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. 2010 Jan-Feb;1(1):96-108.
doi: 10.4161/nucl.1.1.10680.

Dynamic control of Cajal body number during zebrafish embryogenesis

Magdalena Strzelecka  1 , Andrew C Oates, Karla M Neugebauer
Affiliations

Dynamic control of Cajal body number during zebrafish embryogenesis

Magdalena Strzelecka et al. Nucleus. 2010 Jan-Feb.

Abstract

The Cajal body (CB) is an evolutionarily conserved nuclear subcompartment, enriched in components of the RNA processing machinery. The composition and dynamics of CBs in cells of living organisms is not well understood. Here we establish the zebrafish embryo as a model system to investigate the properties of CBs during rapid growth and cell division, taking advantage of the ease of live-cell imaging. We show that zebrafish embryo CBs contain coilin and multiple components of the pre-mRNA splicing machinery. Histone mRNA 3' end processing factors, present in CBs in some systems, were instead concentrated in a distinct nuclear body. CBs were present in embryos before and after activation of zygotic gene expression, indicating a maternal contribution of CB components. During the first 24 hours of development, embryonic cells displayed up to 30 CBs per nucleus; these dispersed prior to mitosis and reassembled within minutes upon daughter cell nucleus formation. Following zygotic genome activation, snRNP biogenesis was required for CB assembly and maintenance, suggesting a self-assembly process that determines CB numbers in embryos. Differentiation into muscle, neurons and epidermis was associated with the achievement of a steady state number of 2 CBs per nucleus. We propose that CB number is regulated during development to respond to the demands of gene expression in a rapidly growing embryo.

Keywords: Cajal body; pre-mRNA splicing; scaRNA; snRNA; snRNP; zygotic gene expression.

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Figures

Figure 1
Figure 1
Detection of zebrafish coilin in nuclear bodies. (A) Schematic representation of zebrafish coilin indicating the percentage identity to human coilin, the position of the nuclear localization signal (NLS) and the arginine-glycine-rich (RG) box. Coilin self-association occurs via the N-terminal conserved domain (blue), whereas binding of Sm proteins has been mapped to the C-terminal domain (purple). (B) Expression of YFP-tagged zebrafish coilin following injection of mRNA into 1-cell embryos assayed by live-cell confocal imaging at early gastrula stage (5.5 hpf, hours post fertilization), single confocal section in YFP channel and corresponding DIC images. (C) Labeling of fixed late blastula stage embryos (4.5 hpf) with anti-zebrafish coilin (AB 9EA2, Fig. S1), DNA counterstained with Hoechst. (D) Colocalization of endogenous coilin with YFP-tagged zebrafish coilin expressed from in vitro transcribed and injected mRNA is shown by immunostaining of fixed embryos at early gastrula stage (5.5 hpf). Single confocal sections are shown, and arrowheads indicate nuclei magnified in the insets (two-fold magnification in C and D). Scale bars: 10 µm. Additional grayscale insets in (B–D) show embryonic stage at the time of imaging, and the approximate imaged areas (arrows). Scale bars: 250 µm.
Figure 2
Figure 2
Zebrafish nuclear bodies enriched in U7 snRNP are deficient in coilin. Embryos were injected with mRNAs encoding either mRFP-coilin (A) or mRFP-LSm11 (B) and fluorescently labeled U7 snRNA (A and B). Representative confocal sections taken at the blastula stage are shown. Insets show two-fold magnifications of the nuclei (A) or three-fold magnifications of the foci (B) marked by arrowheads. Arrows point to the adjacent coilin- and U7 snRNA-positive foci. Scale bars: 10 µm.
Figure 3
Figure 3
Spliceosomal snRNPs and scaRNAs are CB components in zebrafish embryos. (A) Embryos were fixed at the onset of segmentation (∼10 hpf) and double-stained with antibodies specific for coilin (red) and either tri-methylguanosine cap (TMG) or Sm proteins (green) present on spliceosomal snRNPs. Insets show two-fold magnification of the nucleus indicated with arrowheads. (B) Embryos were injected with either mRNAs encoding mRFP-coilin (red) and SMN-CFP (green) or mRFP-coilin (red) and Alexa488-labeled U85 scaRNA or U4 snRNA, as indicated in the green channel. Representative confocal sections for various stages of development are shown: blastula (upper), segmentation (middle), gastrulation (bottom). Insets show three-fold magnification of the CBs marked by arrowheads. Scale bars: 10 µm.
Figure 4
Figure 4
U4 snRNA positive CBs are present in the nuclei of 4-cell embryos. Embryos were injected at the 1-cell stage with 2.5 femtomole of Alexa-488-labeled, in vitro synthesized U4 snRNA. This embryo was allowed to develop for 1 hour at 28°C and mounted for in vivo imaging in 2% methylcellulose. A single confocal section is shown. Arrowheads indicate regions magnified three-fold in the insets. Scale bar: 10 µm.
Figure 5
Figure 5
Rapid assembly and disassembly of CBs at mitosis. Embryos injected with Alexa488-labeled U4 snRNA were subjected to live cell imaging at the 32-cell stage. Still images of a time-lapse movie (see Suppl. Material) show that U4 snRNA concentrates in nuclear bodies in the early blastula when cell cycles are only ∼15 minutes long. U4-positive nuclear bodies disperse immediately prior to mitosis (after frame: 444 s) and reform in daughter cells within 3 minutes (frame: 184 s). Scale bar: 5 µm.
Figure 6
Figure 6
Regulation of CB number during the cell cycle. (A) Two perspectives of a representative volume-rendered nucleus containing CBs marked by YFP-coilin for quantitation of CB number. Arrowheads point to region magnified in the inset. Major tick: 5 µm. (B) Time series of YFP-coilin expressing embryo from time-lapse confocal imaging. Embryos expressing YFP-coilin imaged at 28°C from 3.25 to 4.25 hpf, confocal sections 1.5 µm apart collected every 50 seconds. Arrowheads: nuclei in the insets. (c) Plot of CB number per nucleus (mean ± SD) from the time-lapse movie shown in (B), quantified as in (A). Each point represents an average number of CBs in the nuclei (N = 4–10 nuclei) present in the imaged volume for a particular time point. No CBs were detected in mitosis. Circles mark the time points shown in (B). Note that single confocal sections (B) do not show all the CBs from each respective nucleus (A). The maximal number of CBs per nucleus in the consecutive interphases is not statistically different (p = 0.22 for the first two interphases, and p = 0.47 for the second and third interphase). Scale bar: 10 µm.
Figure 7
Figure 7
Regulation of CB number during development and differentiation. (A) Number of CBs per nucleus at different stages of embryonic development, mean ± SD (N = 3–9 independent experiments). (B and C) Single confocal sections of striated muscle and motor neurons in live zebrafish embryos at 30 hpf injected with YFP-coilin mRNA (B) or fixed and labeled with an antibody against zebrafish coilin (9EA2; red) and neurolin (zn-8; green) a cell surface molecule on secondary motor neurons (C); hoechst counterstained nuclei are shown in blue. Arrowheads in (B) point to the nuclei magnified in the insets. Magnification is three-fold in (B) and seven-fold in (C). Arrows in the insets point to selected CBs. Scale bars: 10 µm in (B) and 20 µm in (c).
Figure 8
Figure 8
Depletion of SMN leads to dispersal of embryonic CBs. Zebrafish embryos were injected with control morpholino (CtrlMO) or morpholino targeting the 5′-UTR of Smn mRNA (SmnMO) and fixed at the onset of segmentation (∼10 hpf). Reduction in snRNA trimethylation is evident from the overall loss of signal in samples immunostained with anti-TMG (a). Loss of snRNP-containing CBs is also shown by immunostaining with anti-TMG (A) as well as anti-coilin (B). Single confocal sections are shown, and arrowheads indicate nuclei magnified three-fold in the insets. Scale bars: 10 µm.
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