Jack R. Bateman

THE TYROSINE KINASE ABL AND ITS SUBSTRATE ENABLED COLLABORATE WITH THE RECEPTOR PHOSPHATASE DLAR TO CONTROL MOTOR AXON GUIDANCE

Genetic analysis of growth cone guidance choice points in Drosophila identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. We now demonstrate that the Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. We also show that the Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton.

Site-specific transformation of Drosophila via φC31 integrase-mediated cassette exchange

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the ϕC31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, ϕC31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.

The Guanine Nucleotide Exchange Factor Trio Mediates Axonal Development in the Drosophila Embryo

Recent analysis of Rho subfamily GTPases in Drosophila revealed roles for Rac and Cdc42 during axonogenesis. Here, we describe the identification and characterization of the Drosophila counterpart of Trio, a guanine nucleotide exchange factor (GEF) that associates with the receptor phosphatase LAR and regulates GTPase activation in vertebrate cells. Mutants deficient in trio activity display defects in both central and peripheral axon pathways reminiscent of phenotypes observed in embryos deficient in small GTPase function. Double mutant analysis shows that trio interacts with Rac in a dose-sensitive manner but not with Rho. Moreover, reduction of trio activity potentiates the phenotype of mutations in the LAR homolog Dlar, suggesting that these proteins collaborate in orchestrating the cytoskeletal events that underlie normal axonogenesis.

The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium

BACKGROUND Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. RESULTS We find that the receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common beta-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time beta-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Finally, we find that two integrin alpha subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. CONCLUSIONS Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. To our knowledge, this is the first direct demonstration of an RPTPs influence on the actin cytoskeleton.

The twin spot generator for differential Drosophila lineage analysis

In Drosophila melanogaster, widely used mitotic recombination–based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing.

Functions of the ectodomain and cytoplasmic tyrosine phosphatase domains of receptor protein tyrosine phosphatase Dlar in vivo

ABSTRACT The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar−/− lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarΔPTP-D2 and Dlarbypass . Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar −/− genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.

A Simple Polymerase Chain Reaction-Based Method for the Construction of Recombinase-Mediated Cassette Exchange Donor Vectors

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the ΦC31 integrase. This PCR-based strategy employs small attB “tails” on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.

A Genomewide Survey Argues That Every Zygotic Gene Product Is Dispensable for the Initiation of Somatic Homolog Pairing in Drosophila

Studies from diverse organisms show that distinct interchromosomal interactions are associated with many developmental events. Despite recent advances in uncovering such phenomena, our understanding of how interchromosomal interactions are initiated and regulated is incomplete. During the maternal-to-zygotic transition (MZT) of Drosophila embryogenesis, stable interchromosomal contacts form between maternal and paternal homologous chromosomes, a phenomenon known as somatic homolog pairing. To better understand the events that initiate pairing, we performed a genomewide assessment of the zygotic contribution to this process. Specifically, we took advantage of the segregational properties of compound chromosomes to generate embryos lacking entire chromosome arms and, thus, all zygotic gene products derived from those arms. Using DNA fluorescence in situ hybridization (FISH) to assess the initiation of pairing at five separate loci, this approach allowed us to survey the entire zygotic genome using just a handful of crosses. Remarkably, we found no defect in pairing in embryos lacking any chromosome arm, indicating that no zygotic gene product is essential for pairing to initiate. From these data, we conclude that the initiation of pairing can occur independently of zygotic control and may therefore be part of the developmental program encoded by the maternal genome.