Karyotyping and single-gene detection using fl uorescence in situ hybridization on chromosomes of Hydra magnipapillata ( Cnidaria : Hydrozoa )

The fresh water polyp Hydra L., 1758 (Cnidaria, Hydrozoa) plays a key role as a model organism in modern evolutionary and developmental biology. A complete genome sequence has been published recently for Hydra magnipapillata Ito, 1947 and molecular data are rapidly accumulating in the literature, but little information is available on its chromosomes. In this study, an effi cient fl uorescence in situ hybridization (FISH) method is described for H. magnipapillata which not only allows identifi cation of the chromosomes but also visualization of the location of individual genetic loci. Together with cDNA and genomic sequencing this may provide the foundation for increasingly precise genetic and physical mapping in this basal metazoan model organism.


INTRODUCTION
Freshwater hydras (Cnidaria, Hydrozoa, Hydra L., 1758) have long been of general interest since they display fundamental principles that underlie development, differentiation, regeneration and symbiosis (e.g.Bosch et al., 2010;Augustin et al., 2010;Bosch 2007Bosch , 2008; Khalturin et al., 2009).The sequencing of the Hydra magnipapillata Ito, 1947 genome recently has shed light on the evolution and development of complexity of multicellular animals (Chapman et al., 2010) and revealed that these simple multicellular organisms have developed many of the molecular switches that are required for the differentiation of higher organisms.Despite its alleged simplicity, H. magnipapillata has a large, complex genome of 1.0 to 1.5 billion base pairs (Zacharias et al., 2004;Chapman et al., 2010) and around 20,000 protein-encoding genes.The number of genes in H. magnipapillata is considerably higher than that in Drosophila and slightly lower than the number of genes in highly developed organisms such as human, mouse, and pufferfi sh (Tetraodon nigroviridis Marion de Proce, 1822).Similarities between H. magnipapillata and other metazoan species extend beyond gene number and gene product sequence, to include intron-exon structure, higher order chromatin arrangements typical of mammalian cell nuclei (Alexandrova et al., 2003), and conserved genome structure (synteny / gene linkage) (Chapman et al., 2010).However, while molecular data on Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics Hydra species are rapidly accumulating in the literature (e.g.Bosch et al., 2009;Khalturin et al., 2009;Chandramore et al., 2010;Gee et al., 2010;Hartl et al., 2010;Münder et al., 2010), little information is available on their chromosomes.This is to be regretted because considerable information can be obtained by examining karyotypes: for example, sex determination depends in many cases on the presence or absence of sex chromosomes that may be morphologically differentiated.Comparing chromosomes in different species may also shed light on the systematics and evolution of these organisms and provide an even deeper understanding not only of Hydra´s evolutionary history, but also of the structural changes that have shaped genome evolution in this group of basal metazoans.Previous studies have shown that the number of chromosomes is identical in all Hydra species examined (2n = 30).The size of the chromosomes is strictly correlated with the size of the genome, with Hydra viridissima Pallas, 1766 having conspicuously small chromosomes (Zacharias et al., 2004).One of the conserved chromosome features is telomere molecular organization: in basal metazoans including Hydra vulgaris as well as in vertebrates the telomeres consist of a highly conserved telomeric repeat motif (TTAGGG)n (Traut et al., 2007).Whether maintenance of Hydra´s telomeres by telomerase activity is responsible for Hydra´s remarkable immortality (Martinez, 2002) and stem cells which continuously proliferate and thereby generate eternal lineages (Wittlieb et al., 2006;Bosch, 2009) is not known.Studies on longevity and senescence in the jellyfi sh Cassiopea spp.have uncovered telomerase activity in somatic tissues of both the polyp and medusa stages (Ojimi et al., 2009).In several species of coral telomere lengths were greater than 19 kb (Zielke, Bodnar, 2010).Karyotypes in the genus Hydra are poorly understood.Only nine of about 30 Hydra species have been karyologically studied so far.In the majority of cases, karyological data are restricted to chromosome numbers.Previously, methods of classical cytogenetics and differential staining techniques did not reveal clear differences in banding patterns of Hydra chromosomes (Xinbai et al., 1987;Ovanesyan, Kuznetsova, 1995;Anokhin, Kuznetsova, 1999;Anokhin, 2002Anokhin, , 2004;;Anokhin, Nokkala, 2004;Zacharias et al., 2004).The aim of this study was, therefore, to develop fl uorescence in situ hybridization (FISH) to characterize chromosomes in Hydra magnipapillata.In this paper we demonstrate that the "vertebrate" telomere motif (TTAGGG)n was conserved at the end of each H. magnipapillata chromosome.Localization of a number of candidate genes including 18S rDNA, 28S rDNA, Tol2, DMRT and ks1 on distinct chromosome pairs was studied and not only demonstrated the utility of FISH for identifying chromosomes in this species but also provided fi rst evidence that individual genetic loci can be visualized in Hydra magnipapillata.In future this may aid in assembling physical and genetic maps in this widely used model organism.

Specimens
Chromosome and FISH analysis were carried out with Hydra magnipapillata (strain 105).The animals were cultured according to standard conditions at 18 ± 0.5°C.
Chromosome preparations FISH was performed on mitotic plates from cells of asexual H. magnipapillata polyps.Chromosome preparations were obtained using the air-drying method: polyps were treated in a hypotonic 0.4 % sodium citrate solution Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics for 25 min and then fi xed in 3:1 (v/v) ethanolglacial acetic acid for 15-30 min.Fixed polyps were homogenized in 0.1-0.3ml of 70 % acetic acid.The cell suspension was dropped on prewarmed (37-40°C) cleaned slides and dried at 37-40°C.
DMRT sequence has been submitted to GenBank (accession No. HQ687211).
BAC clones containing the 18S rRNA gene (BAC 16C18) and BAC clones without ribosomal genes (BAC 1K4 PPOD, BAC 42P3 PPOD, BAC 16E21, BAC 18A7 ks-1) were selected and picked out from Kiel BAClibraries.BAC DNA was isolated according to the Qiagen Plasmid Midi Kit Protocol.
The 18S rDNA, 28S rDNA, ks1, TOL2 transposable elements genes and telomeric probes were labeled with biotin or digoxigenin by PCR.BAC probes were labeled by random primer labeling with biotin or digoxigenin according to the manufacturer's (Roche) instructions.

Basic chromosome features in Hydra magnipapillata
The diploid karyotype includes 30 chromosomes (Fig. 1).The karyotype is symmetric.The largest chromosome pair bears an achromatic gap in every homologue.The centromere positions are generally diffi cult to distinguish after conventional staining.After DAPI staining followed by C-banding procedure (Fig. 1, D), blocks of constitutive heterochromatin were found only in the centromere regions of the chromosomes.All chromosomes are twoarmed, meta-and submetacentric (Fig. 1, C,  D).No heteromorphic chromosome pair (sex chromosomes) could be identifi ed (Fig. 1).Conventional staining techniques including HOECHST-and Quinacrine-staining did not provide markers for identifi cation of individual chromosomes (Fig. 1, A, B).Since chromosomes in H. magnipapillata represent a regular gradation in size, a preliminary H. magnipapillata karyogram could be produced displayed in decreasing order of size (Fig. 1,  E).
Fluorescence in situ hybridization (FISH) and chromosomal mapping Fluorescence in situ hybridization (FISH) allows identifi cation of the location and abundance of a DNA sequence to be determined by hybridization of a labeled DNA probe to chromosomes and nuclei (Schwarzacher, Heslop-Harrison, 2000).Previously FISH studies in Hydra vulgaris revealed highly repetitive DNA in telomere positions (Traut et al., 2007).To establish FISH in H. magnipapillata and to ascertain the conserved nature of telomeres within the genus Hydra, we have carried out FISH using a (TTAGGG)n probe to test the presence of TTAGGG telomere repeat sequences on H. magnipapillata chromosomes.As shown in Figure 2, signals are visible on all chromosomes ends.The results match those in H. vulgaris (Traut et al., 2007) and support the view that chromosomes are capped by highly conserved telomeres.
For chromosome mapping we used random BAC probes which do not include rDNA genes.Unexpectedly, numerous signals with variable patterns of localization in different mitotic plates were revealed with every BAC probe, whereas single spots were expected.One examples of such hybridization pattern is shown in Fig. 3, A. We speculate that this is most probably due to the presence of abundant repetitive elements in the BAC fragments that can hybridize with complimentary sequences on the chromosomes.Because the abundance of dispersed repetitive elements appears to prevent the direct use of the currently available Hydra BACs as FISH probes, we next tested Tol2-like transposable element probes as a chromosome marker in Hydra magnipapillata.Southern blot hybridization data (Fig. 3,  B) showed the presence of multiple copies of a Tol2 transposable element gene in the H. magnipapillata genome.Consistent with this observation, FISH hybridization with Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics these probes revealed non-identical patterns of numerous signals in the chromosomes (Fig. 3 C, D).

Fluorescence in situ hybridization (FISH) using rDNA and single-gene probes
As alternative to universal markers we next examined the hybridization behavior of a number of selected candidate genes lacking highly repetitive elements.After performing FISH with both ribosomal 18S rDNA and 28S rDNA probes, including BAC-probes (BAC 16C18) containing 18S rDNA fragment, specifi c signals were localized on a single chromosome pair (Fig. 4) which consists of hydra´s largest chromosomes and, therefore, is designated pair #1.The signals obtained correspond to achromatic gaps revealed by routine methods of chromosome staining.

FISH location of ks1 genes on H. magnipapillata chromosomes
Head-specifi c gene ks1 is sensitive to patterning signals along the apical-basal body axis of Hydra and regulated by a complex interaction of inhibitory factors (Weinziger et al., 1994;Endl et al., 1999).ks1 loss-offunction polyps have defects specifi cally during head regeneration, but not foot regeneration, indicating that this gene is functionally involved in head development (Lohmann et al., 1999).To localize ks1 on H. magnipapillata chromosomes, FISH using both the 1.5 kb genomic fragment of H. magnipapillata ks1 gene (Weinziger et al., 1994) and the 10 kb BAC clone 18A7 containing the ks1 gene (Hemmrich, Bosch, unpubl.)resulted in ks1specifi c signals on three pairs of chromosomes (Fig. 5).In some experiments few additional signals were visible.Since ks1 belongs to a rather complex gene family (Hemmrich, Bosch, unpubl.)this probably is the result of hybridization of the ks1 probe with similar sequences of other ks1 gene family sites.After hybridization with BAC clone 18A7 containing ks1 gene sequence (Fig. 5 C, D) we detected additional weak hybridization signals on nearly all chromosomes.This is most probably due to hybridization of additional genomic fl anking sequences of this BAC probe with corresponding sequences on Hydra´s chromosomes.Based on morphology and size of the chromosomes and the localization of ks1 the H. magnipapillata chromosomes, we constructed the karyograms shown in Figure 5.

FISH Location of a DMRT1-related gene on a single chromosome pair
In the Hydra magnipapillata karyotype, no heteromorphic pair of chromosomes could be detected indicating the absence of sex chromosomes.Since in some cases (Marín at al., 2000;Vicoso, Bachtrog, 2009) morphologically distinct sex chromosomes can be traced back to an initially identical chromosome pair, it seems possible that Hydra have a pair of morphologically identical chromosomes bearing sex-linked genes ("sex chromosomes") or a pair of autosomes with clusters of sex linked genes.To address this Comp.Cytogenet., 2010 4(2) Comparative Cytogenetics question, as a fi rst step we decided to isolate a H. magnipapillata member of the DMRT gene family of transcription factors and to determine its chromosomal localization.DMRT genes appear to represent the only factors involved in sexual development that are conserved across the phylum Chordata (Winkler et al., 2004).The fi rst known and prototype member, DMRT1, is implicated in vertebrate male development, although with some speciesspecifi c differences.Invertebrate counterparts implicated in sex determination and differentiation include Drosophila doublesex (dsx) and the Caenorhabditis elegans Mab3 gene (Winkler et al., 2004).
To isolate a DMRT related gene, DMRT-specifi c primers were developed to amplify the corresponding region from the H. magnipapillata genome.Figure 6 shows the structure of the Hydra DMRT gene with its 3 exons.Southern blot analysis using a 1.6 kb fragment as probe suggests that Hydra magnipapillata has a single DMRT gene (Fig. 6 B).For FISH we labelled and detected the 1.6 kb fragment of this gene (Fig. 6 A) with biotin and nanocrystals (Qdots streptavidin conjugate 655).As shown in Figure 6 (E and F), a specifi c signal could be discovered on one pair of middle sized chromosomes.Can this DMRT signal be considered as an evidence for initial stage of evolution of sex chromosomes in an early branching metazoan?It is known that true animal sex chromosomes should contain a region with a cluster of sex-linked genes involved in sex determination (Charlesworth, Charlesworth, 1978;Charlesworth et al., 2005).Therefore, answering this certainly fascinating question awaits both functional characterization of the DMRT gene (Does it trigger sex determination in the Hydra interstitial stem cell system?) and identifying additional sex-controlling genes indicating that this set of chromosomes indeed should be considered as "heteromorphic".

CONCLUSION AND PERSPECTIVE
We report here the localization of several genes and telomeric repeats on the chromosomes of H. magnipapillata using the FISH method.Telomeres signals are located in every chromosome end.Their absence in interstitial positions may point to absence of chromosome fusions in Hydra karyotype evolution.
We also emphasize here that FISH allows the marking of individual chromosomes in H. magnipapillata.The 18S rDNA and 28S rDNA probes appear to mark the fi rst for the fi rst time, comparison of the genome architecture of basal metazoans, hence tracking ancestral genomic changes and providing an attractive tool for reconstruction of ancestral karyotypes.
Our fi ndings also indicate that FISH in H. magnipapillata can detect the localization of single copy genes on Hydra chromosomes.Most interestingly, one sex-related gene, DMRT, was discovered on a single pair of chromosomes (Fig. 6).Does this indicate that DMRT is involved in Hydra magnipapillata sex determination?Hydra´s sex determination remains a mystery.Germ line cells continuously originate from multipotent interstitial stem cells (Bosch, David, 1987).In contrast to other invertebrates and vertebrates where the gonads are the initial determinants of sex, in Hydra sex determination refl ects the sex of the interstitial cell lineage and is independent of the genetic sex of the epithelial (gonadal) cells (Littlefi eld, 1984;Campbell, 1985).We currently assume (Bosch, David, 1986) that the sexual phenotype of Hydra polyps is controlled by the switching rate of male and female stem cells and the repression of female differentiation by male stem cells.The molecular mechanisms controlling the determination of sex in Hydra and other cnidarians remain to be discovered (Fautin, 2002).Localization of DMRT on a single pair of Hydra chromosome (Fig. 6) admits the possibility of a dose-regulated testis (or ovary)-determining gene.
Taken together, we expect that application of FISH karyotyping to cross-species comparisons in the genus Hydra will have a considerable impact on the understanding of chromosome changes that occurred during animal evolution.Characterization of chromosomes in Hydra is certainly as fascinating as the unique biology of this basal metazoan model organism.

Fig. 2 ,
Fig. 2, A, B. FISH in H. magnipapillata mitotic chromosomes with telomere (red signals) and 18S rDNA (green signals) probes.The telomere signals are visible on all chromosomes ends.Two different mitotic plates are shown (A, B).Chromosomes are counterstained blue with DAPI.Bars = 10 μm.

Fig. 3 ,
Fig. 3, A-D'.FISH in H. magnipapillata mitotic chromosomes with two BAC probes.Numerous signals are visible in every chromosome (A).Southern blot of DNA sample of H. magnipapillata digested with HindIII (left lane) and EcoRI (right lane) hybridized with Tol2 transposable element probe (B).FISH in H. magnipapillata mitotic chromosomes with Tol2 transposable element probe.Signals are visible in every chromosome(C-D).Mitotic plates (C, D) and karyograms (C', D') from different FISH experiments.Chromosomes are counterstained blue with DAPI.Bars = 10 μm.

Fig. 5 ,
Fig. 5, A-D'.FISH in H. magnipapillata mitotic chromosomes with ks1 probes.Mitotic plates (A, B) and karyograms (A', B') from different FISH experiments with 1.5 kb size probe.Signals are visible in the same chromosome pairs: 5 th , 8 th and 12 th .Mitotic plate (C, D) and karyogram (C', D') after FISH experiment with 10 kb size BAC probe (clone 18A7) containing ks1 gene fragment.Bright signals are visible in the chromosome pairs 5 th , 8 th and 12 th .Additional weak signals are visible in some other chromosomes.Chromosomes are counterstained blue with DAPI.Bars = 10 μm.