Annette L. Parks

Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development

Analysis of retinal development in Delta (Dl) temperature-sensitive mutants reveals requirements for Delta function in the specification of all retinal cells, including photoreceptors, cone cells, pigment cells and cells that make up interommatidial bristles. In situ hybridization and immunohistochemistry indicate that Delta is expressed dynamically during the specification of different cell types. Comparisons of Delta expression patterns with developmental defects in Dl mutants implies that Delta functions in a cell-nonautonomous manner in the specification of photoreceptors. Delta protein resides predominantly in subcellular vesicles located primarily at the apical ends of developing retinal cells. Localization of Delta protein in Dl and shibire tsl mutants implies that Delta is targeted to the cell surface, but is efficiently removed via endocytosis, resulting in vesicular accumulation.

Molecular analysis of heterochronic changes in the evolution of direct developing sea urchins

The sea urchin Heliocidaris erythrogramma is a direct developer; it progresses directly from the gastrula to the juvenile adult without forming a pluteus larva. No larval skeleton is formed by mesenchyme cells, but formation of the juvenile skeleton is accelerated. We have examined two alterations in mesenchyme cell behavior that accompany this striking change in developmental pattern. 1) Rapid cell proliferation produces 1700–2200 mesenchyme cells by mid‐gastrula, compared to 30–60 primary mesenchyme cells in species with typical larval development. This change may reflect the accelerated production of adult structures in H. erythrogramma. 2) B2C2 is a monoclonal antibody that recognizes primary (Anstrom et al., 1987) and adult mesenchyme cells associated with skeleton formation in typical developers. The altered pattern of B2C2 staining in H. erythrogramma (e.g., a later initial appearance of the B2C2 antigen) suggests that H. erythrogramma has deleted part of a larval program of development and accelerated its adult program of development. These results indicate that cellular and molecular heterochronies accompany the morphological changes in H. erythrogramma development.

The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster

We have examined expression of the neurogenic gene, Delta (Dl), and the regulatory relationships between the Delta-Notch signalling pathway and the proneural gene, achaete, during microchaeta development in Drosophila. Delta is expressed in all microchaeta proneural cells and microchaeta sensory organ precursors (SOPs) and is expressed dynamically in SOP progeny. We find that Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that initial Delta expression in the notum is not dependent on proneural gene function. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is regulated, in part, by Delta-Notch signalling activity. We find that Delta signalling is required for correct delineation of early proneural gene expression in developing nota. Within microchaeta proneural stripes, we demonstrate that Delta-Notch signalling prohibits adoption of the SOP fate by repressing expression of proneural genes.

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

Structure–Function Analysis of Delta Trafficking, Receptor Binding and Signaling in Drosophila

The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta–Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-like repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (DlTD alleles) that encode “trafficking-defective” Delta proteins. We find, unexpectedly, that many DlTD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two DlTD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling.

Safeguarding gene drive experiments in the laboratory

Multiple stringent confinement strategies should be used whenever possible Gene drive systems promote the spread of genetic elements through populations by assuring they are inherited more often than Mendelian segregation would predict (see the figure). Natural examples of gene drive from Drosophila include sex-ratio meiotic drive, segregation distortion, and replicative transposition. Synthetic drive systems based on selective embryonic lethality or homing endonucleases have been described previously in Drosophila melanogaster (1–3), but they are difficult to build or are limited to transgenic populations. In contrast, RNAguided gene drives based on the CRISPR/Cas9 nuclease can, in principle, be constructed by any laboratory capable of making transgenic organisms (4). They have tremendous potential to address global problems in health, agriculture, and conservation, but their capacity to alter wild populations outside the laboratory demands caution (4–7). Just as researchers working with self-propagating pathogens must ensure that these agents do not escape to the outside world, scientists working in the laboratory with gene drive constructs are responsible for keeping them confined (4, 6, 7).

Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers

Presenilin is the enzymatic component of γ-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimers disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for γ-tubulin in the pathway.

Direct Development in the Sea Urchin Phyllacanthus parvispinus (Cidaroidea): Phylogenetic History and Functional Modification

Development in the Australian sea urchin Phyllacanthus parvispinus (Echinoidea: Cidaroidea) is of interest because it has a highly modified, lecithotrophic larva, and because it belongs to an echinoid group whose development has been little studied. This study documents early development and metamorphosis in P. parvispinus and considers the evolution of features unusual in echinoid ontogeny. Some features, such as lack of a vestibule, occur in other cidaroids, and are likely a product of ancestry. Other unusual features, such as larger gametes, an equal fourth cleavage, a wrinkled blastula, and accelerated development of the adult rudiment, are characteristic of other direct developing echinoids, and are probably functional modifications for altered developmental mode. Since the Cidaroidea form the sister group to the more derived Euechinoidea, cidaroid development is critical in assessing the phylogeny of ontogeny among echinoids. The distribution of developmental features among extant echinoids suggests...

New research resources at the Bloomington Drosophila Stock Center

The Bloomington Drosophila Stock Center (BDSC) is a primary source of Drosophila stocks for researchers all over the world. It houses over 27,000 unique fly lines and distributed over 160,000 samples of these stocks this past year. This report provides a brief overview of significant recent events at the BDSC with a focus on new stock sets acquired in the past year, including stocks for φC31 transformation, RNAi knockdown of gene expression, and SNP and quantitative trait loci discovery. We also describe additions to sets of insertions and molecularly defined chromosomal deficiencies, the creation of a new Deficiency Kit, and planned additions of X chromosome duplication sets.

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules.

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.