Irma Nardi

Cytological evidence of transcription of highly repeated DNA sequences during the lampbrush stage in Triturus cristatus carnifex

Highly repeated, or satellite, DNA fractions have been isolated from total Triturus cristatus carnifex DNA by renaturation kinetics, caesium salt centrifugation and restriction endonuclease digestion. We have shown by DNA/DNA in situ hybridisation and autoradiography that all of these probes bind to C-band positive regions on mitotic or lampbrush chromosomes of T.c. carnifex. Under conditions of DNA to RNA-transcript in situ hybridisation labelled satellite DNA binds to nascent RNA transcripts that are still associated with the DNA axes of many lampbrush loops. The majority of the loops that label heavily in these experiments are located on the long arms of chromosome I, a region previously shown to be rich in highly repeated DNA and to have many of the properties of heterochromatin. These satellite DNA probes also label many loops on a comparable chromosome region in T. marmoratus, a species closely related to T. cristatus. However, in DNA/RNA-transcript hybrids to other more distantly related species of Triturus, there are no chromosome regions that have the same concentration of labelled loop pairs as the long arms of T.c.carnifex and T. marmoratus, although some loop pairs do label. We have cloned two satellite sequences in pBR322, and have obtained the same results using these pure probes as we obtained using satellite probes isolated by other techniques. These results demonstrate unequivocally that satellite DNA is transcribed on lampbrush chromosomes during oogenesis in crested newts.

Heterochromatic DNA in Tritmus (Amphibia, Urodela)

We have studied the structure, genome organization, chromosomal location, conservation across species and transcription on lampbrush chromosomes, of an ATrich 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.

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 salamanderHydromantes. The sequence analysis of a 5-kb fragment (Hsr1A) of one member has revealed significant similarities with aminoacidic domains of retroviruses and retrotransposons. The similarity of the reverse transcriptase domain and the gene organization identifies Hsr1A as a member of thegypsy/Ty3 class of retrotransposons. We hypothesize that Hsr1 sequences are vestiges of an invasion of theHydromantes genome that occurred early in the evolutionary history of these European plethodontids. About 106 Hsr1 sequences are present in the largeHydromantes genome. This is the highest number of copies so far discovered for retrotransposon-like elements in eukaryote organisms.

Characterization of the lampbrush chromosomes of the marbled newt Triturus marmoratus (Latreille, 1800).

The maps of the lampbrush chromosomes of Triturus marmoratus oocytes were constructed on the basis of their lengths and major morphological characters such as giant fusing loops, dense matrix loops, lumpy objects, axial granules, lateral globules and reflected fusions; a nucleolus organizing region occurs subterminally on the right side of chromosome X. — Bivalent I appears morphologically asymmetrical, its two partners being of different lengths and bearing heteromorphic loops and other heterozygous structures: this heteromorphism may indicate that the two partners of bivalent I represent the ZW heterochromosomes of the species. Finally, an autoradiographic study has been performed in order to ascertain the pattern of 3H-uridine incorporation shown by the most typical landmarks and nucleoli.

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

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 by 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.

Cytogenetics of the European plethodontid salamanders of the genus Hydromantes (Amphibia, Urodela)

A karyological analysis was carried out on different European species of the genus Hydromantes (Plethodontidae). All the species examined share the same chromosome number (2n=28) and, with the exception represented by pair XIV, morphologically similar karyotypes. While the karyotypes display a similar distribution — mainly centromeric and pericentric — of C-heterochromatin, quantitative variations in pericentric heterochromatin are observed among species. In the continental species Hydromantes italicus and ambrosii as well as in the eastern Sardinian species imperialis, flavus and specie nova, pair XIV consists of heteromorphic sex chromosomes of the XX/XY type. It is proposed that the differentiation of the Y might have taken place through the occurrence of a structural rearrangement, such as a pericentric inversion, starting from a hypothetical, homomorphic pair XIV. A sex-related heteromorphism is not found in the western Sardinian species H. genei. A further karyological differentiation among these species concerns the position of the nucleolus organizing region (NOR), which is located on chromosome XII (H. italicus and ambrosii) or on chromosome X, close to the centromere (H. genei, H. imperialis and H. specie nova), or in an intercalary position (H. flavus). The location and the number of the 5 S DNA sites have been conserved during species divergence. On the basis of these karyological data, as well as of results obtained through a preliminary restriction enzyme analysis of the ribosomal and genomic DNAs, the phyletic relationships among the European Hydromantes species are discussed.

Cloning and developmental expression of 5-HT1A receptor gene in Xenopus laevis

The aim of our work is to investigate the potential involvement of serotonin and its G-protein-coupled receptors in neural differentiation or other developmental processes in Xenopus laevis. By using a RT-PCR strategy, we isolated a cDNA fragment from X. laevis brain showing high amino-acid similarity with the mammalian 5-HT1A receptor. We used this fragment to isolate a cDNA clone containing a single ORF of 408 amino-acids with an overall amino-acid identity of 73% with the human and rat 5-HT1A receptor. This structural similarity suggests that this clone encodes the Xenopus homolog of the mammalian 5-HT1A receptor (X5-HT1A). In order to establish a possible role for this receptor in development, we analyzed the pattern of its gene expression during embryogenesis, larval stages and in adult brain by in situ hybridization. The first signal of mRNA expression appears in the rostral part of brain stem at stage 22, when the first neurons start differentiation [38,21]. In later stages of development, the cells expressing X5-HT1A transcripts appear to correspond to serotonergic neurons. By stage 41, X5-HT1A mRNA is also detected in the inner nuclear layer (INL) of the developing retina. This pattern of expression is maintained until stage 46, i.e. at the beginning of metamorphosis. In adult, additional brain areas express X5-HT1A mRNA, particularly in telencephalon, diencephalon and mesencephalon. On the whole, our data show that the X5-HT1A receptor mRNA is developmentally regulated, with expression first appearing in differentiating serotonergic neurons, where this receptor may mediate, through an autocrine regulatory pathway, the trophic action of serotonin on developing serotonergic system.

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis. 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.

Unraveling new roles for serotonin receptor 2B in development: key findings from Xenopus

The serotonin receptor 5-HT2B has been shown to be critically important during embryogenesis as the knockout of this gene in mice causes heart defects and embryonic lethality that impairs further analyses on other embryonic cell and tissue types. In the present review, we highlight how the use of Xenopus laevis, an alternative vertebrate model suitable for gene loss and gain of function analyses, has contributed to our understanding of the role of 5-HT2B signaling during development. In vivo studies showed that 5-HT2B signaling is not only required for heart development, but that it also has a crucial role in ocular and craniofacial morphogenesis, being involved in shaping the first branchial arch and the jaw joint, in retinogenesis and possibly in periocular mesenchyme development. These findings may be relevant for our understanding of congenital defects including human birth malformations. In addition, 5-HT2B appears to be required for the therapeutic actions of selective serotonin reuptake inhibitors commonly prescribed as antidepressant drugs to pregnant and lactating women. We discuss how the understanding of the molecular basis of serotonin signaling in a suitable animal embryogenesis model may open new lines of investigations and therapies in humans.