Kayo Arita

Maternal Nanos represses hid/skl-dependent apoptosis to maintain the germ line in Drosophila embryos

Nanos (Nos) is an evolutionarily conserved protein essential for the survival of primordial germ cells. In Drosophila, maternal Nos partitions into pole cells and suppresses apoptosis to permit proper germ-line development. However, how this critical event is regulated by Nos has remained elusive. Here, we report that Nos represses apoptosis of pole cells by suppressing translation of head involution defective (hid), a member of the RHG gene family that is required for Caspase activation. In addition, we demonstrate that hid acts in concert with another RHG gene, sickle (skl), to induce apoptosis. Expression of skl is induced in pole cells by maternal tao-1, a ste20-like serine/threonine kinase. Tao-1-dependent skl expression is required to potentiate hid activity. However, skl expression is largely suppressed in normal pole cells. Once the pole cells lack maternal Nos, Tao-1-dependent skl expression is fully activated, suggesting that skl expression is also restricted by Nos. These findings provide the first evidence that the germ line is maintained through the regulated expression of RHG genes.

Isolation of germline cells from Drosophila embryos by flow cytometry

Primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. To understand the molecular basis of germline development, isolation of pure PGC is required. We report here the use of fluorescence‐activated cell sorting (FACS) to isolate pure populations of Drosophila pole cells, which are the presumptive primordial germ cells in flies. In order to fluorescently mark pole cells, we used an EGFP‐vasa transgenic line that expresses green fluorescent protein (GFP) specifically and continuously in the germ line throughout the life cycle. The purity of FACS‐sorted pole cells from embryos was confirmed by microscopic inspection and quantitative polymerase chain reaction. Moreover, by optimizing the sample preparation and the sorting protocol, embryonic gonads could also be isolated. This technique opens the way for genome‐wide transcriptome analysis of germline cells. In a pilot experiment, we generated a cDNA library from purified embryonic gonad and identified a novel germline‐specific gene, RpL22‐like.

Maternal RNAs encoding transcription factors for germline-specific gene expression in "Drosophila" embryos

In early Drosophila embryos, germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm contains the maternal factors required for germline development. It has been proposed that germline-specific gene expression is initiated by the function of maternal factors that are enriched in the germ plasm. However, such factors have remained elusive. Here, we describe a genome-wide survey of maternal transcripts that encode for transcription factors and are enriched in the germ plasm. We isolated pole cells from blastodermal embryos by fluorescence-activated cell sorting (FACS) and then used these isolated cells in a microarray analysis. Among the 835 genes in the Gene Ontology (GO) category transcription regulator activity listed in FlyBase, 68 were found to be predominantly expressed in pole cells as compared to whole embryos. As the early pole cells are known to be transcriptionally quiescent, the listed transcripts are predicted to be maternal in origin. Our in situ hybridization analysis revealed that 27 of the 68 transcripts were enriched in the germ plasm. Among the 27 transcripts, six were found to be required for germline-specific gene expression of vasa and/or nanos by knockdown experiments using RNA interference (RNAi). The identified transcripts encode a transcriptional activator (ovo), components of the transcriptional initiation complexes (Trf2, bip2 and Tif-IA), and the Ccr4-Not complex (CG31716 and l(2)NC136). Our study demonstrates that germ plasm contains maternal transcripts encoding transcriptional regulators for germline-specific gene expression in pole cells.

Expression of the apoptosis inducer gene head involution defective in primordial germ cells of the Drosophila embryo requires eiger, p53, and loki function.

Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage‐activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell‐specific manner by loki/p53 and dpp/egr during embryogenesis.

MAMO, a maternal BTB/POZ-Zn-finger protein enriched in germline progenitors is required for the production of functional eggs in Drosophila.

A hallmark of germline cells throughout the animal kingdom is their ability to execute meiosis. However, despite its prime importance, little is known about how germline progenitors acquire this ability. In Drosophila, the primordial germ cells (PGCs) are characterized by the inheritance of germ plasm, which contains maternal factors that have sufficient ability to direct germline development. Here, we show that a novel maternal factor, MAMO, is autonomously required in PGCs to produce functional gametes. MAMO protein which contains both a BTB/POZ (Broad Complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) domain and C(2)H(2) zinc finger motifs is enriched in PGCs during embryogenesis. The PGCs with reduced maternal MAMO activity are able to undergo oogenesis, but fail to execute meiosis properly. In the resulting oocytes, meiosis-specific chromosomal configurations are impaired. We additionally show that the decondensation of fertilized sperm nuclei is also affected in the eggs. We propose that maternal MAMO activates downstream genes to promote specialized morphological changes of both female meiotic chromosomes and the sperm nucleus, which are critical in zygote formation.