Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.     

Identification of zRAP55,a gene preponderantly expressed in Stages I and II oocytes of zebrafish

In an in silico search for gonand specific expressed genes, we have identified zRAP55 which is enriched in the ovary of zebrafish . zRAP55 encodes a protein of 382 amino acids with a highly conserved Lsm domain. zRAP55 protein shares more than 56% identities with that of other vertebrate species. RT-PCR results show that it is predominantly expressed in the ovary. In situ hybridization and immunohistochemistry studies reveal that zRAP55 is ubiquitously dispersed throughout the cytoplasm of stages I and II oocytes, whereas no expression is observed in stages III and IV oocytes. As an RNA associated protein, zRAP55 might function in the control of protein translation at the early stages of oogenesis in zebrafish.     

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.     

在斑马鱼Ⅰ和Ⅱ期卵母细胞中优势表达基因zRAP55的鉴定(英文)

通过性腺特异性表达基因的筛选,作者发现了一个在斑马鱼卵巢中富集的基因zRAP55。zRAP55蛋白由382个氨基酸组成并含有一个高度保守的Lsm区。zRAP55蛋白与其他脊椎动物一致性在56%以上。RT-PCR结果表明,zRAP55优势表达于卵巢中。原位杂交和免疫组织化学结果表明:在Ⅰ期和Ⅱ期卵母细胞中,zRAP55的阳性信号强烈,均匀地分布于整个细胞质中,但是在Ⅲ期和Ⅳ期卵母细胞中均检测不到信号。作为一个RNA相关蛋白,zRAP55可能在早期卵母细胞中具有调节蛋白质翻译的重要作用。     

Changes of gamma-tubulin expression and distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo

The tubulin gene family is important for individual zebrafish development from the oocyte through to hatching. This involves often rapid, complex changes in the gametes and embryonic cells that are reflected in underlying gene expression changes. Tubulin dynamics, i.e., the interchange of polymeric and soluble forms in zebrafish oogenesis and embryogenesis, is important for microtubule (MT) cellular functions. Nevertheless, our understanding of how tubulin gene expression changes during zebrafish development is not clear. Previous data showed that soluble alpha-tubulin and gamma-tubulin are associated with large molecular weight complexes (>2MDa) which are reduced by the blastula stage, with a concomitant decrease in soluble tubulin amount. Complexes (<2MDa) then increased in the gastrula with an increase in soluble tubulin. Microarray revealed similar patterns of tubulin gene product expression for zebrafish ovary and eggs while both differed from day 4 larva. In situ hybridization with gamma-tubulin oligonucleotide probes revealed diffuse label in oocytes, with a marked localization to the primordial blastodisc upon maturation. These findings, together with recent work on gamma-tubulin ring complexes in other species, suggest that gamma-tubulin (protein complexes) may be involved in regulating tubulin dynamics, thus is important for zebrafish oogenesis and embryogenesis.     

Double-stranded RNA injection produces null phenotypes in zebrafish

Zebrafish is a simple vertebrate that has many attributes that make it ideal for the study of developmental genetics. One feature that has been lacking in this model system is the ability to disable specifically targeted genes. Recently, double-stranded RNA has been used to silence gene expression in the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RNA that is specifically targeted to GFP. To determine that double-stranded RNA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pax6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. Furthermore, Zf-T gene expression could not be detected by in situ hybridization and the message was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish genes sonic hedgehog and floating head was altered in the embryos microinjected with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA targeted to Pax6.1 was associated with depressed expression of Pax6. 1 and resulted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures.     

斑马鱼NUP98基因的克隆及其时空表达图谱

目的：斑马鱼NUP98基因的克隆及其在个体早期发育过程中的表达情况研究。方法：提取斑马鱼胚胎的总RNA,制备地高辛标记的NUP98RNA反义探针,WISH（整体胚胎原位杂交）研究NUP98在斑马鱼早期发育过程中的表达;提取斑马鱼胚胎各时相和成鱼各组织的RNA,实时定量PCR检测斑马鱼胚胎各时相和成鱼各组织中的表达。结果：成功克隆斑马鱼NUP98基因,通过实时定量RT-PCR和原位杂交,获得NUP98基因在斑马鱼早期发育过程中的表达情况：NUP98在2-cell、32．cell、oblong、shield期、12h前普遍性表达（0．75h、1．7h、3．7h、6h、12h）;24h以后在眼部、头部表达较多,特别是在脊索表达较高;斑马鱼NUP98在0、0．5h、6h、12h、24h、48h表达逐渐降低,到72h和96h表达有所增加,但是仍低于24h其表达水平;NUP98在成鱼眼、脑、鳔、肾、肝、睾丸、胆囊、卵巢、鳍、心、肠、肌肉、腮、皮肤的表达中,眼的表达最高,明显高于其他组织,腮、卵巢、肠的表达次之,肌肉、鳔、胆囊、睾丸、皮肤、脑的表达紧随其后,鳍、肝、心、肾的表达最低。结论：NUP98基因可能在个体脑部、脊索及眼部的早期发育过程中起到了重要作用;NUP98基因可能具有抑制肿瘤发生的作用,该基因的调节异常对白血病的发生发展可能有重要影响。这些研究结果为进一步研究NUP98基因在造血系统中的作用,评估其是否适合作为血液系统恶性肿瘤的新的治疗靶点等奠定了理论基础。     

Identification of zebrafish magnetoreceptor and cryptochrome homologs

Magnetoreception is essential for magnetic orientation in animal migration. The molecular basis for magnetoreception has recently been elucidated in fruitfly as complexes between the magnetic receptor magnetoreceptor(Mag R) and its ligand cryptochrome(Cry). Mag R and Cry are present in the animal kingdom. However, it is unknown whether they perform a conserved role in diverse animals. Here we report the identification and expression of zebrafish Mag R and Cry homologs towards understanding their roles in lower vertebrates. A single magr gene and 7 cry genes are present in the zebrafish genome. Zebrafish has four cry1 genes(cry1aa, cry1 ab, cry1 ba and cry1bb) homologous to human CRY1 and a single ortholog of human CRY2 as well as 2 cry-like genes(cry4 and cry5). By RT-PCR, magr exhibited a high level of ubiquitous RNA expression in embryos and adult organs, whereas cry genes displayed differential embryonic and adult expression. Importantly, magr depletion did not produce apparent abnormalities in organogenesis. Taken together, magr and cry2 exist as a single copy gene, whereas cry1 exists as multiple gene duplicates in zebrafish. Our result suggests that magr may play a dispensable role in organogenesis and predicts a possibility to generate magr mutants for analyzing its role in zebrafish.     

Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.     

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.     

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse

During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein--Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis.