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.

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.

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.

Tilapia (Oreochromis niloticus) vitellogenins: development of homologous and heterologous ELISAs and analysis of vitellogenin pathway through the ovarian follicle.

Vitellogenin (VTG) of Oreochromis niloticus was again purified, due to the conflicting results found in the literature. Three purification processes have been used: electrophoresis and electro-elution, double chromatography (gel filtration and ion-exchange chromatography) and single ion-exchange chromatography. Using SDS-PAGE we confirmed in all cases the presence of two polypeptidic forms of plasma VTG of 130 kDa (VTG1) and 170 kDa (VTG2). We raised polyclonal antibodies against each VTG form and we demonstrated the complete cross-reactivity of each antibody with both forms of VTG by Enzyme Immuno-Assay (EIA) and Western blots. The homologous ELISAs developed exhibited a detection limit of 6 ng x ml(-1), equivalent to 60 ng x ml(-1) of plasma VTG and allowed us to quantify the total plasma VTG of O. niloticus with high specificity and sensitivity. Using photonic and electron immunomicroscopy, we followed the pathway of VTG into the ovarian follicle (OF) demonstrating that VTG enters the oocyte at stage 3 of OF development, at the same time as cortical alveoli and lipid globules appear. Heterologous ELISAs performed on other cichlid species allowed us to quantify plasma VTG in Oreochromis aureus and Sarotherodon melanotheron and to detect it in Hemichromis fasciatus, Hemichromis bimaculatus and Tilapia zillii, constituting a reliable tool for monitoring the presence of xeno-estrogens in the environment of these fish species.