Molecular characterization and expression pattern of zona pellucida proteins in gilthead seabream (Sparus aurata)

The developing oocyte is surrounded by an acellular envelope that is composed of 2–4 isoforms of zona pellucida (ZP) proteins. The ZP proteins comprise the ZP1, ZP2, ZP3, and ZPX isoforms. While ZP1 (ZPB) and ZP3 (ZPC) are present in all species, ZP2 (ZPA) is not found in teleost fish and ZPX is not found in mammals. In the present study, we identify and characterize the ZP1, ZP3 and ZPX isoforms of gilthead seabream. Furthermore, by analyzing the conserved domains, which include the external hydrophobic patch and the internal hydrophobic patch, we show that ZP2 and ZPX are closely related isoforms. ZP proteins are synthesized in either the liver or ovary of most teleosts. Only in rainbow trout has it been shown that zp3 has dual transcription sites. In gilthead seabream, all four mRNA isoforms are transcribed in both the liver and ovary, with zp1a, zp1b, and zp3 being highly expressed in the liver, and zpx being primarily expressed in the ovary. However, determination of the ZP proteins in plasma showed high levels of ZP1b, ZP3, and ZPX, with low or non-detectable levels of ZP1a. In similarity to other teleost ZPs, the hepatic transcription of all four ZP isoforms is under estrogenic control. Previously, we have shown that cortisol can potentiate estrogen-induced ZP synthesis in salmonids, and now we show that this is not the case in the gilthead seabream. The present study shows for the first time the endocrine regulation of a teleost ZPX isoform, and demonstrates the dual-organ transcriptional activities of all the ZP proteins in one species

Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal

Prdm1- and Sox6-mediated transcriptional repression specifies muscle fibre type in the zebrafish embryo

The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow-twitch muscle fibres that derive from adaxial cells. Here, we show that Prdm1 functions by acting as a transcriptional repressor and that slow-twitch-specific muscle gene expression is activated by Prdm1-mediated repression of the transcriptional repressor Sox6. Genes encoding fast-specific isoforms of sarcomeric proteins are ectopically expressed in the adaxial cells of ubotp39 mutant embryos. By using chromatin immunoprecipitation, we show that these are direct targets of Prdm1. Thus, Prdm1 promotes slow-twitch fibre differentiation by acting as a global repressor of fast-fibre-specific genes, as well as by abrogating the repression of slow-fibre-specific genes

A Balance of BMP and Notch Activity Regulates Neurogenesis and Olfactory Nerve Formation

Although the function of the adult olfactory system has been thoroughly studied, the molecular mechanisms regulating the initial formation of the olfactory nerve, the first cranial nerve, remain poorly defined. Here, we provide evidence that both modulated Notch and bone morphogenetic protein (BMP) signaling affect the generation of neurons in the olfactory epithelium and reduce the number of migratory neurons, so called epithelioid cells. We show that this reduction of epithelial and migratory neurons is followed by a subsequent failure or complete absence of olfactory nerve formation. These data provide new insights into the early generation of neurons in the olfactory epithelium and the initial formation of the olfactory nerve tract. Our results present a novel mechanism in which BMP signals negatively affect Notch activity in a dominant manner in the olfactory epithelium, thereby regulating neurogenesis and explain why a balance of BMP and Notch activity is critical for the generation of neurons and proper development of the olfactory nerve

Developmental and reproductive regulation of NR5A genes in teleosts

In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The fushi tarazu factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation

Zebrafish sex determination and differentiation: Involvement of FTZ-F1 genes

Abstract Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.</p

The zebrafish HGF receptor met controls migration of myogenic progenitor cells in appendicular development

The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. Cmet has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates

Pax7 is required for establishment of the xanthophore lineage in zebrafish embryos

The pigment pattern of many animal species is a result of the arrangement of different types of pigment-producing chromatophores. The zebrafish has three different types of chromatophores: black melanophores, yellow xanthophores, and shimmering iridophores arranged in a characteristic pattern of golden and blue horizontal stripes. In the zebrafish embryo, chromatophores derive from the neural crest cells. Using pax7a and pax7b zebrafish mutants, we identified a previously unknown requirement for Pax7 in xanthophore lineage formation. The absence of Pax7 results in a severe reduction of xanthophore precursor cells and a complete depletion of differentiated xanthophores in embryos as well as in adult zebrafish. In contrast, the melanophore lineage is increased in pax7a/pax7b double-mutant embryos and larvae, whereas juvenile and adult pax7a/pax7b double-mutant zebrafish display a severe decrease in melanophores and a pigment pattern disorganization indicative of a xanthophore-deficient phenotype. In summary, we propose a novel role for Pax7 in the early specification of chromatophore precursor cells

Genetic compensation between Pax3 and Pax7 in zebrafish appendicular muscle formation

Background: Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3). Results: In this study, we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a, and pax7b, we determined that there is a feedback system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins. Conclusions: We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the premigratory progenitor cells