Planarians, a tale of stem cells.

Planarians possess amazing abilities to regulate tissue homeostasis and regenerate missing body parts. These features reside on the presence of a population of pluripotent/totipotent stem cells, the neoblasts, which are considered as the only planarian cells able to proliferate in the asexual strains. Neoblast distribution has been identified by mapping the cells incorporating bromodeoxyuridine, analyzing mitotic figures and using cell proliferation markers. Recently identified molecular markers specifically label subgroups of neoblasts, revealing thus the heterogeneity of the planarian stem cell population. Therefore, the apparent totipotency of neoblasts probably reflects the composite activities of multiple stem cell types. First steps have been undertaken to understand how neoblasts and differentiated cells communicate with each other to adapt the self-renewal and differentiation rates of neoblasts to the demands of the body. Moreover, the introduction of molecular resource database on planarians now paves the way to renewed strategies to understand planarian regeneration and stem cell-related issues.

An MCM2-related gene is expressed in proliferating cells of intact and regenerating planarians.

The minichromosome maintenance (MCM2-7) gene family encodes conserved proteins, which are essential for DNA replication licensing in eukaryotes. They are abundant in proliferating cells, and specific MCM transcripts undergo cell cycle-dependent oscillations. Here we report the characterization of a planarian MCM2 homologue, DjMCM2, which represents the first molecular marker for detecting proliferating cells in planarians. DjMCM2-expressing cells are broadly distributed in the mesenchymal space of the body, with the exception of the cephalic region, and are preferentially accumulated in the peripheral area of the dorso-lateral mesenchyme, along the anteroposterior axis. During regeneration, no DjMCM2 transcripts are observed within the blastema, according to the current view that this structure is not a proliferation site in planarians. Spatio-temporal changes in DjMCM2 RNA expression pattern in the stump parallel blastema growth, coordinately with the orientation of the cut. X-ray irradiation results in the disappearance of DjMCM2 expression, thus confirming that these transcripts are detected specifically in proliferating cells, visualized as neoblasts by in situ hybridization in dissociated cells. In addition to neoblasts, rare large DjMCM2-expressing cells are observed in macerates of tissues excised just below the wound, suggesting that cell types other than neoblasts may be sporadically recruited for proliferation in planarians.

Expression of DjY1, a protein containing a cold shock domain and RG repeat motifs, is targeted to sites of regeneration in planarians.

Planarians are well-known for their exceptional regenerative abilities. This investigation focuses on the involvement of a Y-box protein, defined by the presence of a cold-shock domain, in regeneration-specific processes. Previous studies have shown that developmentally expressed Y-box proteins bind to mRNA molecules and regulate the timing of their translation. We have isolated and characterized a planarian Y-box gene, DjY1, which is specifically expressed at the site of regeneration, the blastema. DjY1 transcripts appear rapidly at the site of cutting and increase in number as the blastema grows. The timing and level of expression is similar irrespective of the orientation of the cut: in anterior, posterior, and lateral regenerative tissue. As regeneration nears completion, there is a general decrease in transcript level except in structures which are still differentiating, specifically in the auricles where new DjY1 transcripts are produced. A similarly modulated temporal pattern of expression throughout regeneration is seen in assaying the DjY1 protein. Within the population of blastemal cells, a subset of differentiating cells is specifically immunostained using antibodies to DjY1. The DjY1 protein contains a cold-shock domain and RG-repeat motifs, both of which are associated with RNA-binding properties: in vitro binding studies using recombinant DjY1 show that the preferred template is single-stranded RNA of heterogeneous sequence. These data provide the first direct evidence that a Y-box protein is involved in the regeneration process in planarians and implicate DjY1 in the translational regulation of differentiation-specific mRNAs.

H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida).

Two clones, obtained by polymerase chain reaction from macronuclear DNA of the unicellular ciliated protist Blepharisma japonicum, were isolated and sequenced. They correspond to fragments of two different putative H4 histone genes. The existence of multiple H4 histone genes was also suggested by Southern blot hybridisation experiments employing one of the obtained clones as a probe. Two B. japonicum H4 protein fragments, which were directly sequenced, show differences in the amino acid sequences too. The comparison of the obtained B. japonicum H4 partial amino acid sequences with each other, and with H4 from other ciliates and from representative microbial and multicellular organisms, highlights the larger histone heterogeneity of lower eukaryotes compared to that observed in higher organisms.

Gypsy/Ty3-like elements in the genome of the terrestrial Salamander hydromantes (Amphibia, Urodela).

We have studied a family of long repetitive DNA sequences (Hsr1) interspersed in the large genome of the European plethodontid salamander Hydromantes. The sequence analysis of a 5-kb fragment (Hsr1A) of one member has revealed significant similarities with amino acidic domains of retroviruses and retrotransposons. The similarity of the reverse transcriptase domain and the gene organization identifies Hsr1A as a member of the gypsy/Ty3 class of retrotransposons. We hypothesize that Hsr1 sequences are vestiges of an invasion of the Hydromantes genome that occurred early in the evolutionary history of these European plethodontids. About 10(6) Hsr1 sequences are present in the large Hydromantes genome. This is the highest number of copies so far discovered for retrotransposon-like elements in eukaryote organisms.

A tandemly repeated DNA family originated from SINE-related elements in the European plethodontid salamanders (Amphibia, Urodela).

We have characterized a highly repetitive family, named Hy/Pol III, in the genome of the European salamanders Hydromantes (Plethodontidae). This family consists of short, tandemly repeated sequences organized in clusters, scattered through the genome as shown both by in situ hybridization to chromosomes and by Southern blot hybridization. The repeat unit is about 200 bp in length and it is a composite element since it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats. The whole element itself is bordered by two other direct repeats. The sequence data suggest that two elements, presumably derived from polymerase III transcripts, have been inserted one into the other, giving rise to the observed composite structure. During evolution the Hy/Pol III family was then amplified by tandem duplication at the DNA level. The inferred relationships between Hy/Pol III members from three representative species of the European Hydromantes suggests that a subfamily structure characterizes the evolutionary history of this family.

Molecular cytogenetics of the ribosomal (18S + 28S and 5S) DNA loci in primitive and advanced urodele amphibians.

In the present work we performed a cytogenetic analysis of the ribosomal (18S + 28S and 5S) loci in amphibian species belonging to the advanced family Salamandridae (genera Triturus, Salamandra, and Salamandrina) and in the primitive hynobiid Salamandrella keyserlingii (family Hynobiidae). In each analyzed karyotype the 5S rDNA sites appear to be stable, and definite in number, while an intraspecific variability both in number and chromosomal location of the 18S + 28S rDNA loci has been found in some Triturus species. In particular, an evolutionary trend toward a large intraspecific variability of the 18S + 28S rDNA loci has been found in the T. vulgaris species group. A structural analysis of the ribosomal repetition units demonstrates the occurrence of a length polymorphism within the 18S + 28S rDNA repeats in the examined species of the family Salamandridae; however, this polymorphism is rather limited, even in those Triturus species characterized by high intragenomic variability of the ribosomal sites. We show that in T. vulgaris meridionalis the variant repetition units actually segregate with individual chromosomes. This implies that they are not intermingled in the ribosomal clusters.

A centromeric satellite DNA of the unisexual Bacillus atticus (Insecta: Phasmatodea).

In the parthenogenetic Bacillus atticus atticus, widespread over most of the Mediterranean basin, a highly repeated satellite DNA (BaB300) has been detected and analysed. BgI II, Taq I and Alu I restriction enzymes cut 316 bp long repeating units from the BaB300 family. These are non-coding, AT rich and well conserved in the three isolated populations studied. By in situ hybridization, the satellite has been located in the centromeric heterochromatin of a subset of medium- and small-sized chromosomes, including the Xs. Related sequences have been found in the bisexual B. grandii grandii and in its specific hybrid B. whitei (=B. rossius/grandii grandii), while they are absent from B. rossius redtenbacheri. These data support the genetic affinity relationships within the genus Bacillus inferred from morphological, allozymic and karyological data. They also encourage further comparisons among Bacillus hybrids, in order to trace their parental genomes in the hybrid nuclei, and within the B. atticus complex (i.e. additional samples of B. a. atticus and diploid/triploid cytotypes of B. a. carius) to study repetitive DNA turnover in unisexual organisms.

A centromeric satellite DNA in the European plethodontid salamanders (Amphibia, Urodela).

A highly repeated satellite DNA (Hy500) located in the centromeric heterochromatin of the European plethodontid salamander Speleomantes (formerly Hydromantes) was studied. The Hy500 family represents about 1% of the Speleomantes supramontis genome and has a major repeating unit of about 500 base pairs, which may have evolved from the progressive amplification of shorter sequences. This centromeric satellite is conserved in all the Speleomantes species, which nevertheless show distinct patterns of chromosomal distribution, which are of relevance as to their phylogenetic relationships.

Two dispersed highly repeated DNA families of Triturus vulgaris meridionalis (Amphibia, Urodela) are widely conserved among Salamandridae.

Two BamHI families of repeated sequences were characterized from the genome of the Italian smooth newt, Triturus vulgaris meridionalis (Amphibia, Urodela). The first family, which is divided into subfamilies, consists of tandemly arranged arrays whose basic repeat is around 398 bp long; these arrays are dispersed throughout the entire chromosome sets of the various species of Triturus tested. Moreover the family is widely conserved among Salamandridae, being detected by genomic DNA blotting of Notophthalmus viridescens, Taricha granulosa, Salamandrina terdigitata and Euproctus platycephalus. The second BamHI family is represented by a cloned sequence of 419 bp, which is dispersed in the chromosome set of several species of Triturus. The sequence is also conserved in S. terdigitata and in E. platycephalus but is not detectable in N. viridescens or T. granulosa. The cloned sequence is most probably only part of a longer unit interspersed within the Triturus genome.

Heterochromatic DNA in Triturus (Amphibia, Urodela) II. A centromeric satellite DNA.

The MspI family of highly repeated sequences is a centromeric satellite DNA representing about 1% of the genome of the Italian smooth newt, Triturus vulgaris meridionalis. We have studied the structure, genomic organization, chromosomal localization and conservation across species of this family. MspI sequences are around 197 bp long, as shown by sequencing of three cloned units. The family is organized in large clusters of tandemly arrayed units, present at almost all the centromeres of T.v. meridionalis, and is well conserved in the T.v. vulgaris subspecies. Conserved MspI sequences are also present in the related species T. helveticus, where they appear to be clustered at the centromeres of only a few chromosomes. MspI sequences are not found in other Triturus species analysed. The correlation of these sequences with the overall distribution pattern of heterochromatin and the extent of their conservation within the genus Triturus, are discussed.

Evolution of highly repeated DNA within the genusTriturus (Amphibia, Urodela).

Highly repeated DNA is a main feature of urodele amphibian genomes. InTriturus this class of DNA consists of several sequence families differently arranged at both the molecular and the chromosomal level, showing varying degrees of conservation across species. Present data on highly repeated DNA inTriturus are here summarized and discussed with regard to the evolution and possible functional role of these sequences.

Chromosome banding in amphibia. XI. Constitutive heterochromatin, nucleolus organizers, 18S + 28S and 5S ribosomal RNA genes in Ascaphidae, Pipidae, Discoglossidae and Pelobatidae.

The karyotypes of 14 species of Anura from 9 genera of the suborders Amphicoela, Aglossa, Opisthocoela and Anomocoela were analysed with various banding techniques and conventional cytogenetic methods. The 18S + 28S and 5S ribosomal RNA genes were localized by means of in situ hybridization. No Q-, R- and G-banding patterns in the euchromatic segments of the metaphase chromosomes could be demonstrated in any of the species; this does not seem to be caused by a higher degree of spiralization of the amphibian chromosomes, but by the special DNA organization in these organisms. In most karyotypes, constitutive heterochromatin is present at centromeres, telomeres and nucleolus organizer regions (NORs), but rarely in interstitial positions. The heterochromatic regions are either quinacrine positive and mithramycin negative or vice versa. All species examined possess only one homologous pair of NORs: these display the brightest mithramycin fluorescence in the karyotypes. Many specimens exhibited unequal labelling of the two NORs both after silver and mithramycin staining as well as after in situ hybridization with 3H-18S + 28S rRNA. In four species, between one and six chromosome pairs with homologous 5S rRNA sites could be identified. The 5S rRNA genes and the 18S + 28S rRNA genes are closely linked in two species. In the male meiosis of the Amphicoela and Opisthocoela, there are intersitial, subterminal and terminal chiasmata in the bivalents, whereas only terminal chiasmata are observed in the bivalents of the Aglossa and Anomocoela. No heteromorphic sex-specific chromosomes could be demonstrated in any of the species. The differential staining techniques revealed that the chromosomal structure in these four suborders is largely the same as in the highly evolved anuran suborders Procoela and Diplasiocoela.

Heterochromatic DNA in Triturus (Amphibia, Urodela). I. A satellite DNA component of the pericentric C-bands.

We have studied the structure, genome organization, chromosomal location, conservation across species and transcription on lampbrush chromosomes, of an AT-rich satellite DNA component of the newt, Triturus vulgaris meridionalis. The satellite (Sat G), originally isolated by gradient centrifugation, represents about 2% of the vulgaris genome and comprises a highly repetitive sequence family (HindIII family), whose monomers have been cloned. The repeat units are about 330 bp long, as measured on gels, and a cloned unit (pTvm1) is 310 bp long, as shown by sequencing. Abundant clusters of the HindIII family sequences are located within the pericentric heterochromatin (i.e. the C-bands placed at both sides of, and at a certain distance from, the centromeres) in most chromosomes. Both the sequence family and its overall pattern of chromosomal distribution are conserved within the genus Triturus, despite a few species-specific differences. The great majority of the HindIII family sequences are unexpressed on lampbrush chromosomes; they reside within pericentric, condensed segments of the chromosome axis ("loopless bars"). Only a few sequences are transcribed on some loops, suggesting that transcription promotion does not depend on the satellite sequences themselves.

Molecular organization of ribosomal RNA genes clustered at variable chromosomal sites in Triturus vulgaris meridionalis (Amphibia, Urodela).

The ribosomal RNA genes of Triturus vulgaris meridionalis (Amphibia, Urodela) show the peculiar feature of being clustered not only at the nucleolar organizer, present in the species at a definite chromosome location, but also at "additional ribosomal sites" which are highly variable in number and chromosomal distribution among individuals. The additional ribosomal sites are most often found at specific chromosome regions, such as telomeres, C-bands and centromeres, in virtually all the chromosomes. With increasing numbers of additional clusters, the genomic dosages of ribosomal RNA genes are found to increase over a tenfold range, though not linearly. At a molecular level, the ribosomal DNA repeats differ in size because of discrete variations in the length of the non-transcribed spacers. However, the resulting length heterogeneity of the gene family is rather limited within a single genome as well as within the species. Many of the ribosomal loci appear to be internally homogeneous with respect to the repeat length. Moreover, separate clusters from distant genomic regions can share the same size class of ribosomal repeats even in the same specimen. The nucleolar organizer is mostly endowed with "shorter" ribosomal repeating units, ranging in size from 13.7 X 10(3) to 15.2 X 10(3) base-pairs. The additional ribosomal sites are characterized by the occurrence of "longer" repeats, ranging in size from 16.2 X 10(3) to 19.7 X 10(3) base-pairs. The "shorter" class of ribosomal repeats is always detected in the amplified ribosomal DNA, suggesting that the nucleolar organizer locus is involved in the amplification process in most oocytes. "Longer" ribosomal repeats are also detectable in the amplified ribosomal DNA of a few females.

Chromosomal localization of 18S + 28S and 5S Ribosomal RNA genes in evolutionarily diverse anuran amphibians.

The chromosomal locations of the 18S + 28S and 5S ribosomal RNA genes have been analyzed by in situ hybridization in ten anuran species of different taxonomic positions. The chosen species belong to both primitive and evolved families of the present day Anura. Each examined species has 18s + 28S rRNA genes clustered in one locus per haploid chromosome set: this locus is placed either in an intercalary position or proximal to the centromere, or close to the telomere. The 5S rRNA genes are arranged in clusters which vary in number from one to six per haploid set. The 5S rDNA sites are found in intercalary positions, at the telomeres, and at, or close to, the centromeres. Microchromosomes and small chromosomes in primitive karyotypes have been found to carry 5S rDNA sequences. The results are discussed in relation to ideas on the karyological evolution of Amphibia.

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela). III. Inheritance of the chromosomal sites for 18S + 28S ribosomal RNA.

In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.--The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia, Urodela). II. Intraspecific variability in number and position of the chromosome loci for 18S + 28S ribosomal RNA.

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with 3H 18S + 28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.