piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells

piwi represents the first class of genes known to be required for stem cell self-renewal in diverse organisms. In the Drosophila ovary, piwi is required in somatic signaling cells to maintain germline stem cells. Here we show that piwi encodes a novel nucleoplasmic protein present in both somatic and germline cells, with the highly conserved C-terminal region essential for its function. Removing PIWI protein from single germline stem cells significantly decreases the rate of their division. This suggests that PIWI has a second role as a cell-autonomous promoter of germline stem cell division. Consistent with its dual function, over-expression of piwi in somatic cells causes an increase both in the number of germline stem cells and the rate of their division. Thus, PIWI is a key regulator of stem cell division - its somatic expression modulates the number of germline stem cells and the rate of their division, while its germline expression also contributes to promoting stem cell division in a cell-autonomous manner.     

The role of PIWI and the miRNA machinery in Drosophila germline determination

BACKGROUND: The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. RESULTS: We report the function of Drosophila PIWI, DICER-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. PIWI is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect OSK or VASA expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases OSK and VASA levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, PIWI forms a complex with dFMRP and DICER-1, but not with DICER-2, in polar-granule-enriched fractions. Depleting DICER-1, but not DICER-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. CONCLUSIONS: Because DICER-1 is required for the miRNA pathway and DICER-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, our data implicate a crucial role of the PIWI-mediated miRNA pathway in regulating the levels of OSK, VASA, and possibly other genes involved in germline determination in Drosophila.     

zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline

RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. Here we report the identification of Drosophila zucchini (zuc) and squash (squ), which function in germline RNAi processes. Zuc and Squ contain domains with homologies to nucleases. Mutant females are sterile and show dorsoventral patterning defects during oogenesis. In addition, Oskar protein is ectopically expressed in early oocytes, where it is normally silenced by RNAi mechanisms. Zuc and Squ localize to the perinuclear nuage and interact with Aubergine, a PIWI class protein. Mutations in zuc and squ induce the upregulation of Het-A and Tart, two telomere-specific transposable elements, and the expression of Stellate protein in the Drosophila germline. We show that these defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs.     

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identification of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrafish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression.     

Expression pattern of Siaz gene during the zebrafish embryonic development

Siah is a mammalian homolog of Drosophila seven in absentia (SINA). Here we report the identification of a new member of the SINA/Siah gene family. This new gene, designated Siaz, was found in zebrafish, and its product is predicted to share extensive amino acid sequence homology with Drosophila SINA. Siaz is maternally inherited, with zygotic expression in all blastomeres starting at the mid-blastula transition. After the 20-somite stage, Siaz expression occurs in a stage-specific manner in particular regions, including the brain, eye, cranial cavity, otic vesicle, optic chiasm and gut.     

Midline signals regulate retinal neurogenesis in zebrafish

In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.     

鹌鹑gcl基因BTB/POZ结构域编码序列的克隆及组织中的表达

Germ cell-less(GCL)是与原始生殖细胞发生相关的重要因子,果蝇、斑马鱼、青鳉鱼和小鼠的GCL蛋白都含有一个进化上保守的BTB/POZ结构域。本研究应用简并PCR克隆技术,在鹌鹑(Coturnix coturnix)中扩增得到319bp的gcl基因(包含BTB/POZ结构域编码序列)保守序列。通过与线虫、果蝇、斑马鱼、小鼠和人gcl同源序列的比对,发现该片段与它们的同源性分别为52.4%、55.4%、84.6%、79.6%和76.8%。采用RT-PCR和mRNA整体原位杂交方法,研究了gcl基因在不同组织和原条期胚胎中的表达。结果显示,该基因在卵巢、精巢和肝中表达;gcl mRNA存在于原条期胚胎的生殖新月区。     

Vasa protein expression and localization in the zebrafish

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.     

Heterotypic piRNA Ping-Pong requires qin, a protein with both E3 ligase and Tudor domains

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example, nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.     

pc1 and psc1, zebrafish homologs of Drosophila Polycomb and Posterior sex combs,encode nuclear proteins capable of complex interactions

Drosophila Polycomb group proteins are thought to form multimeric nuclear complexes that are responsible for stable transmission of repressed states of gene expression during the proliferation of differentiating embryos. In this study, we cloned, sequenced, and characterized two Polycomb group homologs, designated pc1 and psc1, in zebrafish. Amino acid sequence analyses determined that pc1 is a structural homolog of Drosophila Polycomb and that psc1 is a homolog of Drosophila Posterior sex combs. Northern blots and whole-mount in situ hybridization revealed that pc1 and psc1 had overlapping expression patterns at successive stages of embryogenesis. Immunocytochemistry localized both Pc1 and Psc1 protein in blastomere nuclei. Pull-down assays and two-hybrid system deletion analyses showed that these proteins were capable of homotypic and heterotypic interactions and identified the regions required for these interactions. The evidence supports the idea that zebrafish Polycomb group proteins, like those of other species, form nuclear complexes with compositions that may vary in a spatio-temporal manner during development.     

PIWI/piRNA“机器”与雄性生殖细胞发育

PIWI(P-element-induced wimpy testis)蛋白在动物生殖系细胞中特异性表达,为动物生殖细胞发育分化所必需。piRNA(PIWI-interacting RNAs)是最近在动物生殖系细胞中发现的一类非编码小分子RNA,这类小RNA特异性地与PIWI家族蛋白相互作用。PIWI/piRNA"机器"通过沉默转座元件和调控编码mRNA等方式在动物生殖细胞发育分化过程中发挥重要作用。本文围绕PIWI/piRNA"机器"的生物学功能及分子机制,对近期取得的相关研究进展进行了系统性总结。