Nuclear Gems and Cajal (Coiled) Bodies in Fetal Tissues: Nucleolar Distribution of the Spinal Muscular Atrophy Protein, SMN

Experimental Cell Research 265, 252–261 (2001)doi:10.1006/excr.2001.5186, available online at http://www.idealibrary.com on

Nuclear Gems and Cajal (Coiled) Bodies in Fetal Tissues: NucleolarDistribution of the Spinal Muscular Atrophy Protein, SMN

P. J. Young, T. T. Le,* M. Dunckley,† Nguyen thi Man, A. H. M. Burghes,* and G. E. Morris1

MRIC Biochemistry Group, North East Wales Institute, Mold Road, Wrexham LL11 2AW, United Kingdom; *Department of MedicalBiochemistry, Ohio State University, Columbus, Ohio 43210; and †Neuromuscular Unit, Imperial College School of Medicine,

Hammersmith Hospital, London, United Kingdom

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SMN, the affected protein in spinal muscular atro-phy (SMA), is a cytoplasmic protein that also occurs innuclear structures called “gems” and is involved insnRNP maturation. Coilin-p80 is a marker protein fornuclear Cajal bodies (coiled bodies; CBs) which arealso involved in snRNP maturation, storage or trans-port. We now show that gems and CBs are present inall fetal tissues, even those that lack gems/CBs in theadult. Most gems and CBs occur as separate nuclearstructures in fetal tissues, but their colocalization in-creases with fetal age and is almost complete in theadult. In adult tissues, up to half of all gems/CBs areinside the nucleolus, whereas in cultured cells theyare almost exclusively nucleoplasmic. The nucleolarSMN is often more diffusely distributed, comparedwith nucleoplasmic gems. Up to 30% of cells in fetaltissues have SMN distributed throughout the nucleo-lus, instead of forming gems in the nucleoplasm. Theresults suggest a function for gems distinct from Cajalbodies in fetal nuclei and a nucleolar function forSMN. Spinal cord, the affected tissue in SMA, behavesdifferently in several respects. In both fetal and adultmotor neurons, many gems/CBs occur as larger bodiesclosely associated with the nucleolar perimeter.Uniquely in motor neurons, gems/CBs are more nu-merous in adult than in fetal stages and colocalizationof gems and CBs occurs earlier in development. Theseunusual features of motor neurons may relate to theirspecial sensitivity to reduced SMN levels in SMA pa-tients. © 2001 Academic Press

INTRODUCTION

Childhood spinal muscular atrophy (SMA) is an au-tosomal recessive disorder characterized by the loss ofalpha motor neurons of the spinal cord [1]. The diseasedetermining protein, SMN (survival of motor neuron),is a 40-kDa ubiquitously expressed protein [2, 3]. Thereare two inverted copies of the SMN gene on chromo-

1 To whom correspondence should be addressed. Fax: 44-1978-
r290008. E-mail: [email protected].
2520014-4827/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

some 5q13, SMN1 and SMN2 [3]. The two genes differby only 11 nucleotides, none of which alter the codingsequence [3, 4]. However, one of these differences al-ters a putative exon splicing enhancer in exon 7, en-suring that the main product of SMN2 is a truncatedtranscript lacking exon 7 [4, 5]. The severity of thedisease is directly correlated to the levels of functionalfull-length SMN produced from the SMN2 gene [2, 6].Deletions of the SMN1 gene produce the disease phe-notype, but increased copy number of the SMN2 genecan modify the disease severity [3, 7, 8].

SMN localizes in the cytoplasm and in subnuclearstructures termed gemini of coiled bodies, or “gems”[9]. SMN has been shown to play an important role inthe cytoplasmic assembly of spliceosomal U snRNPs[10–12]. Pellizzoni et al. [13] have suggested that nu-lear SMN may be involved in recycling/regeneration of

snRNPs and Meister et al. [14] have proposed thatMN may play an active role in pre-mRNA splicing byltering the Sm core protein components of the UnRNPs. However, the nuclear role of SMN may not beestricted to splicing with several other possible func-ions suggested, including regulation of gene transcrip-ion [15–17] and nucleocytoplasmic transport [18].

The close relationship between nuclear gems andajal bodies suggests that they may have related func-

ions. Cajal bodies contain snRNA specific transcrip-ion factors, PTF and TATA-box binding protein (TBP),nd have been shown to accumulate around the U1 and2 gene clusters on chromosomes 1 and 17, respec-

ively, in interphase cells, leading to the suggestionhat they mediate U1 and U2 transcription [19]. Inhi-ition of dephosphorylation using okadaic acid resultsn the relocation of Cajal bodies to the nucleolus [20].he identification of low levels of coilin-p80 and UnRNPs in cultured HeLa cell nucleoli under normalonditions suggests that U snRNPs may undergo aaturation process within the nucleolus [20–22],hile the phosphorylation state of coilin-p80 may di-

ect U snRNPs to and from the nucleolus [20]. Thus,ajal bodies may be involved in the production, matu-
ation, and storage of U snRNPs [20–24]. Although

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253NUCLEAR GEMS AND CAJAL BODIES IN FETAL TISSUES

gems were first described as adjacent to Cajal bodies[9], more recent studies suggested that they may be thesame structure [23, 25] and our own studies of SMNand coilin-p80 colocalization in adult tissues could beinterpreted as supporting this view [26]. By studyingfetal tissues, however, we now show that gems andCajal bodies are predominantly separate early in de-velopment and colocalization increases with develop-mental age.

Although SMN and coilin-p80 are ubiquitously ex-pressed, gems and Cajal bodies are absent from somecell types, including smooth and cardiac muscle, endo-thelial cells, dermal and epidermal cells, myoepithelialcells, stomach parietal and peptic cells, and spleenparenchymal cells [26]. Total SMN levels are high incardiac muscle, so we suggested that, although cyto-plasmic SMN levels may influence gem formation, ad-ditional cell-specific factors may be required for theformation of gems and Cajal bodies [26]. The absence ofboth structures from several cell types suggests that,although both SMN and coilin-p80 are ubiquitous pro-teins, visible Cajal bodies and gems are not essentialfor viability. Previous studies have found a drop intotal SMN levels between fetal and adult tissues [27].In the present study, we have addressed the questionof whether the elevated SMN levels in fetal tissuesaffect nuclear gem and Cajal body formation and dis-tribution. We also demonstrate a novel nucleolar local-ization of SMN and report several unique characteris-tics of nuclear gems in motor neruons of the spinal cordthat may aid the understanding of the disease pathol-ogy.

EXPERIMENTAL PROCEDURES

Immunohistochemistry. Human fetal (10–18 weeks) and adultig (2 months) tissues were embedded in tissue freezing medium androzen in isopentane cooled in liquid nitrogen. Cryostat sections (5

mm) were mounted on microscope slides and fixed for 1 min with 50%methanol/50% acetone followed by a PBS wash. Previously reportedanti-SMN mouse monoclonal antibodies (MANSMA1, -3, and -5 [26]),an anti-p80 rabbit polyclonal (Ab204 [28]), and an anti-fibrillarinhuman auto-anti-serum [29] were used during this study. Mabs werediluted 1 1 4 with PBS (ca. 5 mg/ml final concn) and protein local-zation was revealed using FITC-conjugated horse anti-(mouse Ig).uclear localization was confirmed using ethidium bromide or DAPIs counterstain. For double labeling experiments, fixed sections werencubated with the mouse mAb for 1 h, followed by the rabbit poly-lonal antibody (diluted 1 in 250 PBS) for 1 h. The mAb was detectedith a FITC-conjugated goat anti-(mouse Ig) (Sigma: diluted 1 in 80BS) and the polyclonal antibody with a TRITC-conjugated goatnti-(rabbit Ig) (Sigma: diluted 1 in 128 PBS). For triple labelingxperiments, sections were exposed to mouse mAb and rabbit poly-lonal antibodies as stated above, followed by the human auto-anti-erum against fibrillarin (diluted 1 in 1000) for 1 h. The humannti-serum was detected using an AMCA-conjugated goat anti-(hu-an Ig) (Chemicon: diluted 1 in 200 PBS). TRITC and FITC (L4)lter sets and a 63X oil immersion objective were used with the LeicaMRB photomicroscope. The TRITC filter was also used for
thidium bromide and a UV filter for AMCA and DAPI. Images were
aptured under standard and equivalent conditions using an inte-rating video camera (JVC) and image grabber (Neotech).

RESULTS

Distribution of SMN, SIP-1, and Coilin-p80in Human Fetal Tissues

Although SMN and coilin-p80 are ubiquitously ex-pressed, we have shown previously that neither gemsnor Cajal bodies are ubiquitous nuclear structures [26],since both are absent from some adult cell types, in-cluding smooth and cardiac muscle, endothelial cells,dermal and epidermal cells, myoepithelial cells, stom-ach parietal and peptic cells, and spleen parenchymalcells [26]. When Cajal bodies and gems were present inadult tissues, SMN was always found to colocalize withcoilin-p80 [26]. Most studies on adult tissues were per-formed with pig or rabbit tissues because of restrictedavailability of adult human tissues. However, humanskeletal muscle biopsy sections gave similar quantita-tive results to pig muscle and the tissues that lackedCajal bodies and gems were the same in both pig andrabbit [26].

Total SMN levels are higher in fetal tissues than inadult tissues [26, 27] and Burlet et al. [27] found nu-clear gems in all fetal tissues they examined, includingbrain, heart, kidney, skeletal muscle, and thymus.Studies on cultured skin fibroblasts from SMA patientssuggest that nuclear gem formation may be inhibited(or decreased) by reduced SMN levels [2, 26]. To deter-mine whether the elevated SMN levels in fetal tissuesare associated with an increase in nuclear gem andCajal body formation, quantitative counts of nucleargems and Cajal bodies were performed on human fetaltissues using anti-SMN mAbs, MANSMA1 (exon 2 spe-cific) and MANSMA3 (exon 5 specific), and an anti-p80mAb, 5P10 (Table 1).

Nuclear Cajal bodies and gems were present in alltissues tested at all fetal ages (Table 1), includingheart, skin, and spleen (Fig. 1), which were negativefor both structures in adult tissues [26]. For all fetaltissues tested, there was an increase in the averagenumber of gems per nucleus with fetal age (Table 1).Cajal bodies were also present in all fetal tissues andwere more numerous than in adult tissues, except inmotor neurons (Table 1). In general, nuclear gems weremore abundant in the CNS than in non-CNS tissues,except that very high gem counts were found in fetalskin (Table 1). This is surprising because nuclear gemsare absent from all cell types of adult skin (Table 1; Fig.1 in Ref. [13]). The number of nuclear gems was higherin motor neurons of adult spinal cord than in fetalspinal cord (Table 1). This unusual developmental ex-pression pattern may signify a special function of nu-
clear SMN in motor neurons. In contrast to nuclear

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254 YOUNG ET AL.

gems, the highest levels of Cajal bodies were foundoutside the central nervous system, in heart, lung, andskin, none of which contain Cajal bodies in adult tis-sues (Table 1).

To determine the exact relationship between nuclearSMN and coilin-p80 in fetal tissues, double labelingexperiments were performed using MANSMA1 andAb204 [28]. Figure 2 shows that in fetal cerebral cor-tex, Cajal bodies and nuclear gems occurred as bothindependent and colocalized structures, an expressionpattern more akin to primary skin fibroblast cultures[26] than adult mammalian tissues. In all fetal tissues,colocalization of SMN and coilin-p80 within nuclearbodies increased with increasing fetal age (Table 2).Motor neurons of the spinal cord displayed the highest

TAB

Quantitative Nuclear Gem and Cajal

MANSMA1

NS1. Brain (crebral cortex—molecular neurons)

12.8 weeks 0.87 (0–3, 1613.5 weeks 1.01 (0–4, 20Adult rabbit 1.12 (1–3, 14

2. Spinal cord (anterior horn—motor neurons)10.0 weeks 0.85 (0–2, 1314.8 weeks 1.15 (0–2, 17Adult rabbbit 2.07 (1–4, 54

on-CNS1. Skeletal muscle (muscle nuclei)

10.3 weeks 0.48 (0–2, 1513.3 weeks 0.89 (0–2, 14Adult rabbit 0.44 (0–2, 71

2. Heart (myocardial cells)11.0 weeks 0.39 (0–2, 2012.8 weeks 0.43 (0–2, 16Adult rabbit 0 (200 nucle

3. Liver (parenchymal cells)12.0 weeks 0.31 (0–2, 1213.3 weeks 0.77 (0–2, 20Adult rabbit 0.89 (0–2, 75

4. Lung (myoepithelial cells)11.8 weeks 0.45 (0–2, 2014.8 weeks 0.81 (0–3, 14Adult rabbit 0 (200 nucle

5. Skin (epidermis)11.8 weeks 1.31 (0–2, 1614.8 weeks 1.77 (0–3, 11Adult rabbit 0 (200 nucle

6. Spleen (parenchymal cells)13.0 weeks 0.25 (0–1, 1018.2 weeks 0.65 (0–2, 85Adult rabbit 0 (150 nucle

Note. Data from adult rabbit tissues are shown for comparison. NANSMA1 (exon 2 specific) and MANSMA3 (exon 5 specific) an

nti-coilin-p80 mAb 5P10. The average number of gems per section isounted (when nuclear bodies were absent, the number of nuclei cou

degree of colocalization, with 50% of nuclear bodies at

the age of 14.8 weeks containing both SMN and coilin-p80 (Table 2). SMN and coilin-p80 in fetal motor neu-rons were often localized in larger bodies around thenucleolus (Fig. 3B) as previously described in adult pigmotor neurons [18, 26, 30, 31]. In non-CNS tissues,although the number of independent gems increasedwith age, the majority of gems were independent ofCajal bodies at all ages, with fewer than 25% of iden-tified bodies containing both proteins (Table 2).

Previous reports have identified cytoplasmic dot-structures containing SMN in fetal skeletal muscle[27]. As well as in skeletal muscle (10.3 and 13.3weeks), we observed similar structures in fetal brain(12.8 and 13.5 weeks), lung (11.8 and 14.8 weeks), liver(12.0 and 13.3 weeks), and spleen (13.0 and 18.2

1

dy Counts for Fetal Human Tissues

Nuclear gems Cajal bodies

MANSMA3 MANSIP1A 5P10

0.79 (0–2, 100) 0.79 (0–2, 150) 1.71 (0–6, 200)0.96 (0–2, 100) 0.92 (0–2, 217) 1.64 (0–6, 209)1.01 (0–3, 137) 1.03 (0–3, 100) 1.14 (1–3, 150)

0.96 (0–2, 175) 0.93 (0–2, 156) 1.18 (0–3, 150)1.21 (0–2, 100) 1.26 (0–2, 134) 1.21 (0–3, 160)2.25 (0–6, 45) 2.03 (0–6, 57) 2.15 (1–4, 61)

0.45 (0–2, 100) 0.47 (0–1, 76) 1.00 (0–2, 160)0.96 (0–2, 154) 0.86 (0–2, 132) 1.30 (0–3, 130)0.41 (0–2, 57) 0.39 (0–2, 59) 0.46 (0–2, 50)

0.34 (0–2, 115) 0.29 (0–1, 89) 3.56 (1–8, 250)0.39 (0–2, 95) 0.41 (0–2, 145) 2.51 (1–4, 150)0 (150 nuclei) 0 (276 nuclei) 0 (145 nuclei)

0.42 (0–2, 150) 0.37 (0–1, 90) 1.54 (0–3, 150)0.82 (0–3, 100) 0.76 (0–2, 145) 1.98 (0–3, 170)0.95 (0–3, 60) 0.97 (0–2, 58) 0.85 (0–2, 121)




lear gems were recognized using anti-SMN monoclonal antibodiesnti-SIP1A mAb MANSIP1A. Cajal bodies were identified by theowed, in parentheses, by the range and the number of nuclear bodiesed is shown).

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corresponding adult tissues, suggesting a real differ-ence between fetal and adult tissues. SMN and SIP-1colocalized in both nuclear and cytoplasmic bodies infetal cells from all ages (results not shown).

SMN Colocalizes with Fibrillarin in the Nucleolusin Fetal Tissues

SMN was also observed in nucleoli in all fetal tissuesexamined. The nucleolar localization was obtained us-ing three monoclonal antibodies against different SMNepitopes (MANSMA1, -3, and -4) and anti-SMN rabbit

FIG. 1. Distribution of SMN and coilin-p80 in fetal human andadult pig tissues. Visible gems and Cajal bodies were not detected inadult heart, spleen, and skin, but were detected in the correspondingfetal tissues. SMN was identified using MANSMA1 and a FITC-conjugated horse anti-(mouse Ig). Coilin-p80 was identified usingAb204 and a FITC-conjugated goat anti-(rabbit Ig). Nuclei werecounterstained with ethidium bromide. The bar represents 30 mm.

serum, thus ruling out the possibility of a cross-reac-

tion with another nucleolar protein (results notshown).

Fibrillarin was used as a nucleolar marker and triplelabeling was performed on fetal human and adult pigtissues, using the mouse mAb MANSMA1, anti-coilin-p80 rabbit sera 204 Ab, and human anti-fibrillarin sera(Fig. 3).

In fetal liver (13.0 weeks), skeletal muscle (13.3weeks, Fig. 3A), spinal cord (14.8 weeks, Fig. 3B), skin(11.8 weeks), and spleen (18.2 weeks), fibrillarin waspredominantly localized in the nucleolus (Table 3). Inall tissues, the majority of nucleoli stained only forfibrillarin (66–70%), but SMN colocalized with fibrilla-rin in 24–31% of the nucleoli (Table 3, Fig. 3A). In alltissues except spinal cord, those cells with SMNthroughout the nucleolus did not express nuclear gems(Fig. 3A) and vice versa (results not shown). Motorneurons of the spinal cord were the only cell type thatsometimes displayed both nucleoplasmic gems andSMN colocalized with fibrillarin (Fig. 3B). Cajal bodies,with or without SMN, were observed in less than 3% ofnucleoli in fetal tissues (Table 3), but some coilin-p80in diffuse form was detectable in the SMN-positivenucleoli of fetal tissues (Figs. 3A and 3B).

Gems and Cajal Bodies in the Nucleolusin Adult Tissues

Double and triple labeling with the fibrillarin auto-antibody revealed that nuclear gems and Cajal bodiesin adult tissues were commonly found as discrete struc-tures within nucleoli, as well as in the nucleoplasm(Fig. 3A and Table 4).

Fibrillarin staining was restricted to the nucleolus inadult tissues (Figs. 3A and 3B). This was confirmed byviewing labeled cells under phase contrast (results notshown). Colocalization of SMN and coilin-p80 was notmediated by fibrillarin, since most gems/CBs contain-ing both proteins were outside the nucleolus and werenot stained by the anti-fibrillarin antibody (Table 4;Fig. 3B). Those adult pig tissues that lack both Cajalbodies and gems also showed no nucleolar stainingwith either MANSMA1 or 204 Ab (results not shown).

As previously reported [26], the majority of Cajalbodies in adult pig tissues also contain SMN (Figs. 2and 3, Table 4). This increase in colocalization appearsto be linked with an increase in nucleolar association ofCajal bodies, with 38–46% of Cajal bodies in pig cere-bral cortex, skeletal muscle (Figs. 3A and 3C), and liver(Fig. 3C) localizing within areas of intense fibrillarinstaining (Table 4). The possibility that the CBs arelying on top of, rather than inside, the nucleoli can onlybe ruled out completely by 3D reconstruction experi-ments. In motor neurons, however, of 48 Cajal bodiesclosely associated with the nucleolus, only 1 was within
the nucleolar boundary (Fig. 3B, Table 4). In adult

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256 YOUNG ET AL.

tissues, SMN distribution was sometimes intermediatebetween the diffuse nucleolar staining pattern charac-teristic of fetal tissues (Figs. 3A–3C) and the concen-tration into sharply defined nucleoplasmic gems typi-cal of cultured cell lines. This may best be seen in adultliver parenchymal cells which often contain largepatches of SMN staining within larger, fibrillarin-pos-itive nucleoli (Fig. 3C). Within these patches of nucle-olar SMN staining, coilin-p80 staining is usually moreconcentrated in structures of similar size to nucleoplas-mic Cajal bodies (Fig. 3C). These were counted as “p80/SMN” bodies in Table 4.

DISCUSSION

A number of novel observations have emerged fromthe present study. First, the association between gemsand CBs is developmentally regulated, since they occurmainly as separate structures in fetal tissues but arealmost invariably colocalized in adult tissues. Second,the strong SMN staining throughout the nucleolus infetal tissues has not been reported previously, al-though nucleolar staining by anti-SMN antisera hasbeen reported in the adult central nervous system [31].Third, we found that, in most adult tissues examined,colocalized gems/CBs occur very frequently inside nu-cleoli, as well as in the nucleoplasm. Finally, spinalcord motor neurons were found to differ from other celltypes in several ways that may be related to the spe-cific pathogenic effects on this tissue when SMN levelsare reduced in SMA patients.

It was known from earlier studies [26, 27] that SMNlevels are higher in fetal tissues compared with adult

FIG. 2. Distribution of SMN and coilin-p80 by double labeling onsing MANSMA1 and a FITC-conjugated horse anti-(mouse Ig). Cointi-(rabbit Ig). Nuclei were counterstained with DAPI (blue). Indeprrows), and colocalized SMN and coilin-p80 (green arrows) are ind

tissues and our quantitative studies have now con- t

firmed that this is reflected in higher numbers of gemsand CBs in the nucleus (Table 1). Lafarga et al. [32]lso showed that CBs in cerebellar Purkinje cell nucleiecreased from 1.33 in newborn rats to 0.47 in adults.vidence that CB numbers are higher in dividing cellsnd metabolically active cells has been available forome time [33–38] and the higher CB numbers in fetalissues are consistent with this. In an earlier study ofdult tissues [26], however, we noted that many tissuesad no visible gems or CBs and these were not alwaysissues perceived as nondividing or “inactive” (e.g., epi-ermis and spleen). This suggested that additional tis-ue-specific factors might be needed for gem/CB forma-ion. SMN and coilin-p80 are always present in someorm in all tissues, but this is not established for all thether known components of gems and CBs. Cytoplas-ic dot-like structures containing SMN have previ-

usly been reported in fetal skeletal muscle [27]. Inddition to fetal skeletal muscle, we have seen similartructures in fetal brain, lung, spleen, and liver, butot in the corresponding adult pig tissues or humankeletal muscle (results not shown). This signifies aeal difference between adult and fetal tissues withegard to the cytoplasmic accumulation of SMN.We have shown that gems and CBs occur mainly as

eparate structures in fetal cells but show increasingolocalization during fetal development (Table 2), al-hough only a limited period of 10–18 weeks gestationould be studied with available tissues. One possiblexplanation is that a linker protein is up-regulateduring development. Fibrillarin was considered as aandidate, since it interacts with SMN in the yeast

al human and adult pig cerebral cortex. SMN (green) was identifiedp80 (red) was identified using Ab204 and a TRITC-conjugated goatdent nuclear gems (white arrow), independent Cajal bodies (yellowed. The bar represents 30 mm.

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wo-hybrid system [9] and has been detected in Cajal


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258 YOUNG ET AL.

bodies [9], but this was ruled out by its absence frommany colocalized gems/CBs in our study (Fig. 3B). An-other possibility is that some posttranslational modifi-cation (e.g., phosphorylation) of a component of gems orCBs enables their colocalization. Colocalization offibrillarin with SMN was only observed when SMN(but not coilin-p80) occurred throughout the nucleolusin 25–30% of most fetal cells studied, instead of form-ing nucleoplasmic gems (Table 3). It is not yet clearwhether these two different distributions of SMN cor-

TAB

Quantitative Counts of SMN and Coilin-p80 Colocalizatio

Tissue/age (weeks)Nuclear bo

total cel

1. Brain (cerebral cortex—molecular neurons)12.8 weeks 350/15213.5 weeks 411/209Adult 233/208

2. Spinal cord (anterior horn—motor neurons)10.0 weeks 178/12914.8 weeks 172/156Adult 212/94

1. Skeletal muscle (muscle nuclei)10.3 weeks 163/14013.3 weeks 232/122Adult 68/146Adult human 51/11

2. Heart (myocardial cells)11.0 weeks 277/10212.8 weeks 250/95Adult 0/250

3. Lung (myoepithelial cells)11.8 weeks 579/20414.8 weeks 187/118Adult 0/300

4. Liver (parenchymal cells)12.0 weeks 288/15513.0 weeks 316/128Adult 137/144

5. Skin11.8 weeks 295/8016.1 weeks 76/24Adult 0/150

Note. Counts were also performed on adult human skeletal muscleSMN was identified with MANSMA1 and coilin-p80 was identified w

FIG. 3. Distribution of SMN, coilin-p80, and fibrillarin by triple lafetal and adult pig spinal cord. SMN (red) was identified using MANwas identified using Ab204 and a FITC-conjugated goat anti-(rabbitan AMCA-conjugated goat anti-(human Ig). (A) In fetal skeletal muscIndependent Cajal bodies (white arrows) are indicated. In adult skelenucleolus (broken green arrows) is shown. (B) Cajal bodies containi(green arrows) and within the nucleoplasm (broken white arrows) arbright SMN staining (red) within the nucleoli (blue) is larger thancoilin-p80 staining (green outside nucleoli, or white when colocalizedin muscle as well as liver. (c) Three Cajal bodies, two inside a nucle
30 mm and the black bar in C represents 10 mm.
respond to different cell types or different metabolicstates, but the cells with nucleolar SMN staining didnot appear to form any recognizable structure in thefetal tissues. Although SMN dispersed throughout nu-cleoli was not observed in adult tissues as a specificstaining pattern, SMN was present throughout thecytoplasm and nucleus (including nucleoli) in motorneurons (Fig. 3B), so our data are not inconsistent withother reports of SMN in adult motor neuron nucleoli[31]. A high proportion of gems/CBs in adult tissues

2

n Nuclear Bodies in Fetal Human and Adult Pig Tissues

s/p80 SMN (%) p80 (%) SMN (%)

67 (19.1) 207 (59.1) 76 (21.8)159 (38.6) 201 (48.9) 51 (12.5)217 (93.1) 16 (6.9) 0 (0)

71 (39.8) 74 (41.5) 33 (18.7)86 (50.0) 62 (36.1) 24 (13.9)

212 (100) 0 (0) 0 (0)

19 (11.6) 106 (65.0) 38 (23.4)39 (16.8) 118 (50.8) 75 (32.4)62 (91.2) 6 (8.8) 0 (0)48 (92.3) 4 (7.7) 0 (0)

12 (4.3) 241 (87.0) 24 (8.7)52 (20.8) 120 (48.0) 78 (31.2)

n/a n/a n/a

27 (4.7) 497 (85.8) 55 (9.5)19 (10.2) 100 (53.4) 68 (36.4)

n/a n/a n/a

53 (18.4) 208 (72.2) 27 (9.4)79 (25.0) 149 (47.1) 88 (27.9)

129 (94.2) 8 (5.8) 0 (0)

34 (11.5) 194 (65.7) 67 (22.8)16 (21.1) 32 (42.1) 28 (36.8)

n/a n/a n/a

etermine whether the adult human and pig counts were comparable.Ab204. n/a, not applicable.

ling on (A) human fetal and adult pig skeletal muscle and (B) humanA1 and a TRITC-conjugated goat anti-(mouse Ig). Coilin-p80 (green)Fibrillarin (blue) was identified using a human auto-antiserum andMN colocalized with fibrillarin within the nucleolus (yellow arrows).muscle, a Cajal body containing both SMN and coilin-p80 within theboth SMN and coilin-p80 adjacent to, but not within, the nucleolusdicated. (C) Expanded views of nucleoli in adult pig tissues. (a) Thepical gems but does not fill the nucleolus as in fetal tissue, whileith SMN) is less dispersed. (b) SMN staining within nucleoli occurss and one in the nucleoplasm. The white bars in A and B represent

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were inside nucleoli (Table 4) but the nucleolar SMNwas sometimes more diffuse than is typical of gems(Fig. 3C), although less diffuse than in the fetal nucle-oli. Nucleolar CBs have been reported in cultured cellsbut only as 1–2% of the total, although the proportionwas greatly increased by okadaic acid, which inhibitsprotein dephosphorylation, or by transfection with mu-tants which mimic phosphorylated coilin-p80 [20]. Thissuggests that nucleolar versus nucleoplasmic localiza-tion of proteins can be influenced by their state ofphosphorylation. Whether the localization of SMN isaffected by SMN phosphorylation has not been estab-lished. A nucleolar localization has also been estab-lished for another protein component of isolated gems,gemin4 [39]. This protein interacts with SMN onlyindirectly through gemin3 (dp103) [15, 40, 41] and it is

TAB

Localization of SMN, Coilin-p80, and Fi

Tissue (cell) Fib (%)

1. Liver 13.3 weeks (parenchyma) 87 (66.0)2. Skeletal Muscle 13.3 weeks 69 (72.6)3. Spinal cord 14.8 weeks

(Alpha-motor neurons)(Nonneuronal cells)

98 (85.2)103 (66.4)

4. Spleen 18.2 weeks (parenchyma) 106 (69.3)5. Skin 11.8 weeks (epidermis) 134 (70.2)

Note. The number of nucleoli containing fibrillarin alone (Fib), SMNand Cajal bodies containing SMN (SMN/p80) is indicated. Coilin-p80gems were not seen in nucleoli.

TABLE 4

Localization of SMN and Coilin-p80 in the Nucleoplasmand Nucleolus of Adult Pig Tissues

Tissue (cell)

Nucleoplasm Nucleolus

p80 (%)p80/SMN

(%) p80 (%)p80/SMN

(%)

1. Spinal cord(motor neurons) 0 48 (98.0) 0 1 (2.0)

2. Cerebral cortex(molecular neurons) 1 (1.0) 49 (52.0) 1 (1.0) 43 (46.0)

3. Liver (parenchyma) 2 (3.5) 29 (50.0) 1 (1.0) 26 (44.8)4. Skeletal muscle 4 (8.5) 25 (53.2) 0 18 (38.3)Human skeletal muscle 7 (10.0) 38 (54.3) 1 (1.4) 24 (34.3)

Note. The number of Cajal bodies lacking SMN (p80) and contain-ing both SMN and coilin-p80 (SMN/p80) in the nucleoplasm andnucleolus is indicated. Counts were also performed on adult humanskeletal muscle to determine whether the adult human and pigcounts were comparable. An anti-fibrillarin human autoantibodywith an AMCA-conjugated goat anti-(human Ig) secondary was used
to reveal nucleoli.
not clear whether it has any role in regulating nucleo-lar distribution of SMN.

The observed distribution of SMN (cytoplasmic, nu-cleolar, gems/CBs in nucleoplasm or nucleolus, nucle-oplasmic gems without coilin-p80) may be stages in onepathway, some of which only become highly populatedwhen a bottleneck arises. Gall et al. [21], have sug-gested that gems/CBs may be part of a complex path-way for assembly of the RNA transcription and pro-cessing machinery. SMN is believed to be involved inthe maturation of snRNPs in the cytoplasm and theirtransport into the nucleus [10–13]. Both gems and CBshave been proposed to be involved in the assembly,storage, or transport of components of the RNA splic-ing machinery [13, 23]. It has been suggested that UsnRNPs may undergo an additional maturation stepwithin the nucleolus [20–22]. Bauer and Gall [42] sug-gested that U snRNPs and coilin-p80 are codependenton each other for CB targeting. The nucleolar proteinNopp140 also appears to be essential for both thistargeting process and the structural integrity of CBsand may pass through CBs before accumulating in thenucleolus [43]. Passage through CBs on the way to thenucleolus has also been reported for U snRNPs [21]and small nucleolar RNAs (snoRNAs) [22]. Low levelsof coilin-p80 and U snRNPs have been identified withinHeLa cell nucleoli under normal conditions and uptakeof CBs into the nucleolus may be controlled by thephosphorylation state of coilin-p80 [20]. It is conceiv-able that some components of gems/CBs, includingSMN, may function in ribosomal RNA transcription/processing in the nucleolus, as well as mRNA tran-scription/processing in the nucleoplasm [21, 44].

It would be of interest for the pathogenesis of SMA ifSMN could be shown to be different in motor neuronsin some specific way that is obviously relevant to theirsurvival. We have found, however, that motor neurons

3

llarin in the Nucleolus of Fetal Tissues

ucleoli Cajal bodies in the nucleolus

SMN/Fib (%) p80 (%) SMN/p80 (%)

39 (29.5) 2 (1.5) 4 (3.0)24 (25.3) 0 2 (2.1)

17 (14.8)48 (31.0)

02 (1.3)

02 (1.3)

45 (29.3) 1 (0.7) 1 (0.7)51 (26.7) 4 (2.1) 2 (1.0)

localized with fibrillarin (SMN/Fib), Cajal bodies lacking SMN (p80),as only present in nucleoli in the form of Cajal bodies. Independent

LE

bri

N

cow

differ from other cell types in many ways. They are the

260 YOUNG ET AL.

only cell type in which we found more gems/CBs inadult than in fetal stages (Table 1). Colocalization ofSMN with coilin-p80 was much greater than in othercell types at all stages of development (Table 2; Fig.3B). The relationship of SMN to the nucleolus was alsodifferent in motor neurons (Tables 3 and 4). We notedin an earlier study that over 20% of gems/CBs in adultmotor neurons were larger and adjacent to the nucle-olus [26]. It is not clear how these are related to“perinucleolar caps” which have been reported in neu-ronal and other cell types [32]. These “caps” at thenucleolar periphery contain coilin-p80 which appearsto exchange with CBs in response to transcriptionalactivity, “caps” occurring under low transcription con-ditions, and CBs under high transcription conditions[37]. Apart from the perinucleolar bodies, gems/CBs inthe interior of the nucleolus were hardly ever observedin adult or fetal motor neurons and the dispersed nu-cleolar distribution of SMN was less common in fetalmotor neurons than in other fetal cell types (Table 3;Fig. 3B). It is unclear how these quantitative differ-ences in motor neurons could render them more sus-ceptible to low SMN concentrations. The possibilitythat SMN may have an additional role in motor neu-rons, outside RNA splicing, has been raised [16, 17].However, the observation that most of the missensemutations that cause SMA are found in regions of SMNinvolved in binding Sm core proteins of snRNPs [10] isconsistent with involvement of a snRNP maturationdefect in SMA pathogenesis.

This work was supported by a research grant from the MuscularDystrophy Association (U.S.A.). We thank Dr. Angus Lamond (Uni-versity of Dundee) for p80-coilin antibodies and Dr. Michael Pollard(Scripps Research Institute, La Jolla, CA) for fibrillarin antibodies.

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Received October 19, 2000Revised version received January 19, 2001

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Nuclear Gems and Cajal (Coiled) Bodies in Fetal Tissues: Nucleolar Distribution of the Spinal Muscular Atrophy Protein, SMN