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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification and purification of a self-associating copper-binding protein from mouse hepatic cytosols.

The proteins which bind copper when it first enters cells and deliver that copper to the cellular sites where copper is incorporated into copper enzymes are unknown. When radiolabeled (64Cu(II)) cytosol proteins were fractionated on Superose columns, two large copper-binding protein fractions were detected which together bound 63% of the total 64Cu recovered from the column. A self-associating, 50-kDa copper-binding protein was identified whose tetramer and dimer appeared to elute in one of the major copper-binding fractions and the monomer eluted in the other. This protein was purified to homogeneity by successive Mono Q, chelating Superose, and phenyl-Superose columns. The concentrated, purified protein showed high amounts of tetramer and monomer plus some dimer, each of which bound copper. Rechromatography of the tetramer-dimer fraction on Superose gave rise to approximately equal amounts of tetramer, dimer, and monomer. Similarly, rechromatography of the monomer fraction gave rise to dimer and tetramer. Thus, the results with the purified protein confirmed that the 50-kDa protein exists as a tetramer-dimer-monomer equilibrium. The 50-kDa protein apparently makes a major contribution to the total copper-binding activity of the mouse hepatic cytosol and may play a significant role in intracellular copper metabolism.

Purification and properties of a self-associating, 50-kDa copper-binding protein from brindled mouse livers.

The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal 64Cu binding was detected in both the fraction which contained the tetramer plus dimer (approximately 26% less) and the fraction containing the monomer of CuBP (approximately 37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at approximately 20 min after starting the final water elution step, the brindled mouse protein eluted by approximately 5 min. This seemed to be due to abnormal self-association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consistent with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease.

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.

Identification of prosaposin as a neurotrophic factor.

Prosaposin was identified as a neurotrophic factor stimulating neurite outgrowth in murine neuroblastoma (NS20Y) cells and choline acetyltransferase (ChAT) activity in human neuroblastoma (SK-N-MC) cells. The four naturally occurring saposins, which are derived by proteolytic processing of prosaposin, were tested for activity. Saposin C was found to be active, whereas saposins A, B, and D were inactive as neurotrophic factors. Dose-response curves demonstrated that nanomolar concentrations of prosaposin and saposin C stimulated neurite outgrowth and increased ChAT activity. Prosaposin and saposin C exerted activity by a mechanism independent of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3. Binding assays utilizing saposin C as a ligand gave two saturable binding constants, a high-affinity (Kd = 19 pM) and a low-affinity (Kd = 1 nM) constant, with 2000 and 15,000 sites per NS20Y cell, respectively. Phosphorylation stimulation experiments demonstrated that brief treatment with prosaposin or saposin C enhanced phosphorylation of a variety of proteins, some of which contained phosphorylated tyrosine(s). Since both cell lines were also stimulated by ciliary neurotrophic factor (CNTF) as well as prosaposin, inhibition was tested by utilizing an anti-gp130 monoclonal antibody, which specifically inhibited CNTF stimulation; this antibody did not inhibit prosaposin or saposin C stimulation. These results indicate that prosaposin and saposin C are neurotrophic factors which initiate signal transduction by binding to a high-affinity receptor that induces protein phosphorylation.

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.

Six additional mutations in fucosidosis: three nonsense mutations and three frameshift mutations.

The rare lysosomal storage disease, fucosidosis results from an almost complete deficiency of alpha-L-fucosidase (EC 3.2.1.51). We have identified six new potential disease causing mutations detected by PCR amplification and sequencing of all 8 exons of the alpha-L-fucosidase gene FUCA1. (1) A C to T mutation (Q77X) in exon 1 of two Jewish-Italian siblings. This mutation was present in one allele and was found also in the mother who was of Italian origin. (2) A C to A mutation (W382X) in exon 6 in an Italian patient. This mutation was found in one allele and obliterates a unique Hphl site. (3) A C to A mutation (Y211X) in exon 3 in a Belgian patient. This mutation obliterates a unique Rsal site and was present in both alleles. (4) A homozygous single base (C) deletion in exon 2 in an Italian patient. This deletion results in a frameshift mutation (P141fs) and obliterates a unique Eael site. (5) A homozygous single base (C) deletion in exon 5 in a Portuguese patient, which also results in a frameshift mutation (S265fs). (6) A single base (A) deletion in exon 3 in a Canadian-Indian patient, which also results in a frameshift mutation (S216fs). The S216fs mutation was found in only one allele; the mutation in the other allele is not yet known.

Fucosidosis: four new mutations and a new polymorphism.

Fucosidosis is a rare lysosomal storage disease due to a nearly complete deficiency of alpha-L-fucosidase (EC 3.2.1.51). In this study, all 8 exons of the alpha-L-fucosidase structural gene (FUCA-1) were amplified by PCR methods, and the amplified products were subcloned and sequenced. Five patient groups with fucosidosis were selected according to their ethnic backgrounds and haplotypes for RFLPs in FUCA-1. Four presumptive disease causing mutations were detected: 1) A major deletion of DNA containing the last two exons of FUCA-1 in two Algerian siblings. 2) A G to T mutation in exon 6 resulting in an in-frame termination codon (E375X) in eight Hispanic patients from Colorado and New Mexico. 3) A G to A mutation (G60D) in exon 1 in four Italian patients and in three related French-American (Cajun) patients. This G60D mutation creates a unique site for AflIII. 4) A frameshift mutation resulted from a two-base deletion in exon 2 (K151fs) in an Italian patient. This deletion obliterates a unique BstXI site and creates a new BpmI site, and was found in only this patient and in only one allele. The rationale for proposing these defects as disease causing mutations includes pedigree analysis and the predicted consequences of each defect upon the activity and the concentration of the enzyme. An A to G transition (Q281R) in exon 5 was found to be present in homozygous form in affected patients and also in normal subjects; it appears to be a newly identified polymorphism. It causes a charge change and may be responsible for the electrophoretic variant phenotype of fucosidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.