Andrew D. Renault

Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2

In most organisms, primordial germ cells (PGCs) arise far from the region where somatic gonadal precursors (SGPs) are specified. Although PGCs in general originate as a single cluster of cells, the somatic parts of the gonad form on each site of the embryo. Thus, to reach the gonad, PGCs not only migrate from their site of origin but also split into two groups. Taking advantage of high-resolution real-time imaging, we show that in Drosophila melanogaster PGCs are polarized and migrate directionally toward the SGPs, avoiding the midline. Unexpectedly, neither PGC attractants synthesized in the SGPs nor known midline repellents for axon guidance were required to sort PGCs bilaterally. Repellent activity provided by wunen (wun) and wunen-2 (wun-2) expressed in the central nervous system, however, is essential in this migration process and controls PGC survival. Our results suggest that expression of wun/wun-2 repellents along the migratory paths provides faithful control over the sorting of PGCs into two gonads and eliminates PGCs left in the middle of the embryo.

Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin

Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. We show that activation of the G protein–coupled receptor (GPCR) trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila melanogaster germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. We show that uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. Our findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion.

RETRACTED: Metabolism of sphingosine 1-phosphate and lysophosphatidic acid: a genome wide analysis of gene expression in Drosophila

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns.

Giant nuclei is essential in the cell cycle transition from meiosis to mitosis

At the transition from meiosis to cleavage mitoses, Drosophila requires the cell cycle regulators encoded by the genes, giant nuclei (gnu), plutonium (plu) and pan gu (png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu.

Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells.

Metabolism of sphingosine 1-phosphate and lysophosphatidic acid: a genome wide analysis of gene expression in Drosophila

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns.

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration.

Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure

The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure we show that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. We also show that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype.

Spatial and temporal control of mitotic cyclins by the Gnu regulator of embryonic mitosis in Drosophila.

Mutation of the Drosophila maternal cell cycle regulator, Gnu, results in loss of embryonic mitosis and the onset of excessive nuclear DNA replication. The Gnu phosphoprotein is normally synthesized in nurse cells and transported to the developing oocyte. We created a gnuGFP-bcd3′UTR transgene using the gnu promoter and bicoid 3′UTR, that translates GnuGFP only on egg activation from a localized anterior source. This transgene was able to rescue the sterility of gnu mutant females. Gnu is therefore first required after egg activation for polar body condensation and zygotic mitoses. Embryos containing pronounced anterior-posterior gradients of Gnu activity demonstrate that Gnu regulates mitotic activity by promoting cyclin B stability. Our gnuGFP-bcd3′UTR vector provides a novel experimental strategy to analyse the temporal requirement and role of cell cycle regulators including potential sperm-supplied factors in eggs and embryos.

Hedgehog does not guide migrating Drosophila germ cells.

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.