Imaris analysis software, is shown in the right panel. (B) Scheme for testing the contribution of base-pairing to histone transcripts through mutational analysis of the U7 snRNA HDE site is shown. To test the contribution of the Sm-ring, the specialized Sm site of mouse U7 (mU7) was mutated to Sm-OPT, previously shown to target the spliceosomal Sm ring to U7 snRNA. (C-G) Confocal sections showing the localization patterns of wild-type U7 snRNA compared to other markers and mutants, as indicated. Scale bars equal 10 μm. Arrowheads point to foci magnified 2x in the insets.
Figure 5.
Figure 5.
Transcription-dependent changes in embryonic nuclear architecture. Working model showing that between fertilization and ZGA, fibrillarin initially concentrates in CBs while U7 snRNA is not present in foci. Upon transcription activation of histone loci, U7 and SLBP are recruited to HLBs that are separate from CBs in zebrafish embryos. If Pol II transcription is inhibited, U7 accumulates in CBs. Fibrillarin remains associated with CBs until activation of rDNA transcription at 6hpf. Thus, the integrity and composition of each nuclear body depends on transcription.
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