Delta activity independent of its activity as a ligand of Notch

BACKGROUND: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. RESULTS: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. CONCLUSION: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo

The \u3ci\u3eDrosophila\u3c/i\u3e T-box Transcription Factor Midline Functions Within the Notch-Delta Signaling Pathway To Specify Sensory Organ Precursor Cell Fates and Regulates Cell Survival Within the Eye Imaginal Disc

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch-Delta signaling pathway essential for specifying the fates of sensory organ precursor (SOP) cells. These findings complement an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in unique neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch-Delta signaling hierarchy and is essential for maintaining cell viability by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis. © 2013 Elsevier Ireland Ltd

A Phenotypic And Genetic Characterization Of The Cell Adhesion Molecules Echinoid And Friend-Of-Echinoid In The Directed Cell Movements Of Ommatidial Rotation During Drosophila Eye Development.

Correct development of multicellular organisms relies on the precise patterning of cells, which must respond to and interpret specific cues that instruct the cells to differentiate and often undergo directed cell movements and rearrangements to give rise to functional tissues and organs. Differential adhesion between the stationary and mobile cells permits and promotes these cellular movements, effecting patterning of cells and tissues. During Drosophila eye development, groups of cells, the ommatidial precursors, undergo a 90° rotational movement within a matrix of stationary cells, providing the cell motility readout of tissue polarity. The mechanisms that regulate ommatidial rotation are not well understood. In order to better understand how ommatidia coordinate cell signaling and cell adhesion to regulate the directed cell movement of ommatidial rotation, I investigated the roles of two cell adhesion molecules, Echinoid: Ed) and Friend-of-Echinoid: Fred), in this process. Initially, I characterized the misrotation phenotypes resulting from loss-of-function mutations in these two genes, and used a genetic approach to ascertain that they function during larval development and cooperate to regulate rotation. To understand the underlying mechanism by which ed and fred regulate rotation, I performed a row-by-row analysis of Ed and Fred protein localization during ommatidial rotation, and found that these proteins localize in patterns that are consistent with an affect on cell-cell adhesion. This observation led to the hypothesis that different levels of Ed or Fred in rotating vs. nonrotating cells provide a permissive environment for cell movement at the beginning of ommatidial rotation. Beginning midway through ommatidial rotation, equalizing levels of these proteins in the ommatidial cells and the interommatidial cells leads to a restrictive environment, thus slowing ommatidial rotation. In support of this hypothesis, I demonstrate that manipulating levels of these proteins and interfering with the establishment of the early permissive environment slows ommatidial rotation. My work also provides evidence that Ed and Fred may regulate signaling in the slow phase of ommatidial rotation. Mosaic analysis identified a requirement for ed and fred in photoreceptors R1, R6, R7 and the cone cells for proper ommatidial rotation. In addition, I used a genetic approach to identify potential interactors of ed and fred in rotation, and found that both genes interact with two downstream effectors of Egf signaling: the Mapk/Pnt transcriptional output and the Cno cytoskeletal/junctional output. Furthermore, my analysis of the cno loss-of-function phenotype provides the first indication that Cno inhibits ommatidial rotation. Egf signaling promotes ommatidial rotation, although the underlying mechanism is unclear. I hypothesize that Egfr signaling promotes ommatidial rotation by inhibiting Cno activity in the ommatidial cells. As ommatidial rotation slows, Ed and Fred cooperate to regulate the Egf receptor in R1, R6, R7 and the cone cells, and increased inhibition of the Egf receptor as Ed levels rise leads to an increase in Cno activity and the cessation of ommatidial rotation. Using a genetic approach, I also identified the tissue polarity genes as interactors of ed and fred in rotation. Intriguingly, ed and fred specifically modify different subsets of the TP genes. Mosaic analysis of the tissue polarity gene strabismus: stbm) identified a requirement for stbm in photoreceptor R7, thus providing the first indication of a role for a tissue polarity gene outside of photoreceptors R3 and R4 to regulate some aspect of tissue polarity

The Identification of the Mid Transcription Factor Regulatory Network Specifying Cell Fate and Regulating Survival in the \u3ci\u3eDrosophila\u3c/i\u3e Eye

The Drosophila melanogaster T-box transcription factor Midline (Mid) exhibits a high degree of structural and functional conservation with its vertebrate homolog Tbx-20. Both Mid and Tbx-20 regulate cell-fate specification with in diverse tissues including the central nervous system (CNS) and heart. Although, some important studies reported that Tbx-20 transcripts express in a wide range of developing mammalian eye tissues including those of the human fetus, the function of this transcription factor is unknown in the development of eye tissue. This current study is the first attempt to show that Mid and its para log H15 are expressed within distinct ommatidial cell types including photoreceptor neurons and sensory organ precursor (SOP) cells during early stages of pupal eye imaginal disc morphogensis and also identities a Mid transcription factor network regulating eye development. Reducing the expression of mid transcripts within eye disc tissues using RNA interference (mid-RNAi) results in the loss of interommatidial bristles (JOBs) in the adult eye due to the misspecification of sensory organ precursor (SOP) cells and increased levels of apoptosis induced during earlier stages of pupal development. Since the Notch-Delta signaling pathway specifies the SOP cell fate, we sought to place mid within the Notch-Delta genetic hierarchy specifying SOP cell fates. We determined that Mid functions downstream of the Notch receptor and upstream of the Enhancer of Split gene complex [E(Spl)]. We also discovered mid collaborates with two Notch pathway members also implicated in the regulation of cell survival, extramacrochaetae (emc) and senseless (sens). Moreover, toward identifying the Mid regulatory transcription factor network specifying cell fate, we found that mid collaborates with dFOXO. The dFOXO transcription factor is distinct in that it regulates cell proliferation and homeostasis within Insulin regulated stress induced pathway. The culmination of these studies suggests that Mid regulates cell fate specification in collaboration with Notch-Delta signaling pathway and also play important role in cell survival pathways essential for maintaining homeostasis

Sensory Organ Morphogenesis in Caenorhabditis Elegans

Sensory organs are the gates through which information flows into the nervous system. In most animals, such organs consist of sensory neurons, which can transform stimuli into changes in their membrane potential, and glial cells, which establish a niche important for the morphogenesis and function of the neurons. Although similar glial compartments are seen throughout the nervous system, their morphogenesis is poorly understood. In the work presented here, I use the main sensory organ of Caenorhabditis elegans, the amphid, as a model system for understanding how glia form these compartments. First, by the interpretation of electron microscopy reconstructions of the developing amphid, I was able to uncover a role for daf-6/Patched, an established regulator of amphid morphogenesis, in restricting the size of the sensory compartment. Second, I sought to identify genes acting in the opposite direction, in expanding the sensory compartment, by cloning and characterizing suppressors of daf-6. Through this approach I discovered that lit-1/Nlk acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, my genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. I show that actin also lines the sensory compartment, and that WASP is important for compartment expansion, potentially by functioning in the same pathway as LIT-1. These results suggest that the daf-6 and lit-1 glial pathways constitute a rheostat used to control sensory compartment size. Finally, I also identify a role for the retromer complex, a module involved in the recycling of transmembrane proteins and membrane material from the endosomes to the Golgi apparatus, in amphid morphogenesis. Similar to lit-1, mutations of retromer components suppress daf-6, suggesting that the retromer could also act in promoting sensory compartment expansion

Endocytic Regulation of Notch Signaling in Drosophila Melanogaster Neural Progenitor Cells

Notch signaling is a ubiquitously used signaling pathway that is highly conserved and used throughout metazoan development. Understanding the regulation of Notch signaling is becoming increasingly important in determining the mechanism and treatment for the myriad of human Notch-related diseases. In Drosophila. melanogaster, the development of external sensory organs provides a context in which Notch can be manipulated and phenotypes can be easily interpreted. Here, we expand upon the growing field of Notch regulation through endocytic trafficking by examining the role of Numb and Sara endosomes. Numb is a potent Notch inhibitor whose function is conserved in higher organisms, but whose mechanism of action has remained elusive. In this study, we dispel a previous hypothesis that Numb promotes Notch internalization and instead demonstrate that Numb is a suppressor of Notch endocytic recycling. In support of this, we show that Numb is necessary and sufficient for Notch trafficking to late endosomes/lysosomes to promote degradation. We do this by employing a novel technique that is able to distinguish recycled Notch from other populations within the cell. In addition, we show that the cell fate determinant Lethal (2) Giant Larvae, can also suppress Notch recycling, but at a step upstream of Numb. Results from this study help to answer a long-standing questions in the field of Notch signaling, by demonstrating the role of Numb in Drosophila. We also extended our investigation of endocytic Notch regulation by determining the role of a sub-population of early endosomes positive for Sara. We show that these Sara endosomes are trafficked preferentially to Notch activated cells, but do not contain appreciable levels of Notch. While we conclude that the Sara endosomes do not seem relevant to Notch signaling, we show that the mechanism of Sara endosome trafficking is likely tied to global anterior-posterior cues and not related to cell fate determinants. Results from our studies have important implications in the designing of treatments for Notch related dysfunctions that depend on an exquisite understanding of Notch regulation

The role of tumour suppressor fat in the patterning and development of the Drosophila eye.

The atypical cadherin and tumour suppressor, Fat, is a molecule with a well-defined role in planar cell polarity. Specifically, it is one of a number of molecules that provides directional cues to cells in the establishment of tissue polarity in a variety of systems and organisms. Less understood is its function as a tumour suppressor. The work presented here is a characterisation of the fat phenotypes, with a view to understanding the mechanisms underlying Fat's ability to restrict proliferation. Using the Drosophila eye as a model system, I have addressed this problem in three ways. First, I examined the patterns of expression of various cell cycle molecules, as well as markers of various stages of the cell cycle. This analysis led me to propose that mutations in fat result in a de-sensitisation of cells to the signalling cascades that are responsible for triggering a transition from proliferation to differentiation in the course of development. As such, I then looked at the ways in which Fat affects the activities of the main signalling pathways involved in Drosophila eye development: Notch, EGFR, Hedgehog, Dpp, and Wingless. From this analysis I found that Fat function can be connected to EGFR, Hedgehog and Wingless signalling. However, I found no evidence to implicate these pathways in Fat-related overgrowth. Finally, I present evidence of parallels between phenotypes of fat and those of components of the Hippo signalling pathway. I show that Fat is required for localisation of Expanded, the most upstream component of this pathway identified to date. I further demonstrate that this localisation is necessary for the function of this pathway and that compromises to this function contribute to Ft-related overgrowth. I also present evidence that suggests this role in growth suppression is independent of Fat's role in dorsal-ventral polarity in the eye