Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Evolution of the Degenerated Y-Chromosome of the Swamp Guppy, Micropoecilia picta.

The conspicuous colour sexual dimorphism of guppies has made them paradigmatic study objects for sex-linked traits and sex chromosome evolution. Both the X- and Y-chromosomes of the common guppy (Poecilia reticulata) are genetically active and homomorphic, with a large homologous part and a small sex specific region. This feature is considered to emulate the initial stage of sex chromosome evolution. A similar situation has been documented in the related Endler's and Oropuche guppies (P. wingei, P. obscura) indicating a common origin of the Y in this group. A recent molecular study in the swamp guppy (Micropoecilia. picta) reported a low SNP density on the Y, indicating Y-chromosome deterioration. We performed a series of cytological studies on M. picta to show that the Y-chromosome is quite small compared to the X and has accumulated a high content of heterochromatin. Furthermore, the Y-chromosome stands out in displaying CpG clusters around the centromeric region. These cytological findings evidently illustrate that the Y-chromosome in M. picta is indeed highly degenerated. Immunostaining for SYCP3 and MLH1 in pachytene meiocytes revealed that a substantial part of the Y remains associated with the X. A specific MLH1 hotspot site was persistently marked at the distal end of the associated XY structure. These results unveil a landmark of a recombining pseudoautosomal region on the otherwise strongly degenerated Y chromosome of M. picta. Hormone treatments of females revealed that, unexpectedly, no sexually antagonistic color gene is Y-linked in M. picta. All these differences to the Poecilia group of guppies indicate that the trajectories associated with the evolution of sex chromosomes are not in parallel.

5-Methylcytosine-Rich Heterochromatin in Reptiles.

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

Chromosome Banding in Amphibia. XXXVI. Multimorphic Sex Chromosomes and an Enigmatic Sex Determination in Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

A detailed cytogenetic study on the leaf litter frog Eleutherodactylus johnstonei from 14 different Caribbean islands and the mainlands of Venezuela and Guyana revealed the existence of multimorphic XY♂/XX♀ sex chromosomes 14. Their male sex determination and development depends either on the presence of 2 telocentric chromosomes 14 (XtYt), or on 1 submetacentric chromosome 14 (Xsm) plus 1 telocentric chromosome 14 (Yt), or on the presence of 2 submetacentric chromosomes 14 (XsmYsm). The female sex determination and development requires either the presence of 2 telocentric chromosomes 14 (XtXt) or 2 submetacentric chromosomes 14 (XsmXsm). In all individuals analyzed, the sex chromosomes 14 carry a prominent nucleolus organizer region in their long arms. An explanation is given for the origin of the (XtYt)♂, (XsmYt)♂, (XsmYsm)♂, (XtXt)♀, and (XsmXsm)♀ in the different populations of E. johnstonei. Furthermore, the present study gives detailed data on the chromosome banding patterns, in situ hybridization experiments, and the genome size of E. johnstonei.

Chromosome Banding in Amphibia. XXXVII. Y-Autosome Translocations in Anura.

A detailed cytogenetic study on anurans belonging to the unranked taxon Terraranae revealed the existence of microscopically recognizable XY♂/XX♀ or ZZ♂/ZW♀ sex chromosomes in 11 species. Furthermore, in some species Y-autosome translocations were found, of which 5 could be confirmed. The male individuals carrying the Y-autosome translocations still coexist with the males showing the original karyotypes. The present report gives an overview on the mitotic and meiotic structure, staining and banding properties, functional importance, and similarities and differences of these Y-autosome translocations which are very rare in vertebrates. A mathematical model was constructed that calculates the various probabilities of further chromosome rearrangements in these karyotypes with Y-autosome translocations. The localization of the differential segment containing the hypothetical male sex-determining gene in the Y chromosome is discussed.

Chromosome Banding in Amphibia. XXXV. Highly Mobile Nucleolus Organizing Regions in Craugastor fitzingeri (Anura, Craugastoridae).

A 7-year cytogenetic study on the leaf litter frog Craugastor fitzingeri from Costa Rica and Panama revealed the existence of highly mobile nucleolus organizing regions (NORs) in their genomes. Silver (Ag)-staining of the active NORs demonstrated an exceptional interindividual pattern of NOR distribution at the telomeres of the chromosomes. All individuals examined showed a different and specific NOR location in their karyotypes. Furthermore, intraindividual variation in the NOR sites was found. This observation suggested the existence of mobile NORs in C. fitzingeri. Confirmation of this phenomenon was possible by systematic FISH analysis using an 18S + 28S rDNA probe. The extremely variable number and position of the NORs in C. fitzingeri is best explained by highly mobile NORs that move freely between the telomeres of the chromosomes. These transpositions must occur preferentially in premeiotic, meiotic, or postmeiotic stages, but also at a lower incidence in the somatic tissues of the animals. It is hypothesized that transposable (mobile) elements are closely linked to the NORs or are inserted into the major 18S + 28S rDNA spacers of C. fitzingeri. When such transposable elements spread by transpositions, they can carry with them complete or partial NORs. The present study provides detailed information on various differential chromosome banding techniques, in situ hybridization experiments, chromosomal hypermethylation patterns, determination of the genome size, and analyses of restriction fragment length polymorphisms of the DNA.

Demonstration of 5-Methylcytosine-Rich DNA Sequences in Chiroptera.

5-Methylcytosine-rich heterochromatic regions were demonstrated in metaphase chromosomes of 5 species of Chiroptera by indirect immunofluorescence using a monoclonal anti-5-methylcytosine antibody. These species belong to 4 genera and 2 families and are characterized by divergent karyotypes. One species (Glauconycteris beatrix) has an extremely low diploid chromosome number of 2n = 22 with only meta- to submetacentric elements and remarkably large amounts of constitutive heterochromatin located in the centromeric and pericentromeric regions of all chromosome pairs. Two species (G. beatrix and Neoromicia cf. guineensis) possess X-autosome translocations. In all species, the hypermethylated chromosome segments correspond to constitutive heterochromatin, and the numbers and positions of hypermethylated chromosome segments in the karyotypes are constant and species-specific. In some species (Pipistrellus hesperidus, Neoromicia cf. somalicus), there are several smaller chromosome pairs in which the bright anti-5-methylcytosine antibody labeling is not restricted to constitutively heterochromatic regions but is observed along the whole lengths of these chromosomes. The nature of these additional hypermethylated regions is discussed. The analysis of 5-methylcytosine-rich chromosome regions elucidates valuable data for chiropteran cytogenetics and reflects the high pace of evolution of the repetitive DNA fraction in their genomes.

The Hypermethylated Regions in Avian Chromosomes.

Chromosomal locations and amounts of 5-methylcytosine-rich chromosome regions were detected in the karyotypes of 13 bird species by indirect immunofluorescence using a monoclonal anti-5-methylcytosine antibody. These species belong to 7 orders and 10 families of modern (Neognathae) and primitive (Palaeognathae) birds and are characterized by macro- and microchromosomes as well as ZW sex chromosomes. In all 13 species, the hypermethylated chromosome segments are confined to constitutive heterochromatin. The chromosomal locations of hypermethylated DNA regions in the karyotypes are constant and species-specific. There is no general rule with regard to the distribution of these hypermethylated chromosome regions in the genomes of birds. In most instances, hypermethylated segments are located in the centromeric regions of chromosomes, but in the sex chromosomes, these can also be found in telomeric and interstitial postitions. In most of the species studied, the centromeric heterochromatin in many, if not all, of the microchromosomes is hypermethylated. However, in one species, the only detectable hypermethylated heterochromatic regions are located in one pair of macroautosomes and in the Z sex chromosome, but none of the microchromosomes contains visible quantities of 5-methylcytosine. The analysis of 5-methylcytosine-rich chromosome regions can be very helpful for the comparative cytogenetics of closely related species or subspecies. It also reflects the dynamic evolutionary process operating in the highly repetitive DNA of eukaryotic chromosomes.

Multicolor Spectral Analyses of Mitotic and Meiotic Mouse Chromosomes Involved in Multiple Robertsonian Translocations. II. The NMRI/CD and CD/TA Hybrid Strains.

Multicolor spectral analyses (spectral karyotyping) were performed on mitotic chromosomes of NMRI, CD, and TA mice and on male meiotic chromosomes (diakineses) of NMRI/CD and CD/TA hybrids. All chromosomes, including the various centric (robertsonian) fusions, could be unequivocally identified. Apart from the robertsonian translocations, which were previously detected by conventional banding analyses, no other interchromosomal rearrangements were found in these mice. In both the CD and TA mice, the autosomes 19 and the XY sex chromosomes are not involved in robertsonian translocations. In diakineses of male meiosis of the NMRI/CD hybrid, the 9 expected trivalents were present, whereas in those of the CD/TA hybrids a stable large meiotic multivalent, formed by 15 robertsonian fusion chromosomes and 2 terminally located normal chromosomes, was observed. The specific sequential order of the robertsonian fusion chromosomes found within this meiotic chain was as theoretically predicted. In the majority of diakineses of the NMRI/CD and CD/TA hybrids, the free autosomal bivalent 19 and the XY sex bivalent formed noticeable tight spatial associations.

Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura.

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

Chromosome Banding in Amphibia. XXXIV. Intrachromosomal Telomeric DNA Sequences in Anura.

The mitotic chromosomes of 4 anuran species were examined by various classical banding techniques and by fluorescence in situ hybridization using a (TTAGGG)n repeat. Large intrachromosomal telomeric sequences (ITSs) were demonstrated in differing numbers and chromosome locations. A detailed comparison of the present results with numerous published and unpublished data allowed a consistent classification of the various categories of large ITSs present in the genomes of anurans and other vertebrates. The classification takes into consideration the total numbers of large ITSs in the karyotypes, their chromosomal locations and their specific distribution patterns. A new category of large ITSs was recognized to exist in anuran species. It consists of large clusters of ITSs located in euchromatic chromosome segments, which is in clear contrast to the large ITSs in heterochromatic chromosome regions known in vertebrates. The origin of the different categories of large ITSs in heterochromatic and euchromatic chromosome regions, their mode of distribution in the karyotypes and evolutionary fixation in the genomes, as well as their cytological detection are discussed.

Chromosome Banding in Amphibia. XXXII. The Genus Xenopus (Anura, Pipidae).

Mitotic chromosomes of 16 species of the frog genus Xenopus were prepared from kidney and lung cell cultures. In the chromosomes of 7 species, high-resolution replication banding patterns could be induced by treating the cultures with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession, and in 6 of these species the BrdU/dT-banded chromosomes could be arranged into karyotypes. In the 3 species of the clade with 2n = 20 and 4n = 40 chromosomes (X. tropicalis, X. epitropicalis, X. new tetraploid 1), as well as in the 3 species with 4n = 36 chromosomes (X. laevis, X. borealis, X. muelleri), the BrdU/dT-banded karyotypes show a high degree of homoeology, though differences were detected between these groups. Translocations, inversions, insertions or sex-specific replication bands were not observed. Minor replication asynchronies found between chromosomes probably involve heterochromatic regions. BrdU/dT replication banding of Xenopus chromosomes provides the landmarks necessary for the exact physical mapping of genes and repetitive sequences. FISH with an X. laevis 5S rDNA probe detected multiple hybridization sites at or near the long-arm telomeric regions in most chromosomes of X. laevis and X. borealis, whereas in X. muelleri, the 5S rDNA sequences are located exclusively at the long-arm telomeres of a single chromosome pair. Staining with the AT base pair-specific fluorochrome quinacrine mustard revealed brightly fluorescing heterochromatic regions in the majority of X. borealis chromosomes which are absent in other Xenopus species.

5-Methylcytosine-Rich Heterochromatin in the Indian Muntjac.

Two 5-methylcytosine (5-MeC)-rich heterochromatic regions were demonstrated in metaphase chromosomes of the Indian muntjac by indirect immunofluorescence using a monoclonal anti-5-MeC antibody. The metaphases were obtained from diploid and triploid cell lines. A major region is located in the 'neck' of the 3;X fusion chromosome and can be detected after denaturation of the chromosomal DNA with UV-light irradiation for 1 h. It is located exactly at the border of the X chromosome and the translocated autosome 3. A minor region is found in the centromeric region of the free autosome 3 after denaturing the chromosomal DNA for 3 h or longer. The structure and possible function of the major hypermethylated region as barrier against spreading of the X-inactivation process into the autosome 3 is discussed.

Hypermethylated Chromosome Regions in Nine Fish Species with Heteromorphic Sex Chromosomes.

Sites and amounts of 5-methylcytosine (5-MeC)-rich chromosome regions were detected in the karyotypes of 9 Brazilian species of Characiformes fishes by indirect immunofluorescence using a monoclonal anti-5-MeC antibody. These species, belonging to the genera Leporinus, Triportheus and Hoplias, are characterized by highly differentiated and heteromorphic ZW and XY sex chromosomes. In all species, the hypermethylated regions are confined to constitutive heterochromatin. The number and chromosome locations of hypermethylated heterochromatic regions in the karyotypes are constant and species-specific. Generally, heterochromatic regions that are darkly stained by the C-banding technique are distinctly hypermethylated, but several of the brightly fluorescing hypermethylated regions merely exhibit moderate or faint C-banding. The ZW and XY sex chromosomes of all 9 analyzed species also show species-specific heterochromatin hypermethylation patterns. The analysis of 5-MeC-rich chromosome regions contributes valuable data for comparative cytogenetics of closely related species and highlights the dynamic process of differentiation operating in the repetitive DNA fraction of sex chromosomes.

Multicolor Spectral Analyses of Mitotic and Meiotic Mouse Chromosomes Involved in Multiple Robertsonian Translocations. I. The CD/Cremona Hybrid Strain.

Multicolor spectral analysis (spectral karyotyping) was applied to mitotic and male diakinetic chromosomes of hybrid mice carrying a unique system of 18 autosomal Robertsonian translocation chromosomes with alternating arm homologies. Only the autosomes 19 and the XY sex chromosomes are excluded from these Robertsonian translocations. The translocations, previously identified by conventional banding analyses, could be verified by spectral karyotyping. Besides the Robertsonian translocations, no other interchromosomal rearrangements were detected. In diakineses of male meiosis, the 18 metacentric Robertsonian translocation chromosomes form a very large meiotic 'superring'. The predictable, specific order of the chromosomes along this 'superring' was completely confirmed by multicolor spectral analysis. In the majority of diakineses analyzed, the free autosomal bivalent 19 and the XY sex bivalent form a conspicuous complex which tightly associates with the 12;14 Robertsonian translocation chromosome in the 'superring'.