Functional validation of ABHD12 mutations in the neurodegenerative disease PHARC.

ABHD12 mutations have been linked to neurodegenerative PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract), a rare, progressive, autosomal, recessive disease. Although ABHD12 is suspected to play a role in the lysophosphatidylserine and/or endocannabinoid pathways, its precise functional role(s) leading to PHARC disease had not previously been characterized. Cell and zebrafish models were designed to demonstrate the causal link between an identified new missense mutation p.T253R, characterized in ABHD12 from a young patient, the previously characterized p.T202I and p.R352* mutations, and the associated PHARC. Measuring ABHD12 monoacylglycerol lipase activity in transfected HEK293 cells demonstrated inhibition with mutated isoforms. Both the expression pattern of zebrafish abhd12 and the phenotype of specific antisense morpholino oligonucleotide gene knockdown morphants were consistent with human PHARC hallmarks. High abhd12 transcript levels were found in the optic tectum and tract, colocalized with myelin basic protein, and in the spinal cord. Morphants have myelination defects and concomitant functional deficits, characterized by progressive ataxia and motor skill impairment. A disruption of retina architecture and retinotectal projections was observed, together with an inhibition of lens clarification and a low number of mechanosensory hair cells in the inner ear and lateral line system. The severe phenotypes in abhd12 knockdown morphants were rescued by introducing wild-type human ABHD12 mRNA, but not by mutation-harboring mRNAs. Zebrafish may provide a suitable vertebrate model for ABHD12 insufficiency and the study of functional impairment and potential therapeutic rescue of this rare, neurodegenerative disease.

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity.

Triiodothyronine-induced changes in the zebrafish transcriptome during the eleutheroembryonic stage: implications for bisphenol A developmental toxicity.

Thyroid disruption during early development is a current matter of concern due to its significant human health implications. We present here a transcriptome analysis of thyroid hormone-regulated genes in zebrafish during the eleutheroembryonic stage (days 2-5 post fertilization) to detect potential markers of thyroid disruption. Exposure to 3,5,3'-triiodo-l-thyroxine (T3, 50 nM) induced changes in a minor portion (less than 2%) of the zebrafish transcriptome, with a significant fraction of genes involved in the haematopoietic system, eye formation, and ossification/skeletal system, including the thyroid receptor thra gene. Some of the transcriptomic changes were reflected macroscopically, as an allometric decrease of eye size and an increase on thra hybridization signal in the skeletal tissue. Using this information, changes on transcription of three genes (adult alpha globin gene si:ch211-5 k11.6, embryonic globin gene hbae3, and long wavelength cone opsin gene opn1/w1) were analyzed to monitor the effect of the suspected thyroid disrupter bisphenol A (BPA) on the thyroid system during this period of development of zebrafish. BPA acted as a weak T3 agonist when tested alone, but it strongly enhanced the effect of subsaturating concentrations of T3. In thyroxine immunofluorescence quantitative disruption tests (TIQDT), BPA did not prevent the ability of thyroid follicles to synthesize thyroxine, a landmark for direct goitrogens. Our results suggest that BPA potentiates the effect of endogenous T3 in early development and demonstrate the requirement for the use of in vivo, multi-endpoint methods to evaluate thyroid disruption hazards on early developmental processes in vertebrates.

Vitellogenin expression in white adipose tissue in female teleost fish.

In most oviparous animal species, oocyte growth occurs via the uptake of plasma egg yolk precursors, predominantly vitellogenins (Vtg). These glycolipoproteins are members of the large lipid transfer protein superfamily and key players in reproduction. While the vertebrate liver has been demonstrated to synthesize large amounts of Vtg, mostly under 17beta-estradiol control, the ability of other tissues to express significant amounts of Vtg has not been conclusively demonstrated. RT-PCR revealed vtg1 transcripts in female zebrafish and rainbow trout white adipose tissue (WAT). It was also found to coexpress mtp, known to perform the intracellular lipidation of Vtg prior to secretion. The liver and pancreas markers apobb2 and ins, or ela2, respectively, were not expressed in adipocytes. Whole-mount in situ hybridization and in situ RT-PCR tests of histological sections revealed vtg1 signal in adipocytes, whereas no signal was detected in infiltrated pancreatic islets. Transcript expression of vtg1 was induced in WAT of 17beta-estradiol-treated males, and the transcript and corresponding protein were detected in the thin rim of cytoplasm surrounding the adipocyte. Real-time quantitative RT-PCR showed that rainbow trout perivisceral WAT vtg1 transcript levels were high during early compared to late vitellogenesis. Taking normalized mRNA levels and tissue somatic index into account, vtg1 transcript levels at the beginning of oocyte yolk deposition were approximately 45 times lower in WAT than in liver, and these levels were not correlated to plasma Vtg and 17beta-estradiol concentrations. These findings suggest that WAT Vtg is implicated in providing components to the ovary during the early stages of vitellogenesis.

Clofibrate and gemfibrozil induce an embryonic malabsorption syndrome in zebrafish.

Nutrient availability is one of the major non-genetic factors determining embryonic growth and larval or fetal size. Due to the high human consumption of blood lipid regulators, fibrates have recently been reported as pollutants in rivers. Our study investigated the developmental toxicity of fibrates in zebrafish. Treatment with micromolar concentrations of clofibrate or gemfibrozil induced an embryonic malabsorption syndrome (EMS) with very little yolk consumption, resulting in small-sized larvae. This effect was reversible on removing the drug from the water. Clofibrate delayed hatching time and decreased the amount of oil red O lipid staining in the vasculature. It also induced higher density, round-shaped neuromuscular junctions associated with disorganization and less striation of muscular fibers, and pericardial edema, as well as impairing thyroid gland morphogenesis. acox1, apoa1 and mtp hybridization transcript signals were not affected in the yolk syncytial layer (YSL) after clofibrate exposure. Di-(2-ethylhexyl)-phthalate did not slow down yolk resorption, whereas brefeldin A induced EMS. These findings suggest that the inhibition of yolk sac resorption on exposure to fibrate is not at a pre-translational level or peroxisome proliferator-activated receptor alpha dependent and may be due to an inhibition of the YSL constitutive cell secretion. The effects of fibrates and the potential bioconcentration in eggs as well as the additive action of structurally related toxicants warrant an evaluation of the developmental impact of these compounds after long-term exposure at environmentally relevant concentrations. Fibrate-induced EMS in zebrafish seems useful for studying the morphogenetic consequences of impaired nutrient availability during the early stages of vertebrate development.

High transcript level of fatty acid-binding protein 11 but not of very low-density lipoprotein receptor is correlated to ovarian follicle atresia in a teleost fish (Solea senegalensis).

Transcripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly defined teleost fish FABP subfamily that is a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9. RT-PCR revealed high levels of vldlr transcript splicing variants in the ovaries and, to a lesser extent, in somatic tissues, whereas fabp11 was highly expressed in the ovaries, liver, and adipose tissue. In situ hybridization analysis showed vldlr and fabp11 mRNAs in previtellogenic oocytes, whereas no hybridization signals were detected in the larger vitellogenic oocytes. Transcript expression of fabp11 was strongly upregulated in somatic cells surrounding atretic follicles. Real-time quantitative RT-PCR demonstrated that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively. These results suggest that the expression level of vldlr transcripts may be used as a precocious functional marker to quantify the number of oocytes recruited for vitellogenesis and that fabp11 mRNA may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia in fish.

Conserved expression of alternative splicing variants of peroxisomal acyl-CoA oxidase 1 in vertebrates and developmental and nutritional regulation in fish.

The acyl-coenzyme A oxidase 1 (ACOX1) catalyzes the first, rate-limiting step in peroxisomal beta-oxidation of medium to very long straight-chain fatty acids. Zebrafish (Danio rerio) acox1 was characterized and compared with homologs from other sequenced genomes, revealing a remarkable conservation of structure in the vertebrate lineage. Strictly conserved regions of the deduced proteins included acyl-CoA oxidase and FAD binding domains, as well as a COOH-terminal peroxisomal targeting signal. Whole mount in situ hybridization showed that zebrafish acox1 transcripts were diffusely distributed in early-stage embryonic cells, then discreetly expressed in the brain and widely present in the liver and intestine at later stages. An evolutionarily conserved alternative splicing of the corresponding acox1 primary transcript was identified in teleosts and tetrapods including mammals, giving rise, after exon skipping, to two splice variants, ACOX1-3I and ACOX1-3II. Real-time quantitative RT-PCR on zebrafish adult tissues indicated high levels of both variants in the liver, anterior intestine, and to a lesser extent, in the brain. However, the ACOX1-3II transcript variant was expressed seven times more in zebrafish brain than the ACOX1-3I variant. These data suggest a tissue-specific modulation of ACOX1 activity by exchanging exon 3 duplicated isoforms containing amino acid sequences that are potentially implicated in fatty acyl chain specificity. In addition, a significant pretranslational up-regulation of zebrafish and rainbow trout (Oncorhynchus mykiss) acox1 expression was observed in the anterior intestine after feeding. Taken together, these data indicate that ACOX1 alternative splicing isoforms play a key conserved role in the vertebrate fatty acid metabolism.

Epidermal transient down-regulation of retinol-binding protein 4 and mirror expression of apolipoprotein Eb and estrogen receptor 2a during zebrafish fin and scale development.

Very little is known about the molecular control of skin patterning and scale morphogenesis in teleost fish. We have found radially symmetrical epidermal placodes with down-regulation of retinol-binding protein 4 (rbp4) expression during the initial paired fin and scale morphogenesis in zebrafish (Danio rerio). This finding may be related to changes in keratinocyte cytodifferentiation and/or the integument retinoid metabolism. rbp4 transcripts are expressed afterward in the central epidermis of the scale papilla and gradually extend to the epidermis, covering the growing scale, whereas no transcripts were detected in posterior margin epidermis. In contrast, induction of apolipoprotein Eb (apoeb) and up-regulation of estrogen receptor 2a (esr2a) transcripts were observed in the epidermis at initiator sites of zebrafish ectodermal/dermal appendage morphogenesis. This expression was maintained in the posterior margin epidermis of the formed scales. esr2a was also strongly expressed in neuromasts, whereas no rbp4 and apoeb transcripts were detected in these mechanosensory structures. The observed epidermal molecular events suggest that epidermis patterning is due to an activator-inhibitor mechanism operational at epidermal-dermal interaction sites. rbp4 transcript expression was also strongly down-regulated by 1-phenyl-2-thio-urea (PTU). As this inhibitor is commonly used to block obscuring pigmentation during in situ hybridization studies, this finding suggests that PTU should be used with caution, particularly in studying skin development.

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals.

BACKGROUND: The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. RESULTS: Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. CONCLUSION: This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development.

Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio).

Prion diseases are characterized by the accumulation of a pathogenic misfolded form of a prion protein (PrP) encoded by the Prnp gene in humans. In the present study in zebrafish, two transcripts and the corresponding genes encoding prion proteins, PrP1 and PrP2, related to human PrP have been characterized with a relatively divergent deduced amino acid sequence, but a well preserved overall organization of structural prion protein motifs. Whole-mount in situ hybridization analysis performed during embryonic and larval development showed a high level of PrP1 mRNA spatially restricted to the anterior floor-plate of the central nervous system and in ganglia. Transcripts of prp2 were detected in embryonic cells from the mid-blastula transition to the end of the segmentation period. From 24 h postfertilization up to larval stages, prp2 transcripts were localized in distinct anatomical structures, including a major expression in the brain, eye, kidney, lateral line neuromasts, liver, heart, pectoral fins and posterior intestine. The observed differential developmental expression patterns of the two long PrP forms, prp1 and prp2, and the short PrP form prp3, a more divergent prion-related gene previously identified in zebrafish, should contribute to understanding of the phylogenetic and functional relationships of duplicated prion gene forms in the fish genome. Together, the complex history of prion-related genes, reflected in the deduced structural features, conserved amino acid sequence and repeat motifs of the corresponding proteins, and the presence of differential developmental expression patterns suggest possible acquisition or loss of prion protein functions during vertebrate evolution.

Developmental expression and nutritional regulation of a zebrafish gene homologous to mammalian microsomal triglyceride transfer protein large subunit.

The microsomal triglyceride transfer protein (MTP) large subunit is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. We have found a zebrafish mtp homologous gene coding a protein with 54% identity with human MTP large subunit with the most conserved regions distributed in the corresponding predicted alpha-helical and C- and A-sheet domains. In situ hybridizations showed that zebrafish mtp transcripts were distributed in the yolk syncytial layer during early embryogenesis and in anterior intestine and liver from 48 hr postfertilization onward. Real-time quantitative RT-PCR confirmed the developmental regulation and tissue-specificity of mtp expression. A significant pretranslational up-regulation of mtp expression was observed in the anterior intestine after feeding. The nutritional regulation of zebrafish mtp expression observed in the anterior intestine supports the notion that this protein, similar to mammalian MTP large subunit, could be a factor implicated directly or indirectly in large lipid droplets accumulation observed in the fish enterocyte after feeding.

Expression patterns of three estrogen receptor genes during zebrafish (Danio rerio) development: evidence for high expression in neuromasts.

The estrogen receptor (ER) genes encode a group of nuclear enhancer proteins, which are important ligand-activated transcription factors, modulating estrogen-target gene transcription. In this study we analyzed expression patterns of three zebrafish ER genes, esr1, esr2a, and esr2b, during development using whole-mount in situ hybridization. High levels of esr2a and esr2b of maternal origin are inherited and segregated to the blastomers. After the mid-blastula transition, the three genes exhibit similar spatio-temporal patterns of expression. In 24 h postfertilization (hpf) embryos, high levels of esr2a and esr2b and low levels of esr1 mRNAs are detected in the epidermis, pectoral fin buds, hatching gland and, to a lesser extent, developing brain. From 24 hpf onward, the expression of the three genes is down-regulated in the epidermis. By 60 hpf, esr2a mRNA is abundant in mature primary neuromasts of the anterior line system and by 3 days postfertilization (dpf), all mature primary neuromasts in both the anterior and posterior lateral line systems express significant levels of esr2a and esr2b transcripts. Histological sections show a high level of esr2a transcripts in both mechanoreceptive hair cells and supporting cells. The transcripts are still detected after neomycin-induced hair cell death, consistent with the presence of esr2a transcripts in supporting cells. From 6 dpf onward, esr2a and esr2b transcripts are robustly co-expressed in primary neuromasts, branchial arches, pectoral fins, and anal papilla, while slight labeling is observed for esr1 transcripts.