Anthea Letsou

Drosophila Dpp signaling is mediated by the punt gene product: A dual ligand-binding type II receptor of the TGFβ receptor family

Signaling by TGF beta-related factors requires ligand-induced association between type I and type II transmembrane serine/threonine kinases. In Drosophila, the saxophone (sax) and thick veins (tkv) genes encode type I receptors that mediate signaling by decapentaplegic (dpp), a member of the bone morphogenetic protein (BMP) subgroup of TGF beta-type factors. In this report, we demonstrate that the Drosophila punt gene encodes atr-II, a previously described type II receptor that on its own is able to bind activin but not BMP2, a vertebrate ortholog of dpp. Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. Furthermore, punt will bind BMP2 in concert with tkv or sax, forming complexes with these receptors. We suggest that punt functions as a type II receptors for dpp and propose that BMP signaling in vertebrates may also involve sharing of type II receptors by diverse ligands.

The drosophila schnurri gene acts in the Dpp/TGFβ signaling pathway and encodes a transcription factor homologous to the human MBP family

Decapentaplegic (dpp), a TGF beta-related ligand, plays a key role in Drosophila development. Although dpp receptors have been isolated, the downstream components of the signaling pathway remain to be identified. We have cloned the schnurri (shn) gene and show that it encodes a putative zinc finger transcription factor homologous to the human major histocompatibility complex-binding proteins 1 and 2. Mutations in shn affect multiple events that require dpp signaling as well as the transcription of dpp-responsive genes. Genetic interactions and the strikingly similar phenotypes of mutations in shn and the dpp receptors encoded by thick veins and punt suggest that shn plays a downstream role in dpp signaling.

B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster

ABSTRACT Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase Drice. B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.

On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila.

The vitamin K-dependent γ-carboxylation of glutamate to γ-carboxyglutamate was originally well characterized in the mammalian blood clotting cascade. γ-Carboxyglutamate has also been found in a number of other mammalian proteins and in neuropeptides from the venoms of marine snails belonging to the genus Conus, suggesting wider prevalence of γ-carboxylation. We demonstrate that an open reading frame from aDrosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent γ-carboxylase activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian γ-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; inDrosophila there are two much shorter introns but in positions precisely homologous to two of the mammalian introns. In addition, a deletion of 6 nucleotides is observed when cDNA and genomic sequences are compared. In situ hybridization to fixed embryos indicated ubiquitous presence of carboxylase mRNA throughout embryogenesis. Northern blot analysis revealed increased mRNA levels in 12–24-h embryos. The continued presence of carboxylase mRNA suggests that it plays an important role during embryogenesis. Although the model substrate FLEEL is carboxylated by the enzyme, a substrate containing the propeptide of aConus carboxylase substrate, conantokin G, is poorly carboxylated. Its occurrence in vertebrates, molluscan systems (i.e. Conus), and Drosophila and the apparently strong homology between the three systems suggest that this is a highly conserved and widely distributed post-translational modification in biological systems.

Amnioserosa is required for dorsal closure in Drosophila

Dorsal closure in the fruit fly Drosophila melanogaster is a complex morphogenetic process, driven by sequential signaling cascades and involving multiple forces, which contribute to cell movements and rearrangements as well as to changes in cell shape. During closure, lateral epidermal cells elongate along the dorsoventral axis and subsequently spread dorsally to cover the embryonic dorsal surface. Amnioserosal cells, which are the original occupants of the most dorsal position in the developing embryo, constrict during closure; thus, the increase in epidermal surface area is accommodated by a reduction in the amnioserosal surface area. Several of the epidermal requirements for closure have been established in functional assays. In contrast, amnioserosal requirements for closure have remained elusive, in part because laser ablation and clonal approaches are limited to only subsets of amnioserosal cells. Here, we report our use of the UAS‐GAL4 system to target expression of the cell autonomous toxin Ricin‐A to all cells of the amnioserosa. We show that ablation of the amnioserosa leads to clear defects in dorsal closure and, thus, directly demonstrate a role for the amnioserosa in dorsal closure. We also show that DJNK (Drosophila Jun N‐terminal kinase) signaling, an epidermal trigger of closure, is unaffected by amnioserosal ablation. These data, together with our demonstration that amnioserosal ablated and Dpp signaling mutant embryos exhibit shared loss‐of‐function phenotypes, point to a requirement for the amnioserosa in dorsal closure that is downstream of Dpp, perhaps as part of a paracrine response to this signaling cascade. Developmental Dynamics 232:791–800, 2005.

The Drosophila Gene for Antizyme Requires Ribosomal Frameshifting for Expression and Contains an Intronic Gene for snRNP Sm D3 on the Opposite Strand

ABSTRACT Previously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5′ open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5′ ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeelingphenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype.

Head involution in Drosophila: genetic and morphogenetic connections to dorsal closure.

Dorsal closure and head involution are complex morphogenetic processes that occur nearly simultaneously, midway through Drosophila embryonic development. While dorsal closure has been studied extensively in terms of both its morphology and genetics, head involution has not been described comprehensively. A thorough review of the literature nonetheless reveals considerable information regarding the genetic components of head involution. In several instances, authors have made explicit references to head involution in regard to mutant phenotypes; in others, we have made this connection. Here we collect, catalogue, and discuss published head involution studies. In considering and integrating the data, an enhanced appreciation of the molecular mechanisms underlying head involution and its molecular kinship with dorsal closure has emerged. Developmental Dynamics 237:28–38, 2008.

Programmed ribosomal frameshifting in the expression of the regulator of intestinal stem cell proliferation, adenomatous polyposis coli (APC)

A programmed ribosomal frameshift (PRF) in the decoding of APC (adenomatous polyposis coli) mRNA has been identified and characterized in Caenorhabditis worms, Drosophila and mosquitoes. The frameshift product lacks the C-terminal approximately one-third of the product of standard decoding and instead has a short sequence encoded by the -1 frame which is just 13 residues in C. elegans, but is 125 in D. melanogaster. The frameshift site is A_AA.A_AA.C in Caenorhabditids, fruit flies and the mosquitoes studied while a variant A_AA.A_AA.A is found in some other nematodes. The predicted secondary RNA structure of the downstream stimulators varies considerably in the species studied. In the twelve sequenced Drosophila genomes, it is a long stem with a four-way junction in its loop. In the five sequenced Caenorhabditis species, it is a short RNA pseudoknot with an additional stem in loop 1. The efficiency of frameshifting varies significantly, depending on the particular stimulator within the frameshift cassette, when tested with reporter constructs in rabbit reticulocyte lysates. Phylogenetic analysis of the distribution of APC programmed ribosomal frameshifting cassettes suggests it has an ancient origin and raises questions about a possibility of synthesis of alternative protein products during expression of APC in other organisms such as humans. The origin of APC as a PRF candidate emerged from a prior study of evolutionary signatures derived from comparative analysis of the 12 fly genomes. Three other proposed PRF candidates (Xbp1, CG32736, CG14047) with switches in conservation of reading frames are likely explained by mechanisms other than PRF.

Raw Mediates Antagonism of AP-1 Activity in Drosophila

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. Here we show that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. Our studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, we provide the first evidence that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, our mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor.

Small flies—Big discoveries: Nearly a century of Drosophila genetics and development

It was almost 100 years ago, in 1909, that a classically trained embryologist, Thomas Hunt Morgan, chose the fruit fly Drosophila melanogaster as a model organism for an experimental study of evolution. Ever since Morgan’s auspicious choice of the fruit fly as an experimental organism, scientists have been eyewitnesses to the “awesome power” of Drosophila genetics—from the transmission geneticists best exemplified by Thomas Hunt Morgan and his students to the developmental geneticists who have been led by Ed Lewis, Christiane NüssleinVolhard, and Eric Wieschaus. There is no doubt that, even now, in the postgenomic age of the 21st century, we find ourselves ever indebted to our genetics forebearers. Building on the productivity of previous generations, present day Drosophila scientists continue to establish paradigms and achieve technological breakthroughs that help advance not only fly research but many different fields of the life sciences as well. Here, we give a short overview of the history of Drosophila genetics. We hope that an understanding of how we got where we are today and an appreciation of past discoveries will help to place the current excitement about genomes, molecules, and mechanisms within the context of a long-established scientific culture and history of Drosophila experimentation. TRANSMISSION GENETICS IN DROSOPHILA