An assessment of contamination of the Fusaro Lagoon (Campania Province, southern Italy) by trace metals.

The Fusaro Lagoon is a shallow lagoon, located in SW Italy, largely influenced in the last decades by several anthropic impacts. The study examined the pollution status of the lagoon, during year 2011-2012 at nine sampling stations with the aim to find out proper measurements of water lagoon restoration. Concentrations of heavy metals (HMs) (aluminium [Al], barium [Ba], cadmium [Cd], copper [Cu], iron [Fe], manganese [Mn], vanadium [V] and zinc [Zn]) were examined in water, sediments and specimens of the ascidian Ciona intestinalis sp. A. Low levels of dissolved oxygen concentration were detected at many stations, with mean values of 5.2-6.4 mg L(-1). The redox potential of surface waters was also low, -2.7 to 50.7 mV. Sediments possessed high organic matter content, 17.7-29.4%. In sediments, the mean Zn level, 251.4 mg kg(-1), was about sixfold higher than that recorded in year 2000 (38.5 mg kg(-1)) and considerably higher than that recorded in 2007 (191 mg kg(-1)). The mean levels of Cd were outstandingly high, with a mean value of 70.5 mg kg(-1), about 30- and 50-fold higher than those determined in 2000 and 2007, respectively. Cadmium (Cd), Cu and nickel (Ni) appeared in excess with respect to most current guidelines, reaching significant pollution levels. C. intestinalis sp. A was detected only at few stations, with metals accumulated preferentially in the body in respect to the tunic, from 1.2 times for Zn (178 mg kg(-1)) to 4.0 times for V (304 mg kg(-1)). Data suggests the necessity of an immediate action of eco-compatible interventions for environmental restoration.

Localization of BDNF expression in the developing brain of zebrafish.

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease.

Developmental genetics in primitive chordates.

Recent advances in the study of the genetics and genomics of urochordates testify to a renewed interest in this chordate subphylum, believed to be the most primitive extant chordate relatives of the vertebrates. In addition to their primitive nature, many features of their reproduction and early development make the urochordates ideal model chordates for developmental genetics. Many urochordates spawn large numbers of transparent and externally developing embryos on a daily basis. Additionally, the embryos have a defined and well-characterized cell lineage until the end of gastrulation. Furthermore, the genomes of the urochordates have been estimated to be only 5-10% of the size of the vertebrates and to have fewer genes and less genetic redundancy than vertebrates. Genetic screens, which are powerful tools for investigating developmental mechanisms, have recently become feasible due to new culturing techniques in ascidians. Because hermaphrodite ascidians are able to self-fertilize, recessive mutations can be detected in a single generation. Several recent studies have demonstrated the feasibility of applying modern genetic techniques to the study of ascidian biology.

Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish.

Somite formation involves the establishment of a segmental prepattern in the presomitic mesoderm, anteroposterior patterning of each segmental primordium and formation of boundaries between adjacent segments. How these events are co-ordinated remains uncertain. In this study, analysis of expression of zebrafish mesp-a reveals that each segment acquires anteroposterior regionalisation when located in the anterior presomitic mesoderm. Thus anteroposterior patterning is occurring after the establishment of a segmental prepattern in the paraxial mesoderm and prior to somite boundary formation. Zebrafish fss(-), bea(-), des(-) and aei(-) embryos all fail to form somites, yet we demonstrate that a segmental prepattern is established in the presomitic mesoderm of all these mutants and hox gene expression shows that overall anteroposterior patterning of the mesoderm is also normal. However, analysis of various molecular markers reveals that anteroposterior regionalisation within each segment is disturbed in the mutants. In fss(-), there is a loss of anterior segment markers, such that all segments appear posteriorized, whereas in bea(-), des(-) and aei(-), anterior and posterior markers are expressed throughout each segment. Since somite formation is disrupted in these mutants, correct anteroposterior patterning within segments may be a prerequisite for somite boundary formation. In support of this hypothesis, we show that it is possible to rescue boundary formation in fss(-) through the ectopic expression of EphA4, an anterior segment marker, in the paraxial mesoderm. These observations indicate that a key consequence of the anteroposterior regionalisation of segments may be the induction of Eph and ephrin expression at segment interfaces and that Eph/ephrin signalling subsequently contributes to the formation of somite boundaries.

Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development.

The distribution of FMRFamide-like immunoreactive (ir) neurons and fibers was investigated in the central nervous system of developing zebrafish and juvenile sturgeon (sterlet). Adult zebrafish was also studied. In zebrafish embryos FMRFamide-ir elements first appeared 30 h post-fertilization (PF). Ir somata were located in the olfactory placode and in the ventral diencephalon. FMRFamide-ir fibers originating from diencephalic neurons were found in the ventral telencephalon and in ventral portions of the brainstem. At 48 h PF, the ir perikarya in the olfactory placode displayed increased immunoreactivity and stained fibers emerged from the somata. At 60 h PF, bilaterally, clusters of FMRFamide-ir neurons were found along the rostro-caudal axis of the brain, from the olfactory placode to rostral regions of the ventro-lateral telencephalon. At 60 h PF, numerous ir fibers appeared in the dorsal telencephalon, optic lobes, optic nerves, and retina. Except for ir fibers in the hypophysis at the age of 72 h PF, and a few ir cells in the nucleus olfacto-retinalis (NOR) at the age of 2 months PF, no major re-organization was noted in subsequent ontogenetic stages. The number of stained NOR neurons increased markedly in sexually mature zebrafish. In adult zebrafish, other ir neurons were located in the dorsal zones of the periventricular hypothalamus and in components of the nervus terminalis. We are inclined to believe that neurons expressing FMRFamide originate in the olfactory placode and in the ventricular ependyma in the hypothalamus. On the same grounds, a dual origin of FMRFamide-ir neurons is inferred in the sturgeon, an ancestral bony fish: prior to the observation of ir cells in the nasal area and in the telencephalon stained neurons were noted in circumventricular hypothalamic regions.

Zebrafish Hoxa and Evx-2 genes: cloning, developmental expression and implications for the functional evolution of posterior Hox genes.

Vertebrate Hox genes are required for the establishment of regional identities along body axes. This gene family is strongly conserved among vertebrates, even in bony fish which display less complex ranges of axial morphologies. We have analysed the structural organization and expression of Abd-B related zebrafish HoxA cluster genes (Hoxa-9, Hoxa-10, Hoxa-11 and Hoxa-13) as well as of Evx-2, a gene closely linked to the HoxD complex. We show that the genomic organization of Hoxa genes in fish resembles that of tetrapods albeit intergenic distances are shorter. During development of the fish trunk, Hoxa genes are coordinately expressed, whereas in pectoral fins, they display transcript domains similar to those observed in developing tetrapod limbs. Likewise, the Evx-2 gene seems to respond to both Hox- and Evx-types of regulation. During fin development, this latter gene is expressed as the neighbouring Hox genes, in contrast to its expression in the central nervous system which does not comply with colinearity and extends up to anterior parts of the brain. These results are discussed in the context of the functional evolution of Hoxa versus Hoxd genes and their different roles in building up paired appendages.

Teleost HoxD and HoxA genes: comparison with tetrapods and functional evolution of the HOXD complex.

In tetrapods, Hox genes are essential for the proper organization and development of axial structures. Experiments involving Hox gene inactivations have revealed their particularly important functions in the establishment of morphological transitions within metameric series such as the vertebral column. Teleost fish show a much simpler range of axial (trunk or appendicular) morphologies, which prompted us to investigate the nature of the Hox system in these lower vertebrates. Here, we show that fish have a family of Hox genes, very similar in both number and general organization, to that of tetrapods. Expression studies, carried out with HoxD and HoxA genes, showed that all vertebrates use the same general scheme, involving the colinear activation of gene expression in both space and time. Comparisons between tetrapods and fish allowed us to propose a model which accounts for the primary function of this gene family. In this model, a few ancestral Hox genes were involved in the determination of polarity in the digestive tract and were further recruited in more elaborate axial structures.

A molecular approach to the evolution of vertebrate paired appendages.

Over the past few years, genes involved in the ontogenesis of tetrapod limbs have been Isolated and characterized. Some of the developmental mechanisms responsible for the morphogenesis of these complex structures can now be investigated through a new approach. In addition, these genes can serve as tools to re-evaluate some aspects of the long-standing question of the fin-to-limb transition. Comparative molecular developmental biology is providing new insight into the similarities and differences in the morphologies of these homologous structures.

Hox gene expression in teleost fins and the origin of vertebrate digits.

Hox genes are essential for growth and patterning of the tetrapod limb skeleton. Mice mutant for the Hoxd-13 gene have an important delay in morphogenesis owing to reduced proliferation. Based on the appearance of atavisms in such mice, we suggested that modifications of Hox gene regulation may have been a source of morphological variation during the evolution of tetrapod limbs. Pectoral and pelvic fins are homologous to fore- and hindlimbs, respectively. To compare the relative importance of Hox genes during fin versus limb morphogenesis, we cloned zebrafish (Danio rerio) HoxD and HoxA complex genes and analysed their expression during fin development. The results suggest a scheme for the fin-limb transition in which the distal autopods (digits) are neomorphic structures produced by unequal proliferation of the posterior part of an ancestral appendix.

Concordant mitochondrial and nuclear DNA phylogenies for populations of the teleost fish Fundulus heteroclitus.

Molecular phylogenies using mitochondrial DNA and nuclear alleles of the lactate dehydrogenase B locus were found to be concordant for populations of Fundulus heteroclitus ranging from Canada to Florida. Both mitochondrial DNA and lactate dehydrogenase alleles show a clear separation between the northern individuals (from Nova Scotia and Maine) and the southern ones (from Georgia and Florida), with a mixed population found in the geographic intermediate (New Jersey). An historical isolation, possibly as ancient as 0.5-1 million years old, may have played a role in shaping the situation observed today.

Nucleotide sequence of the 18S ribosomal ribonucleic acid gene from two teleosts and two sharks and their molecular phylogeny.

The 18S rRNA sequence was determined for two teleostean fish species, Fundulus heteroclitus and Sebastolobus altivelis, and two sharks, Squalus acanthias and Echinorhinus cookei. To study the molecular phylogeny of these taxa, the sequences were compared with 18S rRNA sequences of the Coelacanth Latimeria chalumnae, the frog Xenopus laevis, and humans. Maximum parsimony analysis of the sequences resulted in a single most parsimonious tree that is in agreement with the expected phylogeny. The correct phylogenetic tree was also found when using S. altivelis alone as the teleost representative. In contrast, the most parsimonious tree found by using F. heteroclitus as the teleost representative presented anomalous groupings (the teleost branch being grouped with humans), matching results previously obtained. However, a bootstrap analysis showed that some branches containing anomalous relationships were not significantly supported. An explanation for this peculiarity, the differences between our tree and previously identified ones, and their phylogenetic implications are discussed.