Melanopsin and Clock Genes: Regulation by Light and Endothelin in the Zebrafish ZEM-2S Cell Line

It is well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In the teleost, Danio rerio, peripheral oscillators can be directly synchronized by light. Danio rerio ZEM-2S embryonic cells respond to light with differential growth: cells kept in constant light exhibited a strong inhibition of proliferation, whereas in cells kept in light:dark (LD) cycles (14L:10D and 10L:14D) or in constant darkness (DD), the doubling times were not statistically different. We demonstrated by RT-PCR followed by PCR that ZEM-2S cells express two melanopsins, Opn4x and Opn4m, and the six Cry genes. The presence of the protein OPN4x was demonstrated by immunocytochemistry. The pattern of temporal expression of the genes Opn4x, Per1, Cry1b, and Clock was studied in ZEM-2S cells kept for five days in 12L:12D or DD. In 12L:12D, the clock genes Per 1 and Cry1b exhibited robust circadian expression, while Opn4x and Clock expression seemed to vary in an ultradian pattern. Both Per1 and Cry1b genes had higher expression during the L phase; Clock gene had an increase in expression coincident with the D phase, and during the subjective night. In DD, the temporal variation of Per1 and Cry1b genes was greatly attenuated but not extinguished, and the higher expressions were shifted to the transition times between subjective day and night, demonstrating that Per and Cry1b were synchronized by the LD cycle. Clock and Opn4x kept the ultradian oscillation, but the rhythm was not statistically significant. As endothelins (ET) have been reported to be a potent stimulator of Per genes in rodents, we investigated the effect of endothelin on ZEM-2S cells, which express ETA receptors. Cells were kept in 12D:12L for five days, and then treated with 10-11 to 10-8M ET-1 for 24h. ET-1 exhibited a biphasic effect on Opn4x expression. At 10-11M, the hormone exerted a highly significant stimulation of Opn4x expression during the L phase and introduced a circadian oscillatory pattern. At 10-10M, a significant increase was seen at ZT21 and ZT0 (i.e., at the end of the D phase and beginning of the L phase), whereas 10-9 and 10-8M ET-1 inhibited the expression of Opn4x at most ZTs. Clock expression was unaffected by 10-8M ET-1; however, in the presence of lower concentrations, the expression was enhanced at some ZTs, strengthening the ultradian oscillation. ET-1 at 10-11 and 10-10M had no effect on Per1 circadian expression; however, 10-9 and 10-8M ET-1 reduced the amplitude of Per1 expression in the beginning of the L phase. ET-1 effects were less evident on Cry 1b. For both genes, the reduction in expression was not sufficient to abolish the circadian oscillatory pattern. Based on these results and data in the literature, a link between ET-1 stimulation of ETA receptors may be established by E4BP4 binding to the promoters and consequent inhibition of gene expression.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L. are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Speed 1 1 is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tiete River Basin (Sao Paulo, Brazil)

The Tiete River and its tributary Pinheiros River receive a highly complex organic and inorganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and teratogenic effects of sediments from selected locations in the Tiete River Basin by means of the sediment contact embryo toxicity assay with Danio rerio, in order to provide a comprehensive and realistic insight into the bioavailable hazard potential of these sediment samples. Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in the samples not only in the vicinity of the megacity Sao Paulo (Billings reservoir and Pinheiros River samples), but also downstream (in the reservoirs Barra Bonita, Promissao and Tres Irmaos). Results confirm that most toxicity is due to the discharges of the metropolitan area of Sao Paulo. However, they also indicate additional sources of pollutants along the river course, probably from industrial, agricultural and agroindustrial residues, which contribute to the degradation of each area. The sediment contact fish embryo test showed to be powerful tool to detect embryo toxicity in sediments, not only by being a sensitive method, but also for taking into account bioavailability. This test provides an ecological highly realistic and relevant exposure scenario, and should therefore be added in ecotoxicological sediment quality assessments. (C) 2011 Elsevier Inc. All rights reserved.

Gene Mapping of 18S and 5S rDNA Genes in the Karyotype of the Three-Spot Gourami Trichogaster trichopterus (Perciformes, Osphronemidae)

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

WNT5A Mutations in Patients With Autosomal Dominant Robinow Syndrome

Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia, and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown; however, the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor. Here, we show that two different missense mutations in WNT5A, which result in amino acid substitutions of highly conserved cysteines, are associated with autosomal dominant Robinow syndrome. One mutation has been found in all living affected members of the original family described by Meinhard Robinow and another in a second unrelated patient. These missense mutations result in decreased WNT5A activity in functional assays of zebrafish and Xenopus development. This work suggests that a WNT5A/ROR2 signal transduction pathway is important in human craniofacial and skeletal development and that proper formation and growth of these structures is sensitive to variations in WNT5A function. Developmental Dynamics 239:327-337, 2010. (C) 2009 Wiley-Liss, Inc.