Drosophila Eye Development Research Articles

Determinants of the Drosophila Odorant Receptor Pattern

In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.

Immunolocalization of Sprouty-1 and Sprouty-2 in Developing Rat Lung

Objective: Sprouty, a common antagonist of fibroblast growth factor (FGF) and epidermal growth factor signaling, is a key player regulating tracheal branching and eye development in Drosophila. Four Sprouty homologs have been identified in vertebrates and all share a cysteine-rich region. However, the physiological function(s) of the individual Sprouty homologs is unknown. mRNA of Sprouty homologs is expressed during mouse lung development. In the present study, we investigated the immunolocalization of Sprouty proteins in rat lung at different stages of development. Methods: Rabbit antibodies were raised against peptides derived from rat Sprouty-1 and Sprouty-2 and were used in Western blot analysis to determine Sprouty distribution in subcellular fractions (pellets and supernatant centrifuged at 5,000 and 20,000 g) and bronchoalveolar lavage fluid (BAL) from adult rat lungs or used in immunohistochemistry. Results: Western blot analysis revealed a 30-kDa Sprouty-1 band and a 34-kDa Sprouty-2 band in the supernatant and pellet fractions centrifuged at 20,000 g. BAL contained a band of approximately 16 kDa with Sprouty-1 antibody derived from proteolytic fragmentation of Sprouty-1. In embryonic day (E) 14 and E16 lungs, Sprouty-1 and Sprouty-2 were expressed both in epithelial and peripheral mesenchymal cells. In adult rat lung, bronchiolar and alveolar type II epithelial cells showed staining for both Sprouty-1 and Sprouty-2. Sprouty-1 expression was also seen in alveolar type I epithelial cells. Conclusion: In light of the proximity of the distribution of Sprouty to that of FGF-10 (peripheral mesenchyme) and its receptor FGFR2IIIb (distal tubular epithelium) in lung development, and the finding that FGF-9, which is expressed in mesothelial cells, upregulates FGF-10, it appears that Sprouty expression in epithelial and mesenchymal cells during branching morphogenesis is closely related to signaling by FGF-9 and FGF-10.

Sarcoglycan complex formation is involved in regulation of EGFR signaling during Drosophila eye development

Abstract Background: Mutations of some components of the Dystrophin Glycoprotein Complex (DGC) are known to cause Muscular Dystrophy in humans. An innovative approach to the further understanding of pathological

The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis

Many human diseases are caused by malfunction of basic types of cellular activity such as proliferation, differentiation, apoptosis, cell polarization, and migration. In turn, these processes are associated with different routes of intracellular signal transduction. A number of model systems have been designed to study normal and abnormal cellular and molecular processes associated with pathogenesis. The developing eye of the fruit fly Drosophila melanogaster is one of these systems. The sequential development of compound eyes of this insect makes it possible to model human neurodegenerative diseases and mechanisms of carcinogenesis. In this paper we overview the program of the eye development in Drosophila, with emphasis on intracellular signaling pathways that regulate this complex process. We discuss in detail the roles of the Notch, Hedgehog, TGFβ, Wnt, and receptor tyrosine kinase signaling pathways in Drosophila eye development and human pathology. We also briefly describe the modern methods of experimentation with this model organism to analyze the function of human pathogenic proteins.

Intracellular trafficking in Drosophila visual system development: A basis for pattern formation through simple mechanisms

Intracellular trafficking underlies cellular functions ranging from membrane remodeling to receptor activation. During multicellular organ development, these basic cell biological functions are required as both passive machinery and active signaling regulators. Exocytosis, endocytosis, and recycling of several key signaling receptors have long been known to actively regulate morphogenesis and pattern formation during Drosophila eye development. Hence, intracellular membrane trafficking not only sets the cell biological stage for receptor-mediated signaling but also actively controls signaling through spatiotemporally regulated receptor localization. In contrast to eye development, the role of intracellular trafficking for the establishment of the eye-to-brain connectivity map has only recently received more attention. It is still poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Yet, the Drosophila visual system provides some of the most striking examples for the regulatory role of intracellular trafficking during multicellular organ development. In this review we will first highlight the experimental and conceptual advances that motivate the study of intracellular trafficking during Drosophila visual system development. We will then illuminate the development of the eye, the eye-to-brain connectivity map and the optic lobe from the perspective of cell biological dynamics. Finally, we provide a conceptual framework that seeks to explain how the interplay of simple genetically encoded intracellular trafficking events governs the seemingly complex cellular behaviors, which in turn determine the developmental product.

Understanding SOS (Son of Sevenless)

Son of Sevenless (SOS) was discovered in Drosophila melanogaster. Essential for normal eye development in Drosophila, SOS has two human homologues, SOS1 and SOS2. The SOS1 gene encodes the Son of Sevenless 1 protein, a Ras and Rac guanine nucleotide exchange factor. This protein is composed of several important domains. The CDC25 and REM domains provide the catalytic activity of SOS1 towards Ras and the histone fold DH/PH (Dbl homology and Pleckstrin homology) domains function, in tandem, to stimulate GTP/GDP exchange for Rac. In contrast to Ras, there have been few studies that implicate SOS1 in human disease and, initially, less attention was given to this gene. However, mutations in SOS1 have been reported recently in Noonan syndrome and in type 1 hereditary gingival fibromatosis. Although, there have been very few studies that focus on the regulation of this important gene by physiological or exogenous factors, we recently found that the SOS1 gene was induced by the environmental toxin, dioxin, and that this effect was mediated by the aryl hydrocarbon receptor (AhR). These recent observations raise the possibility that alterations in the expression of the SOS1 gene and, consequently, in the activity of the SOS1 protein may affect toxicological endpoints and lead to clinical disease. These possibilities, thus, have stimulated much interest in SOS1 recently. In this article, we review the functions of SOS1 and the evidence for its roles in physiology and pathology across species.

Fasciclins 2 and 3 (FAS2 and FAS3) participate in ommatidial patterning during eye development in Drosophila

Fasciclins 2 and 3 (FAS2 and FAS3) participate in ommatidial patterning during eye development in Drosophila

Identification of Novel Ras-Cooperating Oncogenes in Drosophila melanogaster: A RhoGEF/Rho-Family/JNK Pathway Is a Central Driver of Tumorigenesis

We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (Ras(ACT)) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with Ras(ACT) in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance Ras(ACT)-driven hyperplasia. Ras(ACT)-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1(ACT)), RhoGEF2, and pbl cooperated with Ras(ACT), resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with Ras(ACT) upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with Ras(ACT), while in the clonal setting, JNK upregulation was sufficient for Ras(ACT)-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of Ras(V12)-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2(+) human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with Ras(ACT) in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with Ras(ACT).

Cytosolic Ras Supports Eye Development in Drosophila

Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development.

Exon Junction Complex Subunits Are Required to Splice Drosophila MAP Kinase, a Large Heterochromatic Gene

The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns.

Organogenesis and tumorigenesis: Insight from the JAK/STAT pathway in the Drosophila eye

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis. Developmental Dynamics 239:2522–2533, 2010. © 2010 Wiley-Liss, Inc.

Separable transcriptional regulatory domains within Otd control photoreceptor terminal differentiation events

Orthodenticle (Otd)-related transcription factors are essential for anterior patterning and brain morphogenesis from Cnidaria to Mammals, and genetically underlie several human retinal pathologies. Despite their key developmental functions, relatively little is known regarding the molecular basis of how these factors regulate downstream effectors in a cell- or tissue-specific manner. Many invertebrate and vertebrate species encode two to three Otd proteins, whereas Drosophila encodes a single Otd protein. In the fly retina, Otd controls rhabdomere morphogenesis of all photoreceptors and regulates distinct Rhodopsin-encoding genes in a photoreceptor subtype-specific manner. Here, we performed a structure–function analysis of Otd during Drosophila eye development using in vivo rescue experiments and in vitro transcriptional regulatory assays. Our findings indicate that Otd requires at least three distinct transcriptional regulatory domains to control photoreceptor-specific rhodopsin gene expression and photoreceptor morphogenesis. Our results also uncover a previously unknown role for Otd in preventing co-expression of sensory receptors in blue vs. green-sensitive R8 photoreceptors. Sequence analysis indicates that many of the transcriptional regulatory domains identified here are conserved in multiple Diptera Otd-related proteins. Thus, these studies provide a basis for identifying shared molecular pathways involved in a wide range of developmental processes.

Dorsal eye selector pannier ( pnr) suppresses the eye fate to define dorsal margin of the Drosophila eye

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, we found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the dorsal eye margin. We found that pnr acts downstream of Ey and affects the retinal determination pathway by suppressing eya. Further analysis of the “eye suppression” function of pnr revealed that this function is likely mediated through suppression of the homeotic gene teashirt (tsh) and is independent of homothorax (hth), a negative regulator of eye. Pnr expression is restricted to the peripodial membrane on the dorsal eye margin, which gives rise to head structures around the eye, and pnr is not expressed in the eye disc proper that forms the retina. Thus, pnr has dual function, during early developmental stages pnr is involved in axial patterning whereas later it promotes the head specific fate. These studies will help in understanding the developmental regulation of boundary formation of the eye field on the dorsal eye margin.

The gang of four gene regulates growth and patterning of the developing Drosophila eye

We report here the identification of a novel complementation group in the fruit fly Drosophila melanogaster named gang of four (gfr). Mutations in gfr disrupt patterns of cell differentiation in the eye and increase eye size through a proliferative mechanism that can be enhanced by a block in apoptosis. gfr mutant cells show several features of deregulated Ras/MAP kinase activity, including reduced expression of the Capicua growth suppressing transcription factor and synthetically lethality with alleles of the Jun N-terminal kinase phosphatase puckered. gfr alleles also upreguate Notch activity in the eye. Thus, gfr alleles appear to elicit growth and patterning phenotypes via effects on multiple signaling pathways. Moreover, the gfr alleles behave as gain-of-function lesions and overexpress the gene bruno-3 (bru-3), which is located at the genomic region to which gfr lesions map. Genetic reduction of bru-3 suppresses phenotypes caused by gfr alleles, and like gfr alleles, overexpression of bru-3 depresses levels of Cic protein, indicating that overexpression of bru-3 is central to gfr mutant phenotypes.

Chinmo Is a Functional Effector of the JAK/STAT Pathway that Regulates Eye Development, Tumor Formation, and Stem Cell Self-Renewal in Drosophila

The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. Here we show that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs nonautonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts.

Cell cycle arrest by a gradient of Dpp signaling during Drosophila eye development

BackgroundThe secreted morphogen Dpp plays important roles in spatial regulation of gene expression and cell cycle progression in the developing Drosophila eye. Dpp signaling is required for timely cell cycle arrest ahead of the morphogenetic furrow as a prelude to differentiation, and is also important for eye disc growth. The dpp gene is expressed at multiple locations in the eye imaginal disc, including the morphogenetic furrow that sweeps across the eye disc as differentiation initiates.ResultsStudies of Brinker and Dad expression, and of Mad phosphorylation, establish that there is a gradient of Dpp signaling in the eye imaginal disc anterior to the morphogenetic furrow, predominantly in the anterior-posterior axis, and also Dpp signaling at the margins of the disc epithelium and in the dorsal peripodial membrane. Almost all signaling activity seems to spread through the plane of the epithelia, although peripodial epithelium cells can also respond to underlying disc cells. There is a graded requirement for Dpp signaling components for G1 arrest in the eye disc, with more stringent requirements further anteriorly where signaling is lower. The signaling level defines the cell cycle response, because elevated signaling through expression of an activated Thickveins receptor molecule arrested cells at more anterior locations. Very anterior regions of the eye disc were not arrested in response to activated receptor, however, and evidence is presented that expression of the Homothorax protein may contribute to this protection. By contrast to activated Thickveins, ectopic expression of processed Dpp leads to very high levels of Mad phosphorylation which appear to have non-physiological consequences.ConclusionsG1 arrest occurs at a threshold level of Dpp signaling within a morphogen gradient in the anterior eye. G1 arrest is specific for one competent domain in the eye disc, allowing Dpp signaling to promote growth at earlier developmental stages.

Tissue-specific interplay between copper uptake and efflux in Drosophila

The vinegar fly Drosophila melanogaster is proving to be an excellent system to study the in vivo regulation of the essential metal copper. The Ctr1A/B and DmATP7 copper transport proteins have well-established roles in Drosophila copper uptake and efflux, respectively. Both Ctr1A and DmATP7 are essential genes, whereas Ctr1B mutants are viable but die in excess or depleted copper conditions. Less is known about the tissue-specific requirements for these three genes and how they interact to maintain copper homeostasis in different cell types. Here, we use targeted overexpression and suppression of each gene to examine these questions in vivo. We find that in the epidermal cells that form the adult thoracic and abdominal cuticle, Ctr1A plays a major role in copper uptake, whereas Ctr1B plays only a minor supporting role and DmATP7, as previously shown, is essential for transfer of copper to the trans-Golgi network. We also find that the copper chaperone dSco1 appears necessary for supplying the mitochondria with copper in these tissues. In contrast, in the developing Drosophila eye, DmATP7 appears to be non-essential unless copper levels in these cells are artificially elevated. Again, Ctr1A is the main copper uptake gene in the eye, but when ectopically expressed, Ctr1B has greater phenotypic effects than Ctr1A. Furthermore, Ctr1A and Ctr1B show a dramatic synergistic interaction, indicating their relationship is more complicated than a simply additive one and that they may in fact act cooperatively for optimal copper import.

Echinoid regulates Flamingo endocytosis to control ommatidial rotation in theDrosophilaeye

Planar cell polarity (PCP) refers to a second polarity axis orthogonal to the apicobasal axis in the plane of the epithelium. The molecular link between apicobasal polarity and PCP is largely unknown. During Drosophila eye development, differentiated photoreceptors form clusters that rotate independently of the surrounding interommatidial cells (ICs). Here, we demonstrate that both Echinoid (Ed), an adherens junction-associated cell adhesion molecule, and Flamingo (Fmi), a PCP determinant, are endocytosed via a clathrin-mediated pathway in ICs. Interestingly, we found that Ed binds the AP-2 adaptor and is required for the internalization of Fmi into ICs. Loss of ed led to increased amounts of Fmi on the cell membrane of non-rotating ICs and also to the misrotation of photoreceptor clusters. Importantly, overexpression of fmi in ICs alone was sufficient to cause misrotation of the adjacent photoreceptor clusters. Together, we propose that Ed, when internalized by AP-2, undergoes co-endocytosis with, and thereby decreases, Fmi levels on non-rotating ICs to permit correct rotation of ommatidial clusters. Thus, co-endocytosis of Ed and Fmi provides a link between apicobasal polarity and PCP.

The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner βHeavy-spectrin (βH), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of βH (βH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that βH coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.

The cell adhesion molecule Roughest depends on beta(Heavy)-spectrin during eye morphogenesis in Drosophila.

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner beta(Heavy)-spectrin (beta(H)), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of beta(H) (betaH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that beta(H) coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.