# The Nucleolus ofCaenorhabditis elegans

Authors: Li-Wei Lee; Chi-Chang Lee; Chi-Ruei Huang; Szecheng J. Lo
Journal: Journal of Biomedicine and Biotechnology
 (2012)

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## Abstract

Nucleolar size and appearance correlate with ribosome biogenesis and cellular activity. The mechanisms underlying changes in nucleolar appearance and regulation of nucleolar size that occur during differentiation and cell cycle progression are not well understood.Caenorhabditis elegans provides a good model for studying these processes because of its small size and transparent body, well-characterized cell types and lineages, and because its cells display various sizes of nucleoli. This paper details the advantages of using C. elegans to investigate features of the nucleolus during the organism's development by following dynamic changes in fibrillarin (FIB-1) in the cells of early embryos and aged worms. This paper also illustrates the involvement of the ncl-1 gene and other possible candidate genes in nucleolar-size control. Lastly, we summarize the ribosomal proteins involved in life span and innate immunity, and those homologous genes that correspond to human disorders of ribosomopathy.


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## Body

## 1. Introduction

Caenorhabditis eleganslives freely in soil. The organism occurs naturally in two sexes; both sexes have five pairs of autosomes [1]. However, hermaphrodites possess one pair of X chromosomes (XX), while males have a single X chromosome (XO). Mature adults are about 1 mm in length and 80 μm in diameter; they contain approximately 1,000 somatic cells comprising the animal’s minimal systems, including a hypodermis, muscular system, nervous system, digestive organ, and reproductive organ (Figure 1(a)). The life cycle of C. elegans takes about 3 days to complete at 25°C and comprises an embryonic stage, four larval stages (L1 to L4), and adulthood [2]. Adult hermaphrodites can produce about 300 genetically identical progeny by self-fertilization. Progeny carrying various genetic combinations are obtainable by crossing hermaphrodites and males from two genetically different backgrounds [3].Diagram and Nomarski micrographs of adult worm structures. (a) An illustration of the hermaphrodite adult worm showing the major systems and organs, not in precise proportions. Rectangular boxes indicate parts taken using Nomarski optics as shown in (b) (gonad), (c, d) (tail), and Figure2 (head region). (b) A section of a gonad arm under the light microscope. Relative positions in the gonad are indicated as distal, loop, and proximal. White arrowheads indicate the nucleoli in the corresponding cells, germ cells, and oocyte. Note that the nucleolus in the -1 oocyte is absent, and that the diameter ratio of nucleolus to nucleus decreases from germ cells to oocytes. The scale bar represents 10 μm; (c, d) the tail section showing hypodermal cell nucleoli (indicated by arrows). N2 is a wild-type worm and Ncl-1 is a mutant with loss of NCL-1 function. Scale bars indicate 10 μm.
(a)(b)(c)(d)TheC. elegans genome was fully sequenced in 1998 [4] and seven more complete nematode genomes (including Caenorhabditis briggsae, Caenorhabditis angaria, Pristionchus pacificus, and Trichinella spiralis) are now available [5, 6], providing an excellent resource for comparative genomic studies. Genetics continues to be an important tool for studying gene function in C. elegans. Many genetic mutants and knockout strains are available from the Caenorhabditis Genetics Center (CGC, University of Minnesota) for gene function and genetic epistasis experiments [7]. Additionally, whole-genome RNAi (RNA interference) clone libraries are available, providing a convenient and efficient way to study gene function genome wide [8]. Although C. elegans is a simple organism, more than 50% of its genes have human homologues, and many essential biological activities are conserved between humans and C. elegans[9]. Internet resources for worm research are available, including WormBase (http://www.wormbase.org/), WormBook (http://www.wormbook.org/), and WormAtlas (http://www.wormatlas.org/).The invariant cell linage and the transparent body ofC. elegans allows viewing of all 959 somatic cells of hermaphrodites, using Nomarski differential interference contrast (DIC) optics with a light microscope; fluorescent signals are readily detected with a fluorescence microscope for a reporter gene (green fluorescence protein, gfp) observations. The nucleolus is an easily recognizable subcompartment within the nucleus of germline cells and tail hypodermal cells (Figures 1(b)–1(d)). While electron microscopic studies of whole worm sections have been carried out (see WormAtlas), it remains unclear whether the worm nucleolus contains a fibrillar center (FC), a dense fibrillar center (DFC), and a granular component (GC) as discreet regions, as found in higher eukaryotes. In the C. elegans genome, there are approximately 1400 genes known to produce functional noncoding RNA (ncRNA) transcripts. These include about 275 ribosomal RNA (rRNA) genes and around 100 small nucleolar RNA (snoRNA) genes [4, 10–12]. Approximately 55 copies of a 7.2 kb tandem repeat of rDNA is located at the end of chromosome I, which are transcribed to pre-rRNA and then processed into primary 18S, 5.8S, and 26S rRNA subunits. About 110 copies of a 1 kb rDNA unit located on chromosome V are transcribed into a 5S rRNA [4, 13–17].C. elegans has approximately 75 genes coding for the small and large subunits of ribosomal proteins, and hundreds of genes coding for nonribosomal proteins that either play a role in ribosome biogenesis, or are of unknown function in the nucleolus. C. eleganscontains more than 1,000 operons, which are transcribed into polycistronic mRNA and trans-spliced into two to eight mature mRNAs for translation [18]. Genes arranged in operons are ubiquitous, and may be transcribed in all types of cells, explaining why a high percentage of operons is involved in ribosome biogenesis [19]. For example, the operon CEOP5428 encodes fibrillarin (FIB-1) and RPS-16 [20]. FIB-1 is a methyltransferase for pre-rRNA processing and modification, and RPS-16 is a ribosomal protein of the small subunit. Many genes coding important nucleolar proteins found in humans are also conserved in worms, though they are not arranged in operons, examples are Nopp140 (dao-5) and nucleostemin (nst-1). Nopp140 serves as a scaffold protein, binding to rDNA and acrocentric α-satellite DNA in the human nucleolus; it is required for maintenance of nucleolus integrity [21]. Loss of dao-5 in worms results in growth retardation (C. C. Lee and S. J. Lo, unpublished data). The NST-1 protein belongs to a conserved family of nucleolar GTPases and functions to export pre-60S ribosomal subunits from the nucleolus [22, 23]. Global loss of NST-1 results in a larval arrest phenotype; while loss of NST-1 in germline results in animals displaying germline stem cell proliferation arrest [22]. A few other genes that encode nucleolar or nucleolar-associated proteins in humans, such as B23 (nucleophosmin) and coilin (a hallmark protein of the Cajal body), are not found in worms.Many human cancer cells exhibit an increase in both nucleolar size and number because of the high demand for ribosomes in the rapidly dividing cells. This hallmark feature provides a marker for pathological diagnosis [24, 25]. Mutations in the cMyc oncogene and tumor suppressor genes p53and Rb are associated with alteration of nucleolar structure [26, 27]. The ncl-1 gene (abnormal NuCLeolus) was found to regulate nucleolar size in C. elegans, functioning similarly to the p53 and Rb tumor suppressors in vertebrates [28]. The ncl-1gene is a useful tool for mosaic analysis because of the enlarged nucleolus in the ncl-1 mutant, and because it acts cell autonomously [29]. In worms, NCL-1 is a homologue of Brat (brain tumor) in Drosophila[30]. Absence or alteration of the brat gene causes overproliferation of neuron cells and enlarged nucleoli in flies [31]. However, while ncl-1 mutations in worms cause enlargement of the nucleoli in all cells, mutations do not induce proliferation of neurons [28].The association of nucleolar size with genetic content has long been observed in tomato plants [32], and nucleolar-size changes occur in response to seasonal environmental cues and nutrition uptake in fish and experimental rats [33, 34]. In mammalian cells, the target of the rapamycin (TOR) pathway plays a nutrition-sensing role by coupling growth factors and nutrients to protein homeostasis [35, 36]. TOR kinase (let-363) in C. elegans is a homologue of TOR complex 1. Mango and colleagues previously demonstrated that let-363 and ruvb-1, members of the TOR pathway, control nucleolar size and promote localization of box C/D snoRNPs to nucleoli [37].The following sections describe the morphology and variation in nucleoli size in different cells, dynamic patterns of FIB-1 as visualized by fluorescence microscopy, and functionality of nucleoli inC. elegans.


## 2. Morphology ofC. elegans Nucleoli

### 2.1. Visualization ofC. elegans Nucleoli by Nomarski and Fluorescence Microscopy

Nomarski optics (DIC microscopy) provides high-quality images and is commonly used to observe individual cells ofC. elegans [38]. The high refractive index provided by DIC microscopy provides micrographs that clearly reveal the nucleoli of germ cells and growing oocytes in a gonad (Figure 1(b)). An adult hermaphrodite gonad is composed of two U-shaped arms classified as distal and proximal ends, based on their position relative to the uterus (Figure 1(a)). The distal arm contains germ cells in the mitotic zone, and cells in various stages of meiosis. The proximal arm contains growing, mature oocytes, and the spermatheca. The germ cells in the syncytium exhibit a high ratio of nucleolus-to-nucleus diameter; the oocytes have a complete plasma membrane with an enlarged nucleus and exhibit a decrease in the ratio of nucleolus to nucleus diameter (Figure 1(b)). The nucleolar size of germ cells, oocytes, and intestinal nucleoli (see below) are relatively large, with diameters ranging from 3 to 7 μm in worms, while the nucleoli of oocytes, adjacent to the spermatheca (designated as -1 oocyte), are undetectable (Figure 1(b)).The nuclei and nucleoli of the hypodermis at the hermaphrodite tail are easily observable under a DIC microscope (Figures1(c) and 1(d)) because it is the only region of the worm with a single layer of cells [38]. The nucleoli of these hypodermal cells are intermediate sizes, ranging from 2 μm to 3 μm in diameter. As shown in Figures 1(c) and 1(d), nucleolar size differences in hypodermal cells between wild-type (N2) and ncl-1mutant worms are easily identifiable [38]. Although NCL-1 is a negative regulator for rRNA biosynthesis [28], it remains unclear how the protein regulates nucleolus size (see below). The ncl-2(e1896) gene, a mutant with defects in germline nucleoli, has not yet been fully characterized.Observing and determining the size of the nucleoli of neuronal cells, particularly those within the nerve ring—the equivalent to the brain of higher animals is more difficult because neuronal cells are found in a multiple layer zone and are the smallest nucleoli in worms (Figure2(a)). The use of transgenic worms that express fibrillarin, one of the most conserved nucleolar proteins, fused with green fluorescence protein FIB-1::GFP facilitates detection of neuronal nucleoli under a fluorescence microscope (Figure 2(b)). Compared with hypodermal cell nucleoli, the nucleoli of neurons in the nerve ring are smaller and less easily distinguished in DIC micrographs (Figures 2(a) and 2(c)). By contrast, the nucleoli of neurons expressing FIB-1::GFP are instantly recognizable under a fluorescence microscope, despite their being less than one micron in diameter (Figure 2(b)). Nucleolar size usually reflects the cell activity of ribosome biogenesis [24, 25]. We speculate that germ cells and intestinal cells may exhibit greater ribosome production activities than neuronal cells do, presumably because neuron function has less need for protein translation than intestinal cells have.Comparison of nucleolar appearance of cells in the head region in Nomarski and fluorescence micrographs. A transgenic worm expressing nucleolar protein FIB-1::GFP was photographed using Nomarski and fluorescent microscopy. An arrow indicates the nucleolus of neuronal cells located at the nerve ring (as indicated) while arrowheads indicate the nucleoli of the hypodermis in (a) Nomarski micrograph, (b) fluorescence micrograph, and (c) merged (a, b) images. The scale bar indicates 20μm.
(a)(b)(c)


### 2.2.ncl-1 in Nucleolar-Size Control in Worms

Transgenic worms expressing FIB-1::GFP in wild-type (N2) andncl-1 background worms are easily distinguishable because in the former, nucleoli appear as weak GFP foci and are few in number, while the latter have more foci and produce a stronger GFP signal (Figure 3(a)). Absence of NCL-1 causes an increase in FIB-1 expression and an enlargement of nucleoli in the cell; however, the magnitudes of these effects are not the same for each cell (compare the cells indicated by a double arrowhead and single arrowheads in Figure 3(a)). There may be different levels of ncl-1expression in various tissues, for example, the precursor cells of the intestine are the first cell type to lose NCL-1 during embryogenesis [28]. Another interesting feature is the difference in nucleolar size seen in the hypodermis, adjacent to five ventral neurons in worm larva (Figure 3(b), lower panel). The five ventral cord neurons share the same hypodermis precursor cell (Figure 3(b), upper panel). It would be interesting to know whether the hypodermis displays a decrease of NCL-1, or if the ventral neuron expresses greater levels of NCL-1 immediately after precursor cells have completed asymmetric cell division. A recent study on the ASE gustatory neurons revealed that asymmetric ASE cell size, in which the ASER soma is larger than that of ASEL, is under developmental control [39]. ASER and ASEL neuron nucleoli size and number are also different. These are affected by the fib-1 gene, and directly or indirectly are regulated by die-1, which encodes a Zn finger transcription factor [39]. These findings support the hypothesis that asymmetric cell division during development, may alter expression of regulator factors that control nucleolar size.Comparison of the nucleolar size inncl-1 and N2 worms. (a) N2 and ncl-1 background transgenic worms (as indicated) side by side and photographed under a fluorescence microscope to show the head region. The heads of both worms are facing left. Double arrowheads indicate nucleoli where the nucleolar protein (FIB-1::GFP) expression was less affected by NCL-1 than nucleoli marked by a single arrow. (b) Schematic illustration of ventral cord neurons and hypodermal cell lineages (upper panel). The fluorescence micrograph shows that the hypodermis nucleolar size (indicated by double arrowheads) is larger than nucleoli in neuronal cells (indicated by single arrowheads). The worm head is facing left and the ventral side is at the top. (c) A worm gonad arm-section treated with RNAi against the ncl-1 gene shows a similar appearance to the -1 oocyte nucleolus (arrowhead) of ncl-1 mutants. Scale bars indicate 20 μm.
(a)(b)(c)RNAi gene silencing is performed by feeding wormsE. coli, which produces a double-stranded RNA corresponding to the target gene in the worms to be knocked down [8, 40]. N2 worms in which ncl-1 has been silenced by RNAi show enlarged nucleoli and bear the appearance of a nucleolus of the -1 oocytes (Figure 3(c), also compare with Figure 1(b)), a phenotype similar to the ncl-1 mutant animals. The presence of a nucleolus in the -1 oocyte indicates that NCL-1 controls nucleolar size in somatic cells, and regulates nucleolar formation in the -1 oocyte. Since nucleolar size decreases on moving from the -2 oocyte, to the -1 oocyte, and to the ncl-1 worm blastomeres, it is very likely that at least one other gene, besides ncl-1, is a negative regulator of nucleolar size in growing oocytes and blastomeres.


### 2.3. Dynamic Pattern Changes of FIB-1::GFP in Transgenic Worms

Transgenic worms expressing FIB-1::GFP also provide a tool to study nucleolar pattern changes during embryogenesis and aging. In the 40–60 embryo cell stage, FIB-1::GFP appears as one or two small bright dots (less than 0.2μ m diameter) in the nucleolar region, and as a faint distribution in the nucleoplasm (Figure 4(a)). This feature is also present in embryos expressing GFP::NST-1 [22], a nucleolar protein involved in maintenance of worm germ cells. Cell division and nucleogenesis are continuous when the embryo is in the active mitotic stage, and so an electron microscopy investigation is needed to determine if the small diameter foci seen for FIB-1::GFP and GFP::NST-1 represent prenucleolar bodies or small nucleoli.FIB-1::GFP dynamic pattern changes during development. (a) An embryo at the 40–50 cell stage, arrowheads indicate strong dots of nucleoli. Faint GFP outlines are the nucleus. (b) Intestinal cells at the larval stage, arrowheads indicate circles that may represent a GC region. (c) Intestinal cells at the young adult stage, arrowheads indicate GFP foci close to the nucleoli. These may be like the Cajal body or perinucleolar compartment (PNC) in human cells. (d) Intestinal cells of an aged worm, arrowheads indicate FIB-1::GFP fragments, possibly a sign that ribosome biogenesis was aberrant in these cells. The scale bar represents 10μm.
(a)(b)(c)(d)After the completion of mitotic division, larval intestinal cells appear as a ring structure with FIB-1::GFP occupying a few empty holes (Figure4(b)). An electron micrograph displaying a similar ring feature, shows a higher intensity of granules in the periphery than at the center (see WormAtlas). Since FIB-1 is located at the fibrillar center (FC) and dense fibrillar center (DFC) but not the granular component (GC) in other eukaryotes, the non-GFP region is probably a GC in worms. In adults, the FIB-1::GFP occupies the entire nucleolus, and occasionally, a small green dot appears near the nucleolus (Figure 4(c)). This small substructure may correspond to the Cajal body or the perinucleolar compartment (PNC) of higher organisms [41–43], although worms do not have a gene homolog for coilin. It is probable that the small focus expressing the FIB-1::GFP marker protein is a Cajal body based on two observations; fibroblast cells derived from the coilin-gene knock-out mouse have Cajal bodies [44], and fibrillarin and Nopp140 are present in the coilin-null Cajal body [45].Fragmented and irregularly shaped FIB-1::GFP is common in the intestinal nucleoli of aged worms (Figure4(d)), possibly reflecting reduced ribosome biogenesis activity in the intestinal cells of aged worms. This suggestion is supported by reports that aged worms exhibit decreased expression of lamin A, and that lamin A maintains the functional plasticity of nucleoli [46–48]. Additionally, mammalian cells display fragmented fibrillarin distribution when treated with inhibitors, such as actinomycin D and Aapatone [49, 50].


### 2.4. Similar Components for Localization of Nucleolar Proteins in Worm and Mammalian Cells

Proteomic analysis reveals that human nucleoli contain at least 700 proteins, most of which are conserved across species [51] (and references therein). To investigate the similarities in molecular function of worm and mammalian cell nucleoli, one can reciprocally introduce nucleolar genes into worms or mammalian cells. Worms transformed by dao-5, a human homologue of Nopp140, fused with GFP show fluoresce in the nucleoli of most cells (Figure 5(a)) [52]. Conversely, the DAO-5 GFP signal produced by HeLa cells transfected with the dao-5::GFP gene is colocalized with fibrillarin in the nucleoli (Figures 5(b)–5(d)), indicating that the DAO-5 nucleolar localization signal sequence (NoLS) is recognizable by both worms and mammalian factors.GFP::DAO-5 expression in worms and HeLa cells. (a) A transgenic worm expressing GFP::DAO-5 shows an oval distribution of green fluorescence representing cell nucleoli. (b and c) HeLa cells transfected by plasmids containinggfp::dao-5 and co-stained with anti-fibrillarin antibody show the location of nucleoli expressing GFP fusion protein (arrows). (d) The same field of cells stained by a DNA dye and visualized through a UV fluorescence microscope. The scale bar in (a) indicates 100 μm.
(a)(b)(c)(d)Human hepatitis D virus (HDV) is the smallest known RNA virus. HDV has a genome of approximately 1700 nucleotides that encodes two antigen isoforms named SDAg and LDAg, for the small and large delta antigens, respectively [53]. SDAg is required for genome replication while the LDAg is used for virion packaging [54]. Both antigens can target the nucleoli of human cells, although they also shuttle to other locations when posttranslationally modified [55–58]. LDAg is detectable at the nucleoli of transfected human hepatoma cells by using GFP as a reporter of gene expression (Figures 6(a)–6(c)). When transgenic worms coexpress FIB-1::GFP and RFP::LD using a bicistronic vector [59], LDAg is localized at the nucleoli of most cells by FIB-1::GFP (Figures 6(d)–6(f)). Interestingly, in the presence of FIB-1::GFP, RFP::LD is found at the perimeter of the nucleolus in intestinal cells (Figures 6(g)–6(i)). The localization of LDAg at the edge of the nucleolus is uncommon in mammalian cells, which implies that worm intestinal cells have different components to those of mammals. These examples indicate that both organisms’ nucleolar components recognize LDAg and DAO-5 NoLS.Nucleolar localization of the large delta antigen of human hepatitis D virus in human hepatoma cells and in worms. Human hepatoma cells transfected with plasmids expressing GFP::LDAg, (a) visualized by GFP, (b) stained with DNA dye, and (c) the merged picture. (d–i) A transgenic worm carrying a bicistronic vector coexpressing FIB-1::GFP and RFP::LD. A low magnification of worms photographed via (d) the FITC channel to visualize FIB-1::GFP, (e) the rhodamine channel to visualize RFP::LD, and (f) the merged picture. The rectangles in panels (d–f) show four intestinal cells in the same worm. A high magnification of intestinal cells shows (g) FIB-1::GFP, (h) RFP::LD, and (i) the merged picture. Arrows indicate the nucleoli. Note that RFP::LD and FIB-1::GFP are not perfectly colocalized. Scale bars indicate 10μm.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 2.1. Visualization ofC. elegans Nucleoli by Nomarski and Fluorescence Microscopy

Nomarski optics (DIC microscopy) provides high-quality images and is commonly used to observe individual cells ofC. elegans [38]. The high refractive index provided by DIC microscopy provides micrographs that clearly reveal the nucleoli of germ cells and growing oocytes in a gonad (Figure 1(b)). An adult hermaphrodite gonad is composed of two U-shaped arms classified as distal and proximal ends, based on their position relative to the uterus (Figure 1(a)). The distal arm contains germ cells in the mitotic zone, and cells in various stages of meiosis. The proximal arm contains growing, mature oocytes, and the spermatheca. The germ cells in the syncytium exhibit a high ratio of nucleolus-to-nucleus diameter; the oocytes have a complete plasma membrane with an enlarged nucleus and exhibit a decrease in the ratio of nucleolus to nucleus diameter (Figure 1(b)). The nucleolar size of germ cells, oocytes, and intestinal nucleoli (see below) are relatively large, with diameters ranging from 3 to 7 μm in worms, while the nucleoli of oocytes, adjacent to the spermatheca (designated as -1 oocyte), are undetectable (Figure 1(b)).The nuclei and nucleoli of the hypodermis at the hermaphrodite tail are easily observable under a DIC microscope (Figures1(c) and 1(d)) because it is the only region of the worm with a single layer of cells [38]. The nucleoli of these hypodermal cells are intermediate sizes, ranging from 2 μm to 3 μm in diameter. As shown in Figures 1(c) and 1(d), nucleolar size differences in hypodermal cells between wild-type (N2) and ncl-1mutant worms are easily identifiable [38]. Although NCL-1 is a negative regulator for rRNA biosynthesis [28], it remains unclear how the protein regulates nucleolus size (see below). The ncl-2(e1896) gene, a mutant with defects in germline nucleoli, has not yet been fully characterized.Observing and determining the size of the nucleoli of neuronal cells, particularly those within the nerve ring—the equivalent to the brain of higher animals is more difficult because neuronal cells are found in a multiple layer zone and are the smallest nucleoli in worms (Figure2(a)). The use of transgenic worms that express fibrillarin, one of the most conserved nucleolar proteins, fused with green fluorescence protein FIB-1::GFP facilitates detection of neuronal nucleoli under a fluorescence microscope (Figure 2(b)). Compared with hypodermal cell nucleoli, the nucleoli of neurons in the nerve ring are smaller and less easily distinguished in DIC micrographs (Figures 2(a) and 2(c)). By contrast, the nucleoli of neurons expressing FIB-1::GFP are instantly recognizable under a fluorescence microscope, despite their being less than one micron in diameter (Figure 2(b)). Nucleolar size usually reflects the cell activity of ribosome biogenesis [24, 25]. We speculate that germ cells and intestinal cells may exhibit greater ribosome production activities than neuronal cells do, presumably because neuron function has less need for protein translation than intestinal cells have.Comparison of nucleolar appearance of cells in the head region in Nomarski and fluorescence micrographs. A transgenic worm expressing nucleolar protein FIB-1::GFP was photographed using Nomarski and fluorescent microscopy. An arrow indicates the nucleolus of neuronal cells located at the nerve ring (as indicated) while arrowheads indicate the nucleoli of the hypodermis in (a) Nomarski micrograph, (b) fluorescence micrograph, and (c) merged (a, b) images. The scale bar indicates 20μm.
(a)(b)(c)


## 2.2.ncl-1 in Nucleolar-Size Control in Worms

Transgenic worms expressing FIB-1::GFP in wild-type (N2) andncl-1 background worms are easily distinguishable because in the former, nucleoli appear as weak GFP foci and are few in number, while the latter have more foci and produce a stronger GFP signal (Figure 3(a)). Absence of NCL-1 causes an increase in FIB-1 expression and an enlargement of nucleoli in the cell; however, the magnitudes of these effects are not the same for each cell (compare the cells indicated by a double arrowhead and single arrowheads in Figure 3(a)). There may be different levels of ncl-1expression in various tissues, for example, the precursor cells of the intestine are the first cell type to lose NCL-1 during embryogenesis [28]. Another interesting feature is the difference in nucleolar size seen in the hypodermis, adjacent to five ventral neurons in worm larva (Figure 3(b), lower panel). The five ventral cord neurons share the same hypodermis precursor cell (Figure 3(b), upper panel). It would be interesting to know whether the hypodermis displays a decrease of NCL-1, or if the ventral neuron expresses greater levels of NCL-1 immediately after precursor cells have completed asymmetric cell division. A recent study on the ASE gustatory neurons revealed that asymmetric ASE cell size, in which the ASER soma is larger than that of ASEL, is under developmental control [39]. ASER and ASEL neuron nucleoli size and number are also different. These are affected by the fib-1 gene, and directly or indirectly are regulated by die-1, which encodes a Zn finger transcription factor [39]. These findings support the hypothesis that asymmetric cell division during development, may alter expression of regulator factors that control nucleolar size.Comparison of the nucleolar size inncl-1 and N2 worms. (a) N2 and ncl-1 background transgenic worms (as indicated) side by side and photographed under a fluorescence microscope to show the head region. The heads of both worms are facing left. Double arrowheads indicate nucleoli where the nucleolar protein (FIB-1::GFP) expression was less affected by NCL-1 than nucleoli marked by a single arrow. (b) Schematic illustration of ventral cord neurons and hypodermal cell lineages (upper panel). The fluorescence micrograph shows that the hypodermis nucleolar size (indicated by double arrowheads) is larger than nucleoli in neuronal cells (indicated by single arrowheads). The worm head is facing left and the ventral side is at the top. (c) A worm gonad arm-section treated with RNAi against the ncl-1 gene shows a similar appearance to the -1 oocyte nucleolus (arrowhead) of ncl-1 mutants. Scale bars indicate 20 μm.
(a)(b)(c)RNAi gene silencing is performed by feeding wormsE. coli, which produces a double-stranded RNA corresponding to the target gene in the worms to be knocked down [8, 40]. N2 worms in which ncl-1 has been silenced by RNAi show enlarged nucleoli and bear the appearance of a nucleolus of the -1 oocytes (Figure 3(c), also compare with Figure 1(b)), a phenotype similar to the ncl-1 mutant animals. The presence of a nucleolus in the -1 oocyte indicates that NCL-1 controls nucleolar size in somatic cells, and regulates nucleolar formation in the -1 oocyte. Since nucleolar size decreases on moving from the -2 oocyte, to the -1 oocyte, and to the ncl-1 worm blastomeres, it is very likely that at least one other gene, besides ncl-1, is a negative regulator of nucleolar size in growing oocytes and blastomeres.


## 2.3. Dynamic Pattern Changes of FIB-1::GFP in Transgenic Worms

Transgenic worms expressing FIB-1::GFP also provide a tool to study nucleolar pattern changes during embryogenesis and aging. In the 40–60 embryo cell stage, FIB-1::GFP appears as one or two small bright dots (less than 0.2μ m diameter) in the nucleolar region, and as a faint distribution in the nucleoplasm (Figure 4(a)). This feature is also present in embryos expressing GFP::NST-1 [22], a nucleolar protein involved in maintenance of worm germ cells. Cell division and nucleogenesis are continuous when the embryo is in the active mitotic stage, and so an electron microscopy investigation is needed to determine if the small diameter foci seen for FIB-1::GFP and GFP::NST-1 represent prenucleolar bodies or small nucleoli.FIB-1::GFP dynamic pattern changes during development. (a) An embryo at the 40–50 cell stage, arrowheads indicate strong dots of nucleoli. Faint GFP outlines are the nucleus. (b) Intestinal cells at the larval stage, arrowheads indicate circles that may represent a GC region. (c) Intestinal cells at the young adult stage, arrowheads indicate GFP foci close to the nucleoli. These may be like the Cajal body or perinucleolar compartment (PNC) in human cells. (d) Intestinal cells of an aged worm, arrowheads indicate FIB-1::GFP fragments, possibly a sign that ribosome biogenesis was aberrant in these cells. The scale bar represents 10μm.
(a)(b)(c)(d)After the completion of mitotic division, larval intestinal cells appear as a ring structure with FIB-1::GFP occupying a few empty holes (Figure4(b)). An electron micrograph displaying a similar ring feature, shows a higher intensity of granules in the periphery than at the center (see WormAtlas). Since FIB-1 is located at the fibrillar center (FC) and dense fibrillar center (DFC) but not the granular component (GC) in other eukaryotes, the non-GFP region is probably a GC in worms. In adults, the FIB-1::GFP occupies the entire nucleolus, and occasionally, a small green dot appears near the nucleolus (Figure 4(c)). This small substructure may correspond to the Cajal body or the perinucleolar compartment (PNC) of higher organisms [41–43], although worms do not have a gene homolog for coilin. It is probable that the small focus expressing the FIB-1::GFP marker protein is a Cajal body based on two observations; fibroblast cells derived from the coilin-gene knock-out mouse have Cajal bodies [44], and fibrillarin and Nopp140 are present in the coilin-null Cajal body [45].Fragmented and irregularly shaped FIB-1::GFP is common in the intestinal nucleoli of aged worms (Figure4(d)), possibly reflecting reduced ribosome biogenesis activity in the intestinal cells of aged worms. This suggestion is supported by reports that aged worms exhibit decreased expression of lamin A, and that lamin A maintains the functional plasticity of nucleoli [46–48]. Additionally, mammalian cells display fragmented fibrillarin distribution when treated with inhibitors, such as actinomycin D and Aapatone [49, 50].


## 2.4. Similar Components for Localization of Nucleolar Proteins in Worm and Mammalian Cells

Proteomic analysis reveals that human nucleoli contain at least 700 proteins, most of which are conserved across species [51] (and references therein). To investigate the similarities in molecular function of worm and mammalian cell nucleoli, one can reciprocally introduce nucleolar genes into worms or mammalian cells. Worms transformed by dao-5, a human homologue of Nopp140, fused with GFP show fluoresce in the nucleoli of most cells (Figure 5(a)) [52]. Conversely, the DAO-5 GFP signal produced by HeLa cells transfected with the dao-5::GFP gene is colocalized with fibrillarin in the nucleoli (Figures 5(b)–5(d)), indicating that the DAO-5 nucleolar localization signal sequence (NoLS) is recognizable by both worms and mammalian factors.GFP::DAO-5 expression in worms and HeLa cells. (a) A transgenic worm expressing GFP::DAO-5 shows an oval distribution of green fluorescence representing cell nucleoli. (b and c) HeLa cells transfected by plasmids containinggfp::dao-5 and co-stained with anti-fibrillarin antibody show the location of nucleoli expressing GFP fusion protein (arrows). (d) The same field of cells stained by a DNA dye and visualized through a UV fluorescence microscope. The scale bar in (a) indicates 100 μm.
(a)(b)(c)(d)Human hepatitis D virus (HDV) is the smallest known RNA virus. HDV has a genome of approximately 1700 nucleotides that encodes two antigen isoforms named SDAg and LDAg, for the small and large delta antigens, respectively [53]. SDAg is required for genome replication while the LDAg is used for virion packaging [54]. Both antigens can target the nucleoli of human cells, although they also shuttle to other locations when posttranslationally modified [55–58]. LDAg is detectable at the nucleoli of transfected human hepatoma cells by using GFP as a reporter of gene expression (Figures 6(a)–6(c)). When transgenic worms coexpress FIB-1::GFP and RFP::LD using a bicistronic vector [59], LDAg is localized at the nucleoli of most cells by FIB-1::GFP (Figures 6(d)–6(f)). Interestingly, in the presence of FIB-1::GFP, RFP::LD is found at the perimeter of the nucleolus in intestinal cells (Figures 6(g)–6(i)). The localization of LDAg at the edge of the nucleolus is uncommon in mammalian cells, which implies that worm intestinal cells have different components to those of mammals. These examples indicate that both organisms’ nucleolar components recognize LDAg and DAO-5 NoLS.Nucleolar localization of the large delta antigen of human hepatitis D virus in human hepatoma cells and in worms. Human hepatoma cells transfected with plasmids expressing GFP::LDAg, (a) visualized by GFP, (b) stained with DNA dye, and (c) the merged picture. (d–i) A transgenic worm carrying a bicistronic vector coexpressing FIB-1::GFP and RFP::LD. A low magnification of worms photographed via (d) the FITC channel to visualize FIB-1::GFP, (e) the rhodamine channel to visualize RFP::LD, and (f) the merged picture. The rectangles in panels (d–f) show four intestinal cells in the same worm. A high magnification of intestinal cells shows (g) FIB-1::GFP, (h) RFP::LD, and (i) the merged picture. Arrows indicate the nucleoli. Note that RFP::LD and FIB-1::GFP are not perfectly colocalized. Scale bars indicate 10μm.
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3. Functionality of Nucleoli inC. elegans

### 3.1. Ribosome Biogenesis

As with other eukaryotes,C. elegans nucleolus’ main function is as a site for ribosome production, including pre-rRNA transcription, processing, modification, and ribosome assembly [52, 62]. Many methods exist to measure and quantify rRNA biosynthesis activity in cells or organisms. Isotope pulse and chase labeling, and Northern blot analysis are commonly used to reveal dynamic changes during progression of pre-rRNA into mature rRNA. These methods are not applicable at the level of the single cell because of physical limits of detection; in situ demonstration of ribosome biogenesis can be carried out by isotope or BrUTP incorporation to visualize rRNA synthesis activity in individual cells. For example, the signal from BrUTP incorporation into a worm gonad demonstrated that germ cells in the pachytene zone during meiotic division have greater rRNA synthesis activity than germ cells exhibit during the mitotic phase or in the maturing oocyte (Figure 7). Increased rates of ribosome biogenesis occur in the germ cells of many animals in preparation for fast protein-translation during embryogenesis. Currently, the rRNA synthesis activity of individual muscular and neuronal cells in worms remains unclear because of the difficulty of the required analytical techniques.In situ BrUTP incorporation in a gonad to show rRNA synthesis activity. (a) Incorporation of BrUTP was detected by an anti-BrUTP antibody conjugated with fluorescence dye Alexa 594. Arrowheads indicate the signal from BrUTP incorporation. (b) DNA staining of the same gonad. Note the increased BrUTP incorporation in germ cells at the pachytene zone of meiosis. The scale bar indicates 10 μm.
(a)(b)


### 3.2. Worm Ribosome Biogenesis Deficiency Phenotypes

Killian and Hubbard reported theC. elegans “proximal tumor” phenotype, exhibited by the hermaphrodite gonad, this was the first phenotype associated with loss of nucleolar integrity and impaired ribosome biogenesis function [63]. Hubbard mapped this unusual phenotype to progenes (pro-1, pro-2, and pro-3), which encode factors involved in ribosome biogenesis [63, 64]. RNAi screening against other factors involved in ribosome biogenesis revealed a similar gonadogenesis phenotype, suggesting that such phenotypes can result from inefficient ribosome biogenesis.In addition to being a ribosome factory, nucleoli have important roles in other cellular processes such as the cell cycle, stress response, and coordination of the biogenesis of other RNPs species [65, 66]. Ribosomal proteins may act independently to interact with other cellular proteins, for example, RPS-16 and RPL-6 interact with LET-756, one of two C. elegansfibroblast growth factors (EGFs), for the regulation of ribosome biogenesis [67]. Interestingly, a mutation in nol-6, which encodes a nucleolar RNA-associated protein, was found to enhance worm innate immunity against bacterial infection [68]. In mammals, the disruption of ribosomal proteins or nucleolar proteins elicits a p53-mediated response [65]. RNAi screening revealed that knock down of any one of 20 small ribosomal proteins results in increased resistance to p53/CEP-1-dependent bacterial infection [68]. Germ cells have been demonstrated to control both innate immunity and lifespan through distinct signaling pathways [69]. It is unclear whether germ cell nucleoli are involved in these activities, nevertheless, depletion of several ribosomal proteins such as RPS-3, RPS-8, and RPS-16 at the postembryonic stage extended worm life expectancy [70, 71].


### 3.3. Worm Genes Corresponding to Ribosomopathy-Associated Human Disease

Impaired ribosome biogenesis resulting from loss of nucleolar integrity or disruption of rRNA biosynthesis has been described as “nucleolus stress” or “ribosomal stress” [65]. Ribosomopathiesis a clinical pathological term defined as “a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes” [61, 72]. These disorders include Bowen-Conradi syndrome [60], cartilage-hair hypoplasia (CHH) [73], dyskeratosis congenital (DC) [74], Diamond-Blackfan anemia (DBA) [75, 76], Shwachman-Diamond syndrome (SDS) [77], and Treacher-Collin syndrome [78, 79]. Human genes associated with ribosomopathy are also present in C. elegans (Table 1). Most genes encode ribosomal proteins, although some are nucleolar proteins involved in pre-rRNA processing, for example,mrpr-1 encodes for a noncoding RNA. Correlating phenotype with mutations in worms can aid our understanding of the mechanisms of human diseases and so inform drug development for new treatments in the future.Table 1
Mutated human genes involved in the diseases of ribosomopathies and theirC. eleganshomologs.
DiseaseDefective gene*C. eleganshomologChromosomal locationProduct and functionOperonBowen-Conradi syndromeEMG1Y39A1A.14IIIMethyltransferaseTreachere-Collins syndromeTCOF1K06A9.1XThree predicated proteins, containing 825 aa, 1032 aa, and 2232 aa5q-syndromeRPS14rps-14IIISmall ribosomal subunit S14 proteinCartilage hair hypoplasiaRMRPmrpr-1IIRNA component of the endoribonuclease RNase MRPShwachman-Diamond syndromeSBDSW06E11.4IIINucleolar protein required for maturation of 60S ribosomal subunitsT19C3.7Dyskeratosis congenitaDKC1K01G5.5IIIA predicated protein containing 445 aaDiamond-Blackfan anemiaRPS19, RPS24, RPS17, RPL35, RPL5, RPL11, RPS7, RPL36, RPS15, RPS27Arps-19ISmall ribosomal subunit S19 protein*References: Armistead et al., [60]; Narla and Ebert, [61].


## 3.1. Ribosome Biogenesis

As with other eukaryotes,C. elegans nucleolus’ main function is as a site for ribosome production, including pre-rRNA transcription, processing, modification, and ribosome assembly [52, 62]. Many methods exist to measure and quantify rRNA biosynthesis activity in cells or organisms. Isotope pulse and chase labeling, and Northern blot analysis are commonly used to reveal dynamic changes during progression of pre-rRNA into mature rRNA. These methods are not applicable at the level of the single cell because of physical limits of detection; in situ demonstration of ribosome biogenesis can be carried out by isotope or BrUTP incorporation to visualize rRNA synthesis activity in individual cells. For example, the signal from BrUTP incorporation into a worm gonad demonstrated that germ cells in the pachytene zone during meiotic division have greater rRNA synthesis activity than germ cells exhibit during the mitotic phase or in the maturing oocyte (Figure 7). Increased rates of ribosome biogenesis occur in the germ cells of many animals in preparation for fast protein-translation during embryogenesis. Currently, the rRNA synthesis activity of individual muscular and neuronal cells in worms remains unclear because of the difficulty of the required analytical techniques.In situ BrUTP incorporation in a gonad to show rRNA synthesis activity. (a) Incorporation of BrUTP was detected by an anti-BrUTP antibody conjugated with fluorescence dye Alexa 594. Arrowheads indicate the signal from BrUTP incorporation. (b) DNA staining of the same gonad. Note the increased BrUTP incorporation in germ cells at the pachytene zone of meiosis. The scale bar indicates 10 μm.
(a)(b)


## 3.2. Worm Ribosome Biogenesis Deficiency Phenotypes

Killian and Hubbard reported theC. elegans “proximal tumor” phenotype, exhibited by the hermaphrodite gonad, this was the first phenotype associated with loss of nucleolar integrity and impaired ribosome biogenesis function [63]. Hubbard mapped this unusual phenotype to progenes (pro-1, pro-2, and pro-3), which encode factors involved in ribosome biogenesis [63, 64]. RNAi screening against other factors involved in ribosome biogenesis revealed a similar gonadogenesis phenotype, suggesting that such phenotypes can result from inefficient ribosome biogenesis.In addition to being a ribosome factory, nucleoli have important roles in other cellular processes such as the cell cycle, stress response, and coordination of the biogenesis of other RNPs species [65, 66]. Ribosomal proteins may act independently to interact with other cellular proteins, for example, RPS-16 and RPL-6 interact with LET-756, one of two C. elegansfibroblast growth factors (EGFs), for the regulation of ribosome biogenesis [67]. Interestingly, a mutation in nol-6, which encodes a nucleolar RNA-associated protein, was found to enhance worm innate immunity against bacterial infection [68]. In mammals, the disruption of ribosomal proteins or nucleolar proteins elicits a p53-mediated response [65]. RNAi screening revealed that knock down of any one of 20 small ribosomal proteins results in increased resistance to p53/CEP-1-dependent bacterial infection [68]. Germ cells have been demonstrated to control both innate immunity and lifespan through distinct signaling pathways [69]. It is unclear whether germ cell nucleoli are involved in these activities, nevertheless, depletion of several ribosomal proteins such as RPS-3, RPS-8, and RPS-16 at the postembryonic stage extended worm life expectancy [70, 71].


## 3.3. Worm Genes Corresponding to Ribosomopathy-Associated Human Disease

Impaired ribosome biogenesis resulting from loss of nucleolar integrity or disruption of rRNA biosynthesis has been described as “nucleolus stress” or “ribosomal stress” [65]. Ribosomopathiesis a clinical pathological term defined as “a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes” [61, 72]. These disorders include Bowen-Conradi syndrome [60], cartilage-hair hypoplasia (CHH) [73], dyskeratosis congenital (DC) [74], Diamond-Blackfan anemia (DBA) [75, 76], Shwachman-Diamond syndrome (SDS) [77], and Treacher-Collin syndrome [78, 79]. Human genes associated with ribosomopathy are also present in C. elegans (Table 1). Most genes encode ribosomal proteins, although some are nucleolar proteins involved in pre-rRNA processing, for example,mrpr-1 encodes for a noncoding RNA. Correlating phenotype with mutations in worms can aid our understanding of the mechanisms of human diseases and so inform drug development for new treatments in the future.Table 1
Mutated human genes involved in the diseases of ribosomopathies and theirC. eleganshomologs.
DiseaseDefective gene*C. eleganshomologChromosomal locationProduct and functionOperonBowen-Conradi syndromeEMG1Y39A1A.14IIIMethyltransferaseTreachere-Collins syndromeTCOF1K06A9.1XThree predicated proteins, containing 825 aa, 1032 aa, and 2232 aa5q-syndromeRPS14rps-14IIISmall ribosomal subunit S14 proteinCartilage hair hypoplasiaRMRPmrpr-1IIRNA component of the endoribonuclease RNase MRPShwachman-Diamond syndromeSBDSW06E11.4IIINucleolar protein required for maturation of 60S ribosomal subunitsT19C3.7Dyskeratosis congenitaDKC1K01G5.5IIIA predicated protein containing 445 aaDiamond-Blackfan anemiaRPS19, RPS24, RPS17, RPL35, RPL5, RPL11, RPS7, RPL36, RPS15, RPS27Arps-19ISmall ribosomal subunit S19 protein*References: Armistead et al., [60]; Narla and Ebert, [61].


## 4. Conclusion and Prospects

This review illustrates thatC. elegans is a useful animal for studying nucleolus biology. C. elegans has a short life cycle and is very easy to handle in the laboratory. Different cell types become available throughout the worms various stages of development from embryo to adult and during aging, and during the various phases of the cell cycle, including meiotic and mitotic division, asymmetric cell division, and postmitotic stages. Nomarski optics allow the study and analysis of changes in nucleolar size, and used in combination with GFP tagging of nucleolar proteins, transgenic worms can provide information about nucleolar activity. RNAi silencing of specific genes is straightforward to perform in worms and many mutant worms are now available, facilitating the study of nucleoli functionality in worms. C. elegans is a good choice to study nucleolus biology during development, and many important problems remain unresolved, for example, investigating how ncl-1 is regulated temporally and spatially, and determining the functions of the Cajal-like bodies that lack coilin, in worms.



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*Source: 601274-2012-04-19.xml*