Genetica (2009) 137:135–140 DOI 10.1007/s10709-009-9366-y
Chromosomal features of nucleolar dominance in hybrids between the Neotropical fish Leporinus macrocephalus and Leporinus elongatus (Characiformes, Anostomidae) Diogo Teruo Hashimoto Æ Alejandro Laudicina Æ Jehud Bortolozzi Æ Fausto Foresti Æ Fa´bio Porto-Foresti
Received: 13 October 2008 / Accepted: 24 April 2009 / Published online: 9 May 2009 Ó Springer Science+Business Media B.V. 2009
Abstract In the present study, the chromosomal mechanisms of nucleolar dominance were analyzed in the hybrid lineage ‘‘Piaupara,’’ which resulted from crossing the Leporinus macrocephalus female (Piauc¸u) and L. elongatus male (Piapara) fish. The analyses demonstrated that, in the hybrid, the nucleolar region inherited from L. elongatus presented higher activity, with expression in 100% of the cells, whereas the nucleolar region from L. macrocephalus appeared active at a frequency of 11.6%. The FISH technique with an 18S probe showed that the ribosomal DNA of the nucleolar region was not lost in the hybrid, and the results therefore demonstrated invariable marks in two chromosomes, each originating from one parent. An interesting difference between the nucleolar regions of the parental species was the association of the NOR with heterochromatic blocks (repetitive DNA) in L. elongatus, which could act as a determinative element in the establishment of this process. Keywords Epigenetic phenomenon Interspecific hybrid Fish cytogenetic
D. T. Hashimoto J. Bortolozzi F. Porto-Foresti (&) Departamento de Cieˆncias Biolo´gicas, Faculdade de Cieˆncias, Universidade Estadual Paulista (UNESP), Campus de Bauru, Bauru, SP 17033-360, Brazil e-mail:
[email protected] A. Laudicina Escuela de Ciencia y Tecnologı´a, Universidad Nacional de San Martı´n, Campus Miguelete, 1650, San Martı´n, Buenos Aires, Argentina F. Foresti Departamento de Morfologia, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), Campus de Botucatu, Botucatu, SP, Brazil
Introduction Ribosomal DNA (rDNA) is, in higher eukaryotes, organized into two distinct gene classes composed of tandemly repeated units. The major class (45S rDNA) comprises the 18S, 5.8S, and 28S rRNA genes, and the minor class (5S rDNA) is represented by the 5S rRNA gene family (Long and Dawid 1980). Nucleolus organizer regions (NORs) are genetic loci in eukaryotes at which the genes encoding the precursor of the three largest ribosomal RNAs are clustered in hundreds to thousands of copies, collectively spanning millions of base pairs along the chromosome (Wallace and Birnstiel 1966). Nucleolar dominance is an epigenetic phenomenon common in interspecific hybrids, in which a set ribosomal RNA genes inherited from one parent is transcribed more than the other. This was first described in hybrids between plants of the genus Crepis, by Navashin in 1934, and many studies are currently investigating the mechanisms by which the dominance is established (Pikaard 2000a; McStay 2006; Preuss and Pikaard 2007). As in other epigenetic phenomena, modifications in the chromatin silence the rRNA genes for the maintenance of nucleolar dominance. However, the mechanisms that determine which parental rRNA set is dominant or silenced remain obscure (Pikaard 2000a). In general, nucleolar dominance seems to be a consequence of evolutionary divergence of the rRNA genes and/or their transcription regulation mechanisms (Reeder 1985). A predominant theoretical mechanism in nucleolar dominance is found in the ‘‘enhancer imbalance’’ model, first studied in hybrids between the frogs Xenopus laevis and Xenopus borealis (Reeder and Roan 1984; Reeder 1985; Caudy and Pikaard 2002). The rRNA genes of these species have different types and numbers of repetitive
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elements (enhancers) in their intergenic spacers. This suggested a model whereby the more numerous enhancers of X. laevis might titrate an essential transcription factor and sequester it, thus denying the X. borealis rRNA genes access to the factor and causing inactivation. The nucleolar dominance phenomenon has been well characterized in numerous interspecific hybrids of plants (Martini et al. 1982; Chen and Pikaard 1997; Chen et al. 1998; Pikaard 2000b) and, in the same way, important studies have also been performed in some animals, such as in hybrids of Drosophila (Durica and Krider 1978; Oliveira et al. 2006; Commar et al. 2007) and the frog Xenopus (Honjo and Reeder 1973; Reeder and Roan 1984; Caudy and Pikaard 2002), while there are few studies, restricted to only descriptive data, in fish (Ueda and Kobayashi 1990; Gold et al. 1991). Recently, this phenomenon was described in fish by Porto-Foresti et al. (2008) with interspecific hybrids of the Neotropical family Anostomidae, where the main purpose of that work was to search for diagnostic cytogenetic markers for the differentiation between the parental species and hybrids. In these analyses, in hybrid metaphase chromosomes, the NOR-banding by silver nitrate demonstrated variations in the expression of these regions, with more activity in the NOR inherited from one of the parental species, characterizing a previous description of the nucleolar dominance process. In this way, to gain a better understanding about this nucleolar effect, the aim of this study was to cytogenetically analyze the nucleolar dominance phenomenon in artificial interspecific hybrids that resulted from crossing Leporinus macrocephalus females and L. elongatus males.
Materials and methods As the parental lines, 19 specimens of Piauc¸u (L. macrocephalus; 14 males and 5 females) and 20 individuals of Piapara (Leporinus elongatus; 12 males and 8 females) were cytogenetically analyzed. Crosses performed using females of Piauc¸u and males of Piapara resulted in the production of the interspecific hybrid ‘‘Piaupara.’’ The cytogenetic analysis of hybrids comprised 21 individuals of ‘‘Piaupara’’ (8 males and 13 females). All of the analyzed samples were obtained from the stock belonging to the Kabeya Aquaculture (Pena´polis, SP, Brazil), where the hybrids were artificially produced. The parental stocks of L. elongatus and L. macrocephalus, cultivated in this fish culture facility, were captured in the South Pantanal MatoGrossense region. The fish were identified and deposited in the fish collection of the Laboratory of Fish Genetics, UNESP, Bauru (SP), Brazil.
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Before sacrifice, the animals were inoculated with a yeast cell suspension to increase the number of metaphase cells (Oliveira et al. 1988). Chromosome preparations were obtained from gill and kidney tissues using the technique described by Foresti et al. (1981). Silver staining of the nucleolus organizer regions followed the technique of Howell and Black (1980) and C-banding was performed according to Sumner (1972). For fluorescent in situ hybridization (FISH), the method used by Porto-Foresti et al. (2002) was applied with the sex-specific probe (LeSpeI element) isolated from Leporinus elongatus by enzymatic restriction digest (Parise-Maltempi et al. 2007), prepared by Dra. Patricia Pasquali Parise Maltempi, and the 18S rDNA probe extracted from Oreochromis niloticus, prepared by Dr. Cla´udio Oliveira. The probes were labeled through the incorporation of biotin-dATP by the nick translation method TM (BioNick Labeling System; Gibco.BRL). The chromosomes were counterstained with propidium iodide or DAPI. Chromosome morphology was determined based on arm ratio as proposed by Levan et al. (1964) and the chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a).
Results In the parental and hybrid stains, the cytogenetic analysis showed identity in the diploid number of 54 chromosomes and the chromosomal morphology of the meta- and submetacentric types, beyond the occurrence of the ZZ/ZW sexual chromosomal heteromorphism. NOR identification through silver staining in L. macrocephalus and L. elongatus showed only one pair of chromosomes containing ribosomal cistrons. The NOR was situated at the terminal position of a submetacentric chromosomal pair in both species (Fig. 1), although the chromosomal pair in each parental species displayed different sizes and morphologies. The C-banding pattern in chromosomal preparations of L. elongatus showed the presence of heterochromatic blocks associated with the secondary constriction region (NOR), while in L. macrocephalus this situation was not observed (Fig. 2). Therefore, FISH with the LeSpeI repetitive DNA probe was used in the L. elongatus samples, which revealed hybridization signals in these heterochromatic blocks with NOR association, demonstrating homology with the LeSpeI sequence (Fig. 3). Moreover, several different homologous regions were also dispersed in the NOR chromosomes and the W chromosome. The females showed hybridization on the W chromosome in some loci beyond the regions of the NOR-bearing chromosomal pair, but with different signals between the homologous pair; on the other hand, the males also demonstrated hybridization in the NOR chromosomes that were similar in both homologous chromosomes.
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Fig. 1 Metaphase chromosomes from Leporinus macrocephalus (a) and L. elongatus (b) stained with silver. Arrows indicate the NOR-bearing chromosomal pairs
Fig. 2 NOR-bearing chromosomal pairs from Leporinus macrocephalus (1) and L. elongatus (2). a Schematic representation; b silver nitrate stain; c C-banding showing heterochromatin associated to the secondary constriction only in L. elongatus; d FISH demonstrating homology between the LeSpeI repetitive DNA probe and heterochromatin
Fig. 3 Metaphase chromosomes from a Leporinus elongatus male (a) and female (b), submitted to the FISH technique with the LeSpeI probe. Stars show hybridization in the W chromosome and arrows indicate the NORbearing chromosomal pairs marked by the probe, with heterochromatin associated to the secondary constriction (NOR) also showing hybridization
In hybrid ‘‘Piaupara’’ metaphase chromosomes, the nucleolar regions inherited from each parent revealed differences in gene activity by the silver nitrate technique. In 1,710 analyzed cells, 88.4% (1,512 metaphases) showed active NORs in only one chromosome, identified as originating from L. elongatus, while 11.6% (198 metaphases) displayed NOR activity in two chromosomes that were
nonhomologous in relation to morphology, one inherited from L. elongatus and the other from L. macrocephalus (Fig. 4). In the hybrids, no difference in the NOR activity between female and male individuals was observed. Using the FISH method with the 18S rDNA probe, analyses on chromosomal preparations of the hybrid ‘‘Piaupara’’ showed signals at the terminal regions on the
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Fig. 4 Metaphase chromosomes from the hybrid ‘‘Piaupara’’ stained with silver nitrate. In a two chromosomes are stained (one from each parent), and in b only the chromosome from Leporinus elongatus is stained. Dark arrows indicate the NOR chromosomes from L. elongatus and pale arrows from L. macrocephalus
Fig. 5 Metaphase chromosomes from the hybrid ‘‘Piaupara’’ submitted to the FISH technique with the 18S rDNA probe. Regions of 18S rDNA are indicated by white and yellow arrows in the chromosomes from Leporinus elongatus and from L. macrocephalus, respectively (Color figure online)
long arms of two submetacentric chromosomes that displayed differences in relation to morphology, indicating that they are nonhomologous and corresponded to the NOR-bearing chromosomes of each parental species (Fig. 5).
Discussion NORs represent chromosome regions involved in ribosomal gene transcription and can be visualized after silver nitrate staining when the genes are active in interphase in preparation for mitosis (Goodpasture and Bloom 1975; Miller et al. 1976; Howell and Black 1980). The silver nitrate staining detects only the active NORs, since it does not stain the rDNA but rather a set of acidic proteins associated with the process of ribosome production (Howell 1977; Jordan 1987), and is therefore an excellent cytogenetic tool for ribosomal gene activity analysis. Thus, the variations in hybrid ‘‘Piaupara’’ metaphase chromosomes observed after silver staining reflect activity differences of the NORs inherited from each parent, since a
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higher percentage of cells had an active nucleolar region only on the chromosome originating from L. elongatus. Such variations are the result of the epigenetic phenomenon of nucleolar dominance and can also be described by partial dominance, as reported in other cases (Honjo and Reeder 1973; Gold et al. 1991; Oliveira et al. 2006). To verify the ribosomal DNA integrity in the nucleolar formation and determine whether all hybrid cells contained two potentially functional NOR sites, in situ hybridization with the 18S probe was applied to visualize the NOR genomic segment independent of its activity. The result confirmed the nucleolar dominance effect, since signals were observed invariably in two chromosomes by FISH, each proceeding from one parent, even in cells where only one NOR was stained by silver. Consequently, considering that all hybrid cells displayed the ribosomal genes for nucleolar formation from both parent, the nucleolar region inherited from L. elongatus showed dominance and presented higher activity, with gene expression in 100% of the cells, while the nucleolar region from L. macrocephalus appeared to be active at a frequency of 11.6%. In this case, while the NOR of one parent is active, the other is silenced.
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In relation to the mechanisms by which dominance is defined, an interesting observation is that heterochromatic blocks are associated with the ribosomal DNA in L. elongatus. This situation seems to be consistent with the ‘‘enhancer imbalance’’ model, first described in hybrids of the frog Xenopus (Reeder and Roan 1984; Caudy and Pikaard 2002) and found in similar situations in other organisms (Simeone et al. 1985; Reeder 1985; GoodrichYoung and Krider 1989). Due to the fact that heterochromatin generally is composed of highly repetitive DNA (Haaf et al. 1993; Reed and Phillips 1995; Mestriner et al. 2000), the presence of repetitive DNA in the ribosomal intergenic spacers of L. elongatus could serve as enhancers that influence the promoters of the ribosomal genes (Reeder and Roan 1984; Simeone et al. 1985; Caudy and Pikaard 2002) and restrict the access of the L. macrocephalus rRNA genes to transcription factors. Recently, in studies performed by Parise-Maltempi et al. (2007), a new highly repetitive DNA family (LeSpeI element) in the sexual chromosomes of L. elongatus was identified and isolated. When this repetitive DNA was used as a probe in L. elongatus metaphase chromosomes, homology between this sequence and the heterochromatic regions associated to the NOR was seen, beyond the regions in the sexual chromosomes described previously by Parise-Maltempi et al. (2007). From this similarity, studies utilizing molecular techniques should be implemented to potentially identify the relationship and structural organization of these sequences associated with the nucleolar regions in L. elongatus, since these sequences were not observed in L. macrocephalus (Hashimoto et al. 2009). Another interesting fact to be investigated is the presence of rDNA in the W sexual chromosome of L. elongatus, described by Molina and Galetti (2006) in chromosomal preparations previously treated with 5-BrdU. In Drosophila species from the Repleta group mulleri complex, the NOR is found in the microchromosome as well as in the X sexual chromosome, which allows different nucleolar dominance situations between female and male hybrids, probably influenced by the presence or absence of the X chromosome (Durica and Krider 1978; Commar et al. 2007). However, in our data, differences were not observed between ‘‘Piaupara’’ males and females, where the W chromosome in the hybrid females was inherited from L. macrocephalus. Thus, to better elucidate these situations, it would be necessary to verify that L. macrocephalus possesses an NOR in the W chromosome as well as to perform analyses in the reciprocal hybrid ‘‘Piapac¸u.’’ Ribosomal RNA gene transcription is tightly regulated in order to provide the proper amount of rRNA for ribosome assembly (Moss and Stefanovsky 2002; Russell and Zomerdijk 2005). Consequently, nucleolar dominance can
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also be a regulatory process that controls the effective dosage of rRNA genes in pure species (non-hybrid) (Pikaard 2000a). The nucleolar regions can be considered very active loci, accounting for about 40–80% of nuclear transcription in growing cells (Jacob 1995). Thus, a dosage compensation system could explain the partial nucleolar dominance described in the hybrid ‘‘Piaupara.’’ Due to the low level of gene activity of the NOR inherited from L. macrocephalus, a secondary mechanism for dosage compensation could be suggested to supply the cellular need for protein synthesis. A recurrent feature of nucleolar dominance in both plants and animals is that NORs of the same species are always silenced, independent of maternal or paternal effects. This finding suggests that fundamental differences dictate the dominant and repressed sets of rRNA genes in a hybrid, possibly because of species-specific differences in the rRNA genes and RNA polymerase I transcription systems of the progenitors (Reeder 1985; Pikaard 2000a). The choice mechanisms by which whole NORs or rRNA gene subsets are selected for inactivation still remains an intriguing question (Preuss and Pikaard 2007). Recent molecular studies demonstrated that gene silencing in nucleolar dominance frequently resulted from concerted changes of chromatin remodeling, DNA methylation, and specific histone modification (Chen and Pikaard 1997; Chen et al. 1998; Grummt and Pikaard 2003). Nucleolar dominance in Neotropical fish hybrids supplies an excellent model system for analyses of the mechanisms responsible for the establishment and enforcement of this epigenetic effect. Our results serve as framework for the evaluation of epigenetic mechanisms in other hybrids involving different fish species, as well as providing information related to the intense NOR polymorphism in Neotropical fish, described in several cytogenetic works (Foresti et al. 1981; Almeida-Toledo et al. 2000). Acknowledgments This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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