Heredity 75 (1995)10-16 Received24 August 1994

Cytogenetic peculiarities in the Algerian : silver stains not only NORs but also heterochromatic blocks

ANTONIO SANCHEZ, RAFAEL JIMENEZ*, MIGUEL BURGOS, SAIDA STITOU, FEDERICO ZURITA & RAFAEL D1AZ DE LA GUARDIA Departamento de Genética, Facu/tad de Ciencias, Universidad de Granada, 18071 Granada, Spain

Hedgehogsbelong to one of the several mammalian taxa in which karyotype differences are based on variations in heterochromatin content. Furthermore, the number and location of nucleolar organizer regions (NORs) can also vary widely. In the present study these cytogenetic features were investigated in the Algerian hedgehog, (Aethechinus) algirus. The heterochromatin and NOR distribution patterns in the karyotype of this species are new among , whereas the euchromatic regions, including their G-band pattern, are similar to those reported by others. In addition, silver staining revealed a cytogenetic feature exclusive to the heterochromatic blocks of E. algirus: their silver staining with standard cytogenetic procedures. Because no similar phenomenon has been described previously in a mammalian species, several hypotheses about the significance and specificity of silver staining to NOR sites are discussed. Finally, the existence of different types of heterochromatin in the species analysed here, lead us to propose that what hedgehogs have inherited from their common ancestor is a mechanism which permits the accumulation of hetero- chromatin on specific chromosomes, rather than the heterochromatin itself.

Keywords: Erinaceusalgirus, heterochromatinvariation, karyotype evolution, NOR variation, silver staining.

Introduction blocks. In this species, however, their relationship with NORs could not be established, as Ag-staining tech- Hedgehogshave been the subject of several interesting niques were not available when this work was done. cytogenetic studies, mainly devoted to geographical In this paper, we report karyotypic analyses in this variations in the content of autosomal constitutive species with newer, more powerful cytogenetic tech- heterochromatin and their connection with the number niques such as Ag-staining of NORs, C-banding, and localization of nucleolar organizer regions (NOR) fluorescent staining and in situ hybridization, which (for a review, see Zima & Kral, 1984). Several karyo- make it possible to characterize more precisely both typic variants have been described in European species constitutive heterochromatin and NORs, and to estab- of the genus Erinaceus (E. europaeus and E. lish possible relationships between them. We discuss romanicus), which differ in the presence and location several interesting cytogenetic peculiarities of the of large autosomal blocks of constitutive hetero- Algerian hedgehogs such as silver stainability of the chromatin (Gropp, 1969; Mandahl, 1978; Sanchez et heterochromatic blocks and the existence of inter- and al., 1994). The distribution of NORs in these species intraindividual variability for the number and size of was studied by Mandahl (1979), who showed the NORs. existence of intraindividual, interindividual and inter- populational variations for the distribution of these chromosome regions in karyotypes. Materialsand methods The karyotype of the Algerian hedgehog, Erinaceus Atotal of five individuals (two males and three (Aethechinus) algirus, has been analysed by Gropp & females) of the species Erinaceus algirus were trapped Natarajan (1972), who presented evidence for the live in northern Morocco. Chromosome preparations presence in this species of large heterochromatic were made following our standard air-drying proce- dure (Burgos et al., 1986), either directly from bone Correspondence. marrow cells or after culture of spleen lymphocytes.

10 1995The Genetical Society of Great Britain. ing totheprotocolrecommendedbysupplier with digoxigeninandhybridizationweredoneaccord- gene (seeWahietal.,1983).Nicktranslationlabelling somes weusedaSyrianhamsterrDNAprobeconsist- formic acid(Sanchezetal.,1989a). nitrate indeionizedwateradjustedtopH3.1with placed inamoistchamberat60°Candexposedfor Ag-staining, standardchromosomepreparationswere were revealedbythemethodofSumner(1972).For combined method(Burgosetal., Chromosomes wereG-bandedaccordingtoour ment, morphologicaldifferenceswithrespecttoother ing toMandahi(1978)facilitatecomparisons(Fig. (e.g. E.europaeus),karyotypeswerearrangedaccord- somes (2n= Because E.algirushasthesamenumberofchromo- Karyotype Results (Boehringer Mannheim). gene, theinternalspacer,andalmostentire28S ing ofsequencescorrespondingtothe3'end18S 3—5 mmtoa100percent(w/v)solutionofsilver strictions werelocatedcentrallyonthelongarmofpair telocentric) inE.algirus.Inaddition,secondarycon- in pair14(alargesubtelocentric)and21small karyotypic formsdescribedinhedgehogswerefound metacentric Y.Onthebasisofthiskaryotypearrange- sented byameta-submetacentricXanddot-like centric chromosomes.Sexchromosomeswererepre- small telocentric,andpairs2223weremeta- medium-sized submetacentrics;pair21wasavery series ofmetacentricelementsdecreasingsize;pairs G-banding arrangement. although theseauthorspresentedadifferentkaryotype reported byGropp&Natarajan(1972)forthisspecies, metaphase plates. sequently, theywerenotclearlydistinguishableinall secondary constrictionswerevariableinsize,andcon- 13, andnearthecentromereinpairs2122.These 12—14 werelargesubtelocentrics;pairs15—20 1). Autosomalchromosomepairs1—11representeda denoting sensitivity totrypsininthese regions. segments werefoundonchromosome pairs13and14, were found(Fig.2).Interestingly, twolargeG-negative chromosome pairsinwhich morphologicaldifferences and Europeanhedgehogs, except,ofcourse,inthose G-banding For insituhybridizationtofixedmetaphasechromo- This descriptioncoincidesbasicallywiththat The GeneticalSociety ofGreatBritain,Heredity,75, patterns werenearlyidentical inAlgerian 48) as therestofspeciesinthisgenus 1986), and C-bands 10—16. chromosome pair13areclearlyvisible. algirus (2n= Fig. 1ConventionalGiemsa-stainedkaryotypeinErinaceus chromosome. and 14,theentirechromosomeofpairs2123,theY the G-negativeconditionoftwolargesegmentsonpairs13 Fig. 2G-bandingpatternintheAlgerianhedgehog.Note In were alsoentirelyG-negative. Similarly, pairs21and23aswelltheYchromosome showed thestrongestfluorescence, indicatingthatthey chromatic. Thesecondary constrictionsonpair13 positive, despitethefact thattheyarenothetero- chromosome pairs21and 23werealsochromomycin they areC—Grich(Fig. 3b).The0-negativesmall segments arechromomycinpositive,whichimpliesthat Chromomycin F/uorochrome rest ofthechromosomesareC-negative. regions describedaboveforthesechromosomes.The large C-positiveblocks,locateddistallyonpairs13and C-banding are particularly richinC—Gbasepairs. 14 (Fig.3a),andcoincidingwiththelargeG-negative I .1w r',• this species,heterochromatinisclusteredintwo r±; — n is rae ee CYTOGENETICS OFTHEALGERIANHEDGEHOG11 I St Os (fri (MS-s Mae 1 48). a a (a (MaeS staining In thiscell,onlysecondaryconstrictionson A3 showedthattheseheterochromatic a. S. - a. 0I CM

m N (31 — rot N. o, — — a. ro — S O4 a -J .1 C— e —as -s-.1 •t - a m5-s — - -_ -n 3 S.•ø o Wa O ro xa roe — — -— S —< S 0 S _S -(Xe. I. 12 A.SANCHEZETAL.

Fig. 3 Constitutive heterochromatm of *1 . Erinaceus alginis. (a) C-banding show- ing two large C-positive blocks on pairs 1• 13 and 14. (b) Staining with chromo- mycin A3 showing that the hetero- V chromatic blocks on pairs 13 and 14, and the small chromosome pairs 21 r and 23, are rich in C—G base pairs; the 4. ¼ secondary constrictions on pair 13 t showed the brightest fluorescence, suggesting that they are especially rich in C—G base pairs.

Silver staining a As demonstrated by the Ag-staining technique, NORs were observed in chromosome pairs 13, 21 and 23, and in two pairs of submetacentric chromosomes _ which, on the basis of their size, probably correspond etc to pairs 15 and 16 (Fig. 4a). In pairs 13, 21 and 23, -a F') uJ NORs were located interstitially at the secondary constriction, whereas in pairs 15 and 16 they were S located distally at the end of the long arm. Of these 4 NORs, only those located on pair 13 were invariably stained with silver, although inter- and intraindividual size variability was observed. However, staining of the .4 412! rest of the NORs was variable depending on both the V individual and the cell. This variability arose mainly *N\ S

from the NOR-bearing submetacentric chromosomes as WI

there were clear interindividual differences with 4t4 respect to the most frequently found number of these chromosomes that were positive for Ag-staining. In addition to showing the position of NORs (see %%* j'

below), Ag-staining provided another unexpected S finding. In most cells observed in chromosome

preparations treated for NOR detection, the hetero- t. chromatic blocks on pairs 13 and 14 appeared clearly stained by silver, a finding which has not been reported Fig. 4 Silver staining of Erinaceus algirus chromosomes. (a) Location of NORs on chromosome pairs 13, 21 and 23 before in mammalian chromosomes (Fig. 4b). (interstitially), and on pairs 15 and 16 (distally). To compare chromosomal morphology and the position of the secondary Insituhybridization constrictions, conventional Giemsa-stained chromosomes are shown to the right of silver-stained chromosomes. (b) Thistechnique corroborated the position of NORs Metaphase plate showing Ag-staining of the entire hetero- shown by silver staining and demonstrated that varia- chromatic blocks on chromosome pairs 13 and 14 (arrows) bility in Ag-staining may result from the existence of and several stained NORs (arrowheads). polymorphism in the number of ribosomal cistrons in different NOR sites. Thus, heteromorphism for the hybridization signals was frequent, mainly in chromo- addition, in situ hybridization showed that the hetero- some pair 13. Furthermore, one of the individuals chromatic blocks lack ribosomal cistrons in their entire analysed showed only three, instead of the expected length, except in the secondary constriction on pair 13, four submetacentric chromosomes with evident where a NOR is located. Hence, silver stainability can- hybridization signals, suggesting that one of them had not be attributed to a putative NOR condition of these lost most, if not all, of its ribosomal cistrons (Fig. 5). In heterochromatic blocks.

The Genetical Society of Great Britain, Heredity, 75, 10—16. CYTOGENETICS OF THE ALGERIAN HEDGEHOG 13

Table 1 summarizes the results obtained with all Erinaceus. This feature is shared by several other these different teclmiques. mammalian taxa (Fredga & Mandahl, 1973; Dev eta!., 1975; Ando et a!., 1980; Mandahi & Fredga, 1980; Discussion Baverstock et a!., 1982; Yosida & Kodama, 1983; Freitas et a!., 1984; Barros & Patton, 1985). A variety Ourresults consistently support the view (Mandahl, of karyotypic forms has been described for different 1978) that variations in the content and location of species and geographical races of this genus, in which blocks of constitutive heterochromatin are a funda- clear conservation of the euchromatic regions, as mental feature of the karyotypic evolution in the genus shown by the constancy in both chromosomal number and morphology and in the G-band patterns, contrasts strongly with marked variability in the number and location of their C-positive, heterochromatic blocks. On the basis of this variability, five karyotypic forms were established in hedgehogs from Northern Europe -S (Mandahl, 1978), an additional form was established in Southern Europe (Sanchez et a!., 1994), and a new variation was found in the Chinese hedgehog . (Yongshan & Ficin, 1987). The karyotype described here, which basically coincides with that previously reported by Gropp & Natarajan (1972) for E. algirus, 'St represents a further form. In addition to the variability in size and location of the heterochromatic blocks in hedgehogs, there seem to be some qualitative differences between them, at least in E. algirus heterochromatin. This idea is 'I supported by the positive silver staining we have shown and the particular requirements for C-banding (Gropp & Natarajan, 1972). This qualitative heterogeneity may Fig. 5 In situ hybridization with an rDNA probe on imply heterogeneity in the origin of the hetero- chromosomes of Erinaceus algirus. Intraindividual heterom- chromatic blocks, so that the situation in hedgehogs orphism between homologous chromosomes (inset, pair 13) may be similar to that described in several species of and interindividual polymorphism for the number of NOR the genus Microtus: large heterochromatic blocks are sites were frequent; in this cell only three (instead of the expected four) submetacentric chromosomes showed evi- present on their sex chromosomes, and different types of heterochromatin have been found with molecular dent hybridization signals (arrows). (Modi, 1993a,b) and cytogenetic analyses (Sperling et a!., 1985; Burgos et a!., 1988, 1990). Hence, as suggested for Microtus (Burgos et al., 1992) it is Table 1Resultsof the application of different banding possible that some chromosomes in Erinaceus techniques to significant chromosomes or chromosome acquired from a common ancestor the ability to regions of Erinaceus algirus accumulate constitutive heterochromatin, rather than Chr G C CMAAGNORLocationISH the heterochromatin itself. The nature of the heterochromatin in the C-positive 13h — + + + j + blocks of E. algirus deserves further discussion, as it 14h — + + + — — shows a feature unique among , i.e. positive 15 — t + silver staining. This raises two points that have been 16 — t + widely debated for the last several years: the signifi- 21 — — + + 1 + cance of silver staining of mitotic chromosomes, and its 23 — — + + i + specificity for NOR sites. The view that silver specifi- cally stains transcriptionally active NORs, based on 1 3h: Heterochromatic block of the chromosome 13. the clear correlations between the two events (tran- 14h: Heterochromatic block of the chromosome 14. scription activity and silver staining) (see Hubbell, AG: Ag-staining of constitutive heterochromatin. 1985), has repeatedly been contradicted by a number NOR: NOR carrier as detected by Ag-staining. Location: Location of NORs; i, interstitial; t, terminal. of authors, who have suggested that silver stainability ISH: in situ hybridization with rDNA probe. of NORs is exclusively dependent on the decondensed *: Secondary constriction showed the strongest fluorescence. state of NOR chromatin (see Medina et a!., 1983;

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Sánchez-Pina et al., 1984). More recently, Jiménez et NORs are located interstitially, the rest of the species al. (1988) demonstrated that decondensation of NOR showing distally located NORs, generally associated chromatin is necessary but not sufficient for silver with the heterochromatic blocks (Mandahl, 1979). To staining to occur, and suggested that previous tran- explain the marked variability in NOR location in the scriptional activity may also be needed. However, the five karyotypic forms of hedgehogs from Northern fact that silver also stains chromosomal regions other Europe, Mandahl proposed a mechanism of transfer of than NORs, as demonstrated in amphibians (Varley & NORs based on nonhomologous exchange between Morgan, 1978), complicates the problem even further. distal regions of the associated heterochromatin. The fact that this phenomenon has been observed Although a similar mechanism may have operated in E. exclusively in lampbrush chromosomes of amphibian algirus to permit interchromosomal transfer of NORs, oocytes seems to preserve the traditionally accepted additional rearrangements, probably pericentric and/ hypothesis that silver specifically stains NOR sites in or paracentric inversions, would be necessay to explain mammalian mitotic chromosomes (see King, 1980), the intrachromosomal translocation which their inter- but our finding that not only NORs but also hetero- stitial position implies. chromatic blocks in E. algirus are silver stainable Finally, polymorphism for the size of Ag-NORs is a refutes this assumption. common feature in most mammalian species studied so Silver stainability of any biological material is far. Assuming that silver staining reflects previous tran- strongly dependent on the conditions of fixation, Ag- scriptional activity of NORs, Sanchez et a!. (1989b) NOR staining being specific for conventional cytogen- proposed the existence of interference between homo- etic alcohol—acetic acid fixed chromosomes (see logous NORs in the rodent Eliomys quercinus, which Hubbell, 1985). However, silver staining of the hetero- may be responsible for differences in the size and chromatic blocks in E. algirus cannot be attributed to intensity of the Ag-bands. However, in situ hybridiza- this effect, as the conventional methods we used for tion provides additional information, demonstrating both fixation (methanol—acetic acid) and staining led to that these differences may also result from polymorph- no significant modifications. This situation is clearly ism in the number of ribosomal DNA cistrons. Regard- different from that reported by Mandahl & Fredga less of the method used (Ag-staining or in situ (1980) for mustelids, in which silver staining of hetero- hybridization), this type of polymorphism is common chromatin was observed only after the chromosome in , and has been described in a variety of taxa, preparations had been overexposed to the silver nitrate including Drosophila (Ritosa & Spiegelman, 1965), solutions. amphibians (Schmid, 1982), turtles (Bickham & Several investigations have led to the biochemical Rogers, 1985), fishes (Galetti et a!., 1985) and identification of the proteins involved in cytogenetic mammals such as marsupials and humans (Goodpas- silver staining. Hubbell et al. (1979) isolated three ture & Bloom, 1975; Varley, 1977; Merry et al., 1983). different nucleolar proteins, designated C23, B23 and In general, the authors agree that the occurrence of Ag-NOR, although it is not clear which of them, if any, deletions or duplications of the NOR chromatin is the is responsible for specific Ag-staining of mitotic NORs simplest explanation for this polymorphism. Although (Lischwe et a!., 1979; Williams et a!., 1982; Hubbell, these rearrangements probably take place by asymmet- 1985). In any case, because of their nucleolar origin, rical recombination during meiosis, thus giving rise to probably none of these proteins is responsible for the interindividual variability, recombination between heterochromatin silver staining observed in E. algirus homologous NORs and/or sister chromatid exchanges mitotic chromosomes. Since it is clear that silver stains in somatic cells must also occur to give rise to the inter- some kinds of chromosomal proteins —probablythose cellular variability observed in most individuals of an acidic nature (Schwarzacher et al., 1978; Buys & analysed. Associations between homologous and non- Osinga, 1980) —asimilar protein may be responsible homologous NORs which persist until mitotic for the silver staining of heterochromatin we observed, metaphase in European hedgehogs (Mandahl, 1978) and may represent a unique characteristic with respect and in other mammals such as the Spanish mole ( to the other types of heterochromatin studied to date. occidentahs; our unpublished data) are consistent with Further biochemical research will be necessary to test this idea. this hypothesis, and any other considerations on the composition and/or structure of this heterochromatin would be speculative for the moment. Acknowledgements With regard to the position of NOR sites in the Wewould like to thank Dr Thelma Slezyger from the karyotype, E. algirus also shows a feature unique Universidad Simon Bolivar (Venezuela) for providing among hedgehogs: this is the only species in which us with the rDNA probes used in this study, and Ms

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Karen Shashok for revising the English style of the G000PASTURE, C. AND BLOOM, S. E. 1975. Visualization of manuscript. This work was supported by the Spanish nucleolar organizer regions in mammalian chromosomes DGICYT through Project No. PB92-095 1. using silver staining. Chromosoma, 53, 37—50. GROP?, A. 1969. Cytologic mechanism of karyotype evolution in insectivores. In: Benirschke, K. (ed.) Comparative Mammalian Cytogenetics, pp. 247—266. Springer, New References York. GROPP, A. AND NATARAJAN, A. 1972. Karyotype and hetero- ANDO,K., TAGAWA, T. AND UCHIDA, T. A. 1980. C-banding pattern chromatin pattern of the Algerian hedeghog. Cytogenetics, on the chromosomes of the Japanese house , 11,259—269. murinus riukiuanus, and its implication. Experientia, 36, HUBBELL, a it. 1985. Silver staining as an indicator of active 1040—1041. ribosomal genes. Stain Technol., 60, 285—294. BARROS, M. A. AND PATrON, J. L. 1985. Genome evolution in HUBBELL, H. R., ROTHBLUM, L. I. AND HSU, T. C. 1979. 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