RNA interference: mechanisms and applications.

RNA interference (RNAi) is a phenomenon induced by double-stranded RNA (dsRNA) in which gene expression is inhibited through specific degradation of mRNA. The mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets. This process is related to normal defence against viruses and mobilisation of transposons. Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. RNAi has also been demonstrated in several vertebrate species but with lower efficiency. Development of procedures for in vivo production of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.

Topography of different photoreceptor cell types in the larval retina of Atlantic halibut (Hippoglossus hippoglossus).

The identities of single cone cells in the retina of Atlantic halibut (Hippoglossus hippoglossus) larvae were studied by in situ hybridisation using RNA probes for the five different halibut opsins. Four different cone opsins (ultraviolet-, blue-, green- and red-sensitive) are expressed in Atlantic halibut at the end of the yolk-sac period, whereas rod opsin is expressed later in development. Photoreceptor cells expressing ultraviolet-sensitive opsin are found only in the ventral retina, presumably to optimise detection of the downwelling ultraviolet light. The majority of the photoreceptors (approximately 90%) in the retina express green-sensitive opsin and its distribution shows no regional differences. In contrast, blue- and red-sensitive opsins are expressed much less frequently (in approximately 10% of photoreceptors), although these two opsins are also found over the entire retina. The expression patterns of the different visual pigments indicate some form of mosaic expression in the single-coned larval retina, and this is reminiscent of the square mosaic expression found in post-metamorphic Atlantic halibut. These findings suggest plasticity in green-opsin-expressing cells during development, resulting in a square mosaic expression pattern.

Molecular cloning and embryonic expression of Xenopus Six homeobox genes.

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.

Molecular cloning and characterization of five opsin genes from the marine flatfish Atlantic halibut (Hippoglossus hippoglossus).

Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, M(Cone) and M(Rod); and short-wavelength sensitive, S(Blue) and S(Ultraviolet), respectively. In situ hybridization analysis reveals expression in double cone (L and M(Cone)), single cone (S(Blue) and S(Ultraviolet)), and rod (M(Rod)) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

Identification and distribution of nitric oxide synthase in the brain of adult zebrafish.

Nitric oxide (NO) is proposed to be involved in developmental and plastic processes. We investigated the presence and distribution of nitric oxide synthase (NOS) in the zebrafish (Danio rerio) using molecular and histochemical techniques. A partial gene sequence corresponding to the neuronal NOS isoform (nNOS) was identified, and in situ hybridization revealed cellular nNOS mRNA expression throughout the brain of adult zebrafish, distributed in distinct central nuclei and in proliferation zones. NOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase activity partly coincided with the nNOS mRNA expression, however was present also in additional neuronal and non-neuronal cell types. The results indicate the occurrence of different NOS isoforms in the adult brain, of which nNOS may participate in neurotransmission, and in mechanisms related to the continuous growth and neuronal plasticity of the teleost brain.

Characterisation of the promoter region of the zebrafish six7 gene.

The Drosophila homeobox gene sine oculis and its murine homologue Six3 have both been shown to have regulatory functions in eye and brain development. In zebrafish, three Six3-related genes with conserved expression during early eye and head formation have been identified. One of these, six7, is first expressed at the gastrula stage in the involuting axial mesoderm, and later in the overlying neuroectoderm from which the forebrain and optic primordium develop. To elucidate the mechanisms regulating six7 expression, we isolated a 2.7-kb fragment of the 5'-flanking region. Three sequentially deleted fragments of this upstream region were used to produce GFP reporter constructs for analysis of tissue-specific expression in zebrafish embryos. The results show that a 625-bp upstream fragment is sufficient to direct strong expression of the reporter during gastrulation and early neurulation. The proximal part of the promoter contains binding sites for various constitutive transcription factors and an additional upstream element that was shown to be critical in directing expression to the anterior region of the zebrafish brain.

Six class homeobox genes in drosophila belong to three distinct families and are involved in head development.

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

Spectrum of mutations in fucosidosis.

Fucosidosis is a lysosomal storage disorder characterised by progressive psychomotor deterioration, angiokeratoma and growth retardation. It is due to deficient alpha-l-fucosidase activity leading to accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Fucosidosis is extremely rare with less than 100 patients reported worldwide, although the disease occurs at a higher rate in Italy, in the Hispanic-American population of New Mexico and Colorado, and in Cuba. We present here a review study of the mutational spectrum of fucosidosis. Exon by exon mutation analysis of FUCA1, the structural gene of alpha-l-fucosidase, has identified the mutation(s) in nearly all fucosidosis patients investigated. The spectrum of the 22 mutations detected to date includes four missense mutations, 17 nonsense mutations consisting of seven stop codon mutations, six small deletions, two large deletions, one duplication, one small insertion and one splice site mutation. All these mutations lead to nearly absent enzymatic activity and severely reduced cross-reacting immunomaterial. The observed clinical variability is, therefore, not due to the nature of the fucosidosis mutation, but to secondary unknown factors.

Three structurally and functionally conserved Hlx genes in zebrafish.

For the Hlx class, which includes homeodomains (HD) that are similar to Drosophila H2.0, few members have been identified in vertebrates. In this report, we describe three zebrafish genes, hlx1, hlx2 and hlx3, related to the murine Dbx genes. The proteins encoded by hlx1 and hlx2 have about the same sequence identity to Dbx1 (approximately 60%), suggesting that they derive from a duplication in the fish lineage. This is supported by similarities in the embryonic expression patterns and promoter sequence conservation. The zebrafish Hlx3 protein is related to murine Dbx2, but it is apparently too diverged to be orthologous. Our phylogenetic analysis of all the known HD sequences of the Hlx class also shows that it can be divided into at least two distinct families. All the Dbx-like genes have similar expression in the embryonic nervous system. However, the initial expression patterns of the zebrafish hlx genes are quite unique, suggesting that some functional divergence has occurred between fish and mammals.

A zebrafish Six4 homologue with early expression in head mesoderm.

Similar to the Drosophila homeobox gene sine oculis, several of the vertebrate homologues (Six genes) are expressed during eye formation and differentiation. In addition, most of these vertebrate genes show expression in mesodermal derivatives in adults and/or earlier stages of development. We have identified a zebrafish (Danio rerio) gene, six8, which shows the greatest similarity to murine Six4. The deduced proteins of these two genes have an overall sequence identity of 41%, while the homeodomains and Six domains are highly conserved, 90% and 81%, respectively. The spatiotemporal expression pattern of six8 was analyzed by RT-PCR and in situ hybridization. Transcripts were detected in a wide range of embryonic stages and in adults. Notably, the strongest expression was observed in head mesoderm of late gastrula and early neurula stages.

Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation.

Both the Drosophila homeobox gene sine oculis and its murine homologue Six3 have regulatory functions in eye development. In zebrafish, in addition to two previously reported homologues of murine Six3, we have identified a related gene (six7). Although the deduced Six7 protein shares less than 68% sequence identity with the other known zebrafish Six3-like proteins, the embryonic expression patterns have highly conserved features. The six7 transcripts are first detected in involuting axial mesendoderm and, subsequently, in the overlying neurectoderm from which the forebrain and optic primordia develop. Similar to the two other zebrafish Six3 homologues, the expression boundaries of six7 correspond quite closely with the edges of the optic vesicles. Hence, the partially overlapping expression domains of these three six genes probably contribute to anteroposterior specification and in defining the eye primordia.

Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.

The murine homeobox gene Six3 and its Drosophila homologue sine oculis both have regulatory functions in eye development. We report the isolation and characterization of two zebrafish genes, six3 and six6, that are closely related to the murine Six3 gene. Zebrafish six3 may be the structural orthologue, while the six6 gene is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and, subsequently, in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain as reported previously for murine Six3.

Cloning, expression and map assignment of chicken prosaposin.

Prosaposin is the precursor of four small glycoproteins, saposins A-D, that activate lysosomal sphingolipid hydrolysis. A full-length cDNA encoding prosaposin from chicken brain was isolated by PCR. The deduced amino acid sequence predicted that, similarly to human and other mammalian species studied, chicken prosaposin contains 518 residues, including four domains that correspond to saposins A-D. There was 59% identity and 76% similarity of human and chicken prosaposin amino acid sequences. The basic three-dimensional structures of these saposins is predicted to be similar on the basis of the conservation of six cysteine residues and an N-glycosylation site. Identity of amino acid sequences was higher among saposins A, B and D than in saposin C. The predicted amino acid sequence of saposin B matched exactly that of purified chicken saposin B protein. The chicken prosaposin gene was mapped to a single locus, PSAP, in chicken linkage group E11C10 and is closely linked to the ACTA2 locus. This confirms the homology between chicken and human prosaposins and defines a new conserved segment with human chromosome 10q21-q24.

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain.

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.

Fucosidosis with dystonia.

Fucosidosis, a progressive neurodegenerative disease, evident in early childhood, is associated with progressive loss of mental and motor function and increasing spasticity and hyperreflexia. We report a Canadian male, with clinical features similar to previously reported fucosidosis patients, however, since age 5 he has exhibited progressive dystonic posturing, initially unilateral, but recently involving both lower limbs. Extensive study of his cultured lymphoblasts demonstrated that alpha-fucosidase activity and immunoreactive alpha-fucosidase protein were absent. He is homozygous for the Q422X mutation, a C to T transition within exon 8 of the alpha-fucosidase gene which results in loss of an EcoR1 restriction enzyme cut site. Even among the 4 other reported fucosidosis families having one or more individuals homozygous for this same (Q422X) mutation there was no previous report of dystonia.

Identification of the neurotrophic factor sequence of prosaposin.

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.