Biomedical subjects
S L Zipursky
Publications and source records attributed to S L Zipursky.
Mutations disrupting neuronal connectivity in the Drosophila visual system.
The photoreceptor neurons (R cells) of the Drosophila compound eye elaborate a precise array of neuronal connections in the brain. These projections exhibit target specificity and create topographic maps (retinotopy). We have screened histologically for mutations disrupting R cell connectivity in developing tissue. Eighty mutations were isolated from over 6000 ethylmethane sulfonate-mutagenized lines. Characterization of these mutations included genetic mosaic analysis to determine whether the gene is required in the retina or in the optic ganglia. Most mutations were found to affect connectivity indirectly by disrupting development more generally in the eye or brain. Genes were identified as candidates for playing direct roles in R cell connectivity by affecting axonal outgrowth (eddy), target recognition (limbo and nonstop), and retinotopy (limbo).
Homeobox genes and connective tissue patterning.
In vertebrates, limb tendons are derived from cells that migrate from the lateral plate mesoderm during early development. While some of the developmental steps leading to the formation of these tissues are known, little is known about the molecular mechanisms controlling them. We have identified two murine homeobox-containing genes, Six 1 and Six 2, which are expressed in a complementary fashion during the development of limb tendons. Transcripts for both genes are found in different sets of phalangeal tendons. Six 1 and Six 2 also are expressed in skeletal and smooth muscle, respectively. These genes may participate in the patterning of the distal tendons of the limb phalanges by setting positional values along the limb axes.
The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.
Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the R7 photoreceptor cell. Components of the signal transduction pathway activated by Sev in the R7 precursor include proteins encoded by the gap1, drk, Sos, ras1 and raf loci. In this report we present evidence that a Drosophila MAP kinase, ERK-A, is encoded by the rolled locus and is required downstream of raf in the Sev signal transduction pathway.
A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways.
In the Drosophila eye, activation of the sevenless (sev) receptor tyrosine kinase is required for the specification of the R7 photoreceptor cell fate. In a genetic screen for mutations that result in the activation of the sev signaling pathway in the absence of the inducing signal, we identified a gain-of-function mutation in rolled (rlSevenmaker [rlSem]), which encodes a homolog of mitogen-activated protein (MAP) kinase. In addition to the sev pathway, this mutation activates the pathways controlled by torso and the epidermal growth factor receptor homology. The rlSem mutation results in the substitution of a single conserved amino acid in the kinase domain. Activation of MAP kinase by the rlSem mutation is both necessary and sufficient to activate multiple signaling pathways controlled by receptor tyrosine kinases.
The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system.
The transformation of an unpatterned epithelium into a patterned one is a fundamental issue in morphogenesis. This transformation occurs in a dramatic fashion in the developing eye imaginal disc, the primordium of the Drosophila compound eye. Molecular and developmental analyses reveals that the sine oculis (so) locus encodes a homeodomain-containing protein that is expressed and required in the unpatterned epithelium prior to morphogenesis. In mutants, cells undergo apoptosis. These findings argue that so plays an essential role in controlling the initial events of pattern formation in the eye disc. So is also expressed and required for the development of the rest of the fly visual system, including the optic lobes (i.e., those regions of the brain that process visual information). So is expressed in the optic lobe primordium prior to its invagination from the embryonic ectoderm; in so mutants, the optic lobe primordium fails to invaginate.
Determination of neuronal cell fate: lessons from the R7 neuron of Drosophila.
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The Drosophila anachronism locus: a glycoprotein secreted by glia inhibits neuroblast proliferation.
The Drosophila anachronism (ana) locus controls the proliferation of neuroblasts, neuronal stem cells that give rise to the central nervous system. In ana mutants, quiescent postembryonic central brain and optic lobe neuroblasts enter S phase precociously. ana encodes a novel secreted protein of 474 amino acids that is expressed not in the affected neuroblasts, but rather in a subclass of neighboring glial cells. These studies argue for an important role for glia in negatively regulating proliferation of neuronal precursor cells, thereby controlling the timing of postembryonic neurogenesis.
Extracellular domain of the boss transmembrane ligand acts as an antagonist of the sev receptor.
The fate of the R7 photoreceptor cell in the Drosophila compound eye is established by a specific inductive interaction between the R8 photoreceptor neuron and the R7 precursor cell. This induction is mediated by two cell-surface proteins: the ligand, bride of sevenless (boss), and sevenless (sev), a tyrosine-kinase receptor. The structure of boss is unique for a ligand of a tyrosine-kinase receptor. It contains a large extracellular domain, seven transmembrane segments, and a carboxy-terminal cytoplasmic tail. Here we report that: (1) boss activates tyrosine phosphorylation of the sev receptor; (2) the seven transmembrane domain of boss is necessary for its function; and (3) a soluble form of boss acts as an antagonist of the sev receptor both in vivo and in vitro.
Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase.
The extracellular signal-regulated kinases (ERKs) comprise a class of protein-serine/threonine kinases that are activated in response to a wide variety of extracellular signals transduced via receptor tyrosine kinases. Activation of the ERKs requires both threonine and tyrosine phosphorylation suggestive of a key role in mediating intracellular events in response to extracellular cues. To critically assess the role of ERKs in intracellular signaling, a genetically tractable receptor tyrosine kinase system would be invaluable. In this paper we report the identification of a Drosophila homolog of ERK1 and -2, designated DmERK-A. DmERK-A is 80% identical to rat ERK1 and -2 and is rapidly phosphorylated on tyrosine in response to an extracellular signal activating a receptor tyrosine kinase. Biochemical and histological studies reveal its expression in the eye imaginal disc. These studies provide a first step in a genetic analysis of ERK function.
The bride of sevenless and sevenless interaction: internalization of a transmembrane ligand.
During Drosophila retinal development, the R8 photo-receptor neuron induces a neighboring cell to assume an R7 cell fate through cell contact. This is mediated by the transmembrane protein bride of sevenless (boss) on the surface of the R8 cell, which binds the sevenless tyrosine kinase receptor (sev) on the surface of the R7 precursor cell. The boss protein, which contains a large extracellular domain, seven transmembrane segments, and a C-terminal cytoplasmic domain, has an exceptional structure for a ligand of a receptor tyrosine kinase. Using a panel of antibodies directed to various cytoplasmic and extracellular epitopes, we demonstrate that the entire boss protein from its extreme N-terminus to its extreme C-terminus is internalized by sev-expressing tissue culture cells and by the R7 precursor cell in the developing eye imaginal disc. The receptor-mediated transfer of a transmembrane ligand represents a novel mechanism for protein transfer between developing cells.
Induction of the R7 neuron in the Drosophila compound eye: the bride of sevenless and sevenless interaction.
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Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.
The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.
Amphipathic beta structure of a leucine-rich repeat peptide.
Long tandem arrays of a characteristic leucine-rich repeat motif on the order of 24 amino acids in length have been found in the primary structure of an increasing number of proteins. The most striking feature of these repeats is an amphipathic sequence, with leucine as the predominant hydrophobic residue. Based on this amphipathic sequence and the function of the proteins in which they have been found, the repeats have been proposed to be involved in protein-protein and protein-lipid interactions. As a step toward elucidating the structure and biochemical properties of the leucine-rich repeat motif, we have studied a synthetic leucine-rich repeat peptide (LRP32) representing one of the repeats found in Drosophila chaoptin. We have shown that: (i) LRP32 is soluble in aqueous solution but will bind quantitatively to phospholipid vesicles; (ii) LRP32 has a partial beta structure in aqueous solution and is predominantly a beta structure in the presence of phospholipid; (iii) LRP32 integrates into lipid bilayers to form 60-A intramembrane particles as seen using freeze-fracture electron microscopy (these putative oligomeric structures appear to contain a central aqueous core as indicated by their ability to generate conductances in planar lipid bilayers); and (iv) LRP32-lipid complexes generate 2H NMR spectra characteristic of integral membrane proteins. This study is consistent with LRP32 forming an amphipathic beta sheet. We propose that protein segments containing tandem arrays of leucine-rich repeats also may form amphipathic beta sheets.
Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine-kinase receptor.
During development of the Drosophila retina, the R8 photoreceptor neuron induces a neighbouring cell to assume an R7 cell fate. Genetic data suggest that the induction is mediated by two transmembrane proteins encoded by bride of sevenless and sevenless. A direct interaction between these two proteins was demonstrated by the heterotypic aggregation of cell lines expressing them. In the developing eye the sevenless-dependent internalization of bride of sevenless by the R7 precursor cell provides evidence for a direct interaction between these two proteins in vivo.
Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule.
Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules.
The role of cell-cell interaction in the development of the Drosophila visual system.
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Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments.
Previous genetic mosaic studies established that expression of the Drosophila bride of sevenless (boss) gene is required in photoreceptor neuron R8 for the development of photoreceptor neuron R7. This led to the proposal that boss encodes or regulates an R7-specific inductive cue. We have identified the boss gene based on small deletions in mutant alleles and sequenced both cDNAs and corresponding genomic regions. One P element and three X-ray-induced boss alleles show different deletions in the gene ranging in size from 2 to 23 bp, each causing frameshifts leading to premature termination of translation. The boss gene encodes a protein of 896 amino acids with a putative amino-terminal signal sequence, a large extracellular region of 498 amino acids, and seven potential transmembrane domains followed by a carboxy-terminal cytoplasmic tail of 115 amino acids. The putative membrane localization of the boss protein is consistent with a model in which direct interaction between the boss and sevenless proteins specifies R7 cell fate. Another model in which the boss protein functions as a receptor is proposed based on its similarity to the G protein-linked family of membrane receptors.