Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.     

CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.Subject terms: RNA-binding proteins, Infertility     

Comparative analysis of duplicated sox21 genes in zebrafish

Sox21 is thought to function as a counteracting partner of SoxB1 (Sox1, 2, 3) genes and is involved in cell fate determination. In this study, we comparatively analyzed the expression patterns and conserved cis-regulatory elements of the duplicated sox21 genes in zebrafish. In embryogenesis, sox21b is predominantly expressed in the telencephalon, hypothalamus, mesencephalon and lens, and sox21a is solely expressed in the midbrain-hindbrain boundary, olfactory placode and lateral line, while both genes are expressed in the hindbrain, spinal cord and ear. In adult, sox21a is expressed in the brain, skin, ovary and intestine, while sox21b is expressed in the brain and testis. Interestingly, all 16 pan-vertebrate conserved non-coding elements (CNEs) are asymmetrically preserved in the sox21b locus, whereas two fish-specific elements are kept in the sox21a locus, and this is correlated with increased evolutionary rate of the sox21a protein sequence. Transient transgenic reporter analysis revealed that six sox21b CNEs and two sox21a CNEs drove green fluorescent protein (GFP) expression in tissues correlated with the partitioning of expression in two orthologues. These results indicate that sox21a and sox21b have reciprocally lost expression domains of the ancestral gene reflected by degeneration of certain CNEs in their genomic loci and provide clear evidence for evolution of the duplicated sox21 genes by subfunctionalization. In addition, our data suggest that some CNEs-based regulatory pathways have been predominantly preserved in the sox21b locus.     

Heart-targeted overexpression of Nip3a in zebrafish embryos causes abnormal heart development and cardiac dysfunction

We transiently expressed a proapoptotic protein, Nip3a, by a heart-specific BMP4 promoter in zebrafish embryos and generated two variants of embryos with abnormal heart phenotypes (A and B). Embryos with phenotype A heart defects showed hypoplastic or elongated ventricles, elongated or enlarged atriums with no normal cardiac looping resulting a significant longer SV-BA distance, and bradycardia at 48 h post-fertilization (hpf). Embryos with phenotype B heart defects showed an enlarged fluid-filled pericardium, severe hypoplasia, non-contracting ventricles, and elongated or enlarged slowly beating atriums with no normal looping. Histological sections further revealed the absence of a proper atrioventricular boundary and no endocardial cells lining this region in both 48- and 72-hpf Nip3a-overexpressing embryos, implicating defective endocardial cushion formation. These phenotypes are reminiscent of atrioventricular canal defects in humans. In addition, induced apoptotic myocardium cells were clustered in the presumptive atrioventricular boundary as well as in the adjacent ventricle and atrium of 48- and 72-hpf Nip3a-overexpressing embryos. Nip3a expression was readily detected in 80% epiboly BMP4-Nip3a-injected embryos, and defects in heart development were observed in both the linear heart tube and subsequent chamber formation stages. These results showed that myocyte apoptosis is a universal pathogenic factor for congenital heart failure using zebrafish as a model organism.     

Potential role of bone morphogenetic protein-15 in zebrafish follicle development and oocyte maturation

Bone morphogenetic protein (BMP)-15 is a member of the transforming growth factor beta (TGF-beta) superfamily and is closely related to growth and differentiation factor (GDF)-9, both structurally and functionally. In mammals, BMP-15 is predominantly produced by oocytes and exerts important regulatory functions within the ovary, such as promoting early folliculogenesis, preventing premature luteinization and enhancing cumulus cell expansion. The role of BMP-15 in mammalian ovary differs between monoovulatory and polyovulatory species. Recent studies in zebrafish have provided initial evidence that BMP-15 is also an important regulator of ovarian functions. BMP-15 is produced by the zebrafish ovary throughout follicle development and maturation. In vitro studies using zebrafish follicles have revealed that incubation with recombinant human BMP-15 or over-expression of BMP-15 in oocytes results in an inhibition of gonadotropin- and maturation inducing hormone (MIH)-induced oocyte maturation. Conversely, immnunoneutralization with BMP-15 antiserum or silencing of BMP-15 expression using morpholino antisense oligonueclotides enhances oocyte maturation. A key step in BMP-15 action is the sensitivity of follicles to MIH. In vivo injection of BMP-15 antiserum causes a significant decrease in maturation-incompetent (insensitive to MIH) small early growth phase follicles and a concomitant increase in mature follicles. These findings support a role in BMP-15 in preventing precocious oocyte maturation in zebrafish. We propose that the suppression of premature oocyte maturation by BMP-15 may be important to maintain oocyte quality and subsequent ovulation and fertilization.     

A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a; however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.     

sox30: a novel zebrafish sox gene expressed in a restricted manner at the midbrain-hindbrain boundary during neurogenesis

 The Sox family of proteins is thought to act to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of sox30, a novel sox gene from the zebrafish (Danio rerio). In situ hybridization shows that sox30 is expressed in a restricted manner at the boundary between the midbrain and hindbrain during nervous system development. This expression pattern is in direct contrast to that of most other neuronally expressed Sox genes which are expressed throughout the nervous system. Received: 30 October 1998 / Accepted: 1 February 1999     

Ovarian follicle development requires Smad3

Smad3 is an important mediator of the TGF beta signaling pathway. Interestingly, Smad3-deficient (Smad3-/-) mice have reduced fertility compared with wild-type (WT) mice. To better understand the molecular mechanisms underlying the reduced fertility in Smad3-/- animals, this work tested the hypothesis that Smad3 deficiency interferes with three critical aspects of folliculogenesis: growth, atresia, and differentiation. Growth was assessed by comparing the size of follicles, expression of proliferating cell nuclear antigen, and expression of cell cycle genes in Smad3-/- and WT mice. Atresia was assessed by comparing the incidence of atresia and expression of bcl-2 genes involved in cell death and cell survival in Smad3-/- and WT mice. Differentiation was assessed by comparing the expression of FSH receptor (FSHR), estrogen receptor (ER) alpha, ER beta, and inhibin alpha-, beta(A)-, and beta(B)-subunits in Smad3-/- and WT mice. Because growth, atresia, and differentiation are regulated by hormones, estradiol, FSH, and LH levels were compared in Smad3-/- and WT mice. Moreover, because alterations in folliculogenesis can affect the ability of mice to ovulate, the number of corpora lutea and ovulated eggs in response to gonadotropin treatments were compared in Smad3-/- and WT animals. The results indicate that Smad3 deficiency slows follicle growth, which is characterized by small follicle diameters, low levels of proliferating cell nuclear antigen, and low expression of cell cycle genes (cyclin-dependent kinase 4 and cyclin D2). Smad3 deficiency also causes atretic follicles, degenerated oocytes, and low expression of bcl-2. Furthermore, Smad3 deficiency affects follicular differentiation as evidenced by decreased expression of ER beta, increased expression of ER alpha, and decreased expression of inhibin alpha-subunits. Smad3 deficiency causes low estradiol and high FSH levels. Finally, Smad3-/- ovaries have no corpora lutea, and they do not ovulate after ovulatory induction with exogenous gonadotropins. Collectively, these data provide the first evidence that reduced fertility in Smad3-/- mice is due to impaired folliculogenesis, associated with altered expression of genes that control cell cycle progression, cell survival, and cell differentiation. The findings that Smad3-/- follicles have impaired growth, increased atresia, and altered differentiation in the presence of high FSH levels, normal expression of FSHR, and lower expression of cyclin D2, suggest a possible interaction between Smad3 and FSH signaling downstream of FSHR in the mouse ovary.     

Targeted knockout of duox causes defects in zebrafish growth,thyroid development,and social interaction

<正>Reactive oxygen species(ROS) are believed to be inevitable and toxic by-products produced during cellular respiration. However,several lines of evidence show that diverse extracellular signals can activate ROS production and provide important function for various biological processes, as our recent findings in innate     

Monogenic causes of X-linked mental retardation

Mutations in X-linked genes are likely to account for the observation that more males than females are affected by mental retardation. Causative mutations have recently been identified in both syndromic X-linked mental retardation (XLMR) and in the genetically heterogeneous 'nonspecific' forms of XLMR, for which cognitive impairment is the only defining clinical feature. Proteins that function in chromatin remodelling are affected in three important syndromic forms of XLMR. In nonspecific forms of the disorder, defects have been found in signal-transduction pathways that are believed to function during neuronal maturation. These findings provide important insights into the molecular and cellular defects that underlie mental retardation.     

A role for foxd3 and sox10 in the differentiation of gonadotropin-releasing hormone (GnRH) cells in the zebrafish Danio rerio

Gonadotropin-releasing hormone (GnRH) is found in a wide range of vertebrate tissues, including the nervous system. In general, GnRH has two functions: endocrine, acting as a releasing hormone; and neuromodulatory, affecting neural activity in the peripheral and central nervous system. The best understood population of GnRH cells is that of the hypothalamus, which is essential for reproduction. Less well understood are the populations of GnRH cells found in the terminal nerve and midbrain, which appear to be neuromodulatory in function. The GnRH-containing cells of the midbrain are proposed to arise from the mesencephalic region of the neural tube. Previously, we showed that neuromodulatory GnRH cells of the terminal nerve arise from cranial neural crest. To test the hypothesis that neuromodulatory GnRH cells of the midbrain also arise from neural crest, we used gene knockdown experiments in zebrafish to disrupt neural crest development. We demonstrate that decrement of the function of foxd3 and/or sox10, two genes important for the development and specification of neural crest, resulted in a reduction and/or loss of GnRH cells of the midbrain, as well as a reduction in the number of terminal nerve GnRH cells. Therefore, our data support a neural crest origin for midbrain GnRH cells. Additionally, we demonstrate that knockdown of kallmann gene function resulted in the loss of endocrine GnRH cells of the hypothalamus, but not of neuromodulatory GnRH cells of the midbrain and terminal nerve, thus providing additional evidence for separate pathways controlling the development of neuromodulatory and endocrine GnRH cells.     

Experimental increase in fecundity causes upregulation of fecundity and body maintenance genes in the fat body of ant queens

In most organisms, fecundity and longevity are negatively associated and the molecular regulation of these two life-history traits is highly interconnected. In addition, nutrient intake often has opposing effects on lifespan and reproduction. In contrast to solitary insects, the main reproductive individual of social hymenopterans, the queen, is also the most long-lived. During development, queen larvae are well-nourished, but we are only beginning to understand the impact of nutrition on the queens'' adult life and the molecular regulation and connectivity of fecundity and longevity. Here, we used two experimental manipulations to alter queen fecundity in the ant Temnothorax rugatulus and investigated associated changes in fat body gene expression. Egg removal triggered a fecundity increase, leading to expression changes in genes with functions in fecundity such as oogenesis and body maintenance. Dietary restriction lowered the egg production of queens and altered the expression of genes linked to autophagy, Toll signalling, cellular homeostasis and immunity. Our study reveals that an experimental increase in fecundity causes the co-activation of reproduction and body maintenance mechanisms, shedding light on the molecular regulation of the link between longevity and fecundity in social insects.     

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease. Using markers, including phox2b, we investigate the ontogeny of the sox10 ENS phenotype. As previously shown for melanophore development, ENS progenitor fate specification fails in these mutant fish. However, in addition, we trace back the sox10 mutant ENS defect to an even earlier time point, finding that most neural crest cells fail to migrate ventrally to the gut primordium.     

Estrogen actions on follicle formation and early follicle development

Estradiol-17beta (E(2)) affects late follicular development, whereas primordial follicle differentiation and early activation are believed to be independent of E(2). To test this hypothesis we compared numbers of primordial and primary follicles in wild-type and E(2)-deficient, aromatase knockout (ArKO) mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumor 1 (WT-1), and growth differentiation factor (GDF9), which are known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E(2) replacement for 3 wk in 7-wk-old ArKO and wild-type mice on these parameters were also tested. ArKO mice had reduced numbers of primordial and primary follicles compared with wild-type mice (63%, P < 0.001 and 60%, P = 0.062, respectively). This reduction was not corrected by E(2) treatment, suggesting that E(2) affects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared with mice of the wild type. There were no differences in the immunostaining of MIS, WT-1, and PCNA in primordial and primary follicles between wild-type and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells that develop in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E(2) treatment restored the ovarian follicular morphology in ArKO mice, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E(2) has a role in controlling the size of the oocyte and primordial follicle pool in mice.