Drosophila Eye Development Research Articles

Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1–R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.

The Id protein Extramacrochaetae restrains the E protein Daughterless to regulate Notch, Rap1, and Sevenless within the R7 equivalence group of the Drosophila eye.

The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds.

Par3/bazooka binds NICD and promotes notch signaling during Drosophila development

The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.

A cellular tilting mechanism important for dynamic tissue shape changes and cell differentiation in Drosophila

Dynamic changes in three-dimensional cell shape are important for tissue form and function. In the developing Drosophila eye, photoreceptor differentiation requires the progression across the tissue of an epithelial fold known as the morphogenetic furrow. Morphogenetic furrow progression involves apical cell constriction and movement of apical cell edges. Here, we show that cells progressing through the morphogenetic furrow move their basal edges in opposite direction to their apical edges, resulting in a cellular tilting movement. We further demonstrate that cells generate, at their basal side, oriented, force-generating protrusions. Knockdown of the protein kinase Src42A or photoactivation of a dominant-negative form of the small GTPase Rac1 reduces protrusion formation. Impaired protrusion formation stalls basal cell movement and slows down morphogenetic furrow progression and photoreceptor differentiation. This work identifies a cellular tilting mechanism important for the generation of dynamic tissue shape changes and cell differentiation.

A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor.

During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (ShhC) during its release. Although ShhC is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of ShhC during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of ShhC to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that ShhC was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated ShhC requires an N-peptide of a defined minimum length for its signaling function to Ptch.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Dachshund and C-terminal Binding Protein bind directly during Drosophila eye development.

The transcription factor Dachshund (Dac) and the transcriptional co-regulator C-terminal Binding Protein (CtBP) were identified as the retinal determination factors during Drosophila eye development . A previous study established that Dac and CtBP interact genetically during eye development. Co-immunoprecipitation assays suggested that both molecules interact in the Drosophila larval eye-antennal disc. Our present study shows that Dac and CtBP bind each other directly, as determined by GST pull-down assays. Thus, our results demonstrate the molecular mechanism of Dac and CtBP interaction and suggest the direct binding of these two transcription regulators in the cells of the eye disc promotes the Drosophila eye specification.

G.3.2 is a novel allele of the gene connector enhancer of ksr ( cnk ) in Drosophila melanogaster.

Genetic screens in Drosophila melanogaster have long been used to identify genes found in a variety of developmental, cellular, and behavioral processes. Here we describe the characterization and mapping of a mutation identified in a conditional screen for genetic regulators of cell growth and cell division. Within a Flp/FRT system, mutant G.3.2 results in a reduction of mutant tissue and a rough eye phenotype. We find that G.3.2 maps to the gene cnk , providing further support that cnk is a critical gene in Drosophila eye development. This mutant was characterized, mapped and sequenced by undergraduate students within the Fly-CURE consortium.

Cell Cycle Regulation by NF-YC in Drosophila Eye Imaginal Disc: Implications for Synchronization in the Non-Proliferative Region.

Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. Here, we identify the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). Our findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, we identify the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands our understanding of NF-YC's role from cell cycle control to encompass developmental processes.

N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aβ42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aβ42 (GMR > Aβ42) in developing Drosophila eye retinal neurons results in Aβ42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aβ42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aβ42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. We found that gain-of-function of Mnat9 rescues GMR > Aβ42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. Here, we propose a new neuroprotective function of Mnat9 in downregulating the JNK signaling pathway to ameliorate Aβ42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aβ42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9 or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aβ42 and may provide new avenues for targeting neurodegeneration.

Cartesian genetic programming for image analysis of the developing drosophila eye

Automatic feature extraction methods have gained increasing attention in modern image processing. The confocal images of the single-layered epithelium of the developing Drosophila eye may form an excellent model system to develop methods for complex feature extraction. The aim of this work was to explore Cartesian genetic programming for determination of the boundaries of ommatidia, the light-sensitive units in the presumptive eye region. Application of Cartesian genetic programming for the analysis of Fasciclin III expression has shown good results. This opens interesting perspectives for further use of this technology in the automatic analysis of confocal images.

Cartesian Genetic Programming for Image Analysis of the Developing Drosophila Eye

Cartesian Genetic Programming for Image Analysis of the Developing Drosophila Eye

Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye.

The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. Here, we identify a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. Our work expands the range of functions and regulatory mechanisms under Hippo pathway control.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Chromatin remodeler Dmp18 regulates apoptosis by controlling H2Av incorporation in Drosophila imaginal disc development.

Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis. Inhibition of apoptosis suppressed the eye defects caused by Dmp18 deletion. Furthermore, loss of Dmp18 disrupted H2Av incorporation into chromatin, promoted H3K4me3, but reduced H3K27me3 modifications on the TSS regions of pro-apoptotic genes. These results indicate that Dmp18 negatively regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at pro-apoptotic gene loci for transcriptional regulation. Our study uncovers the role of Dmp18 in regulating apoptosis in Drosophila eye and wing development and provides insights into chromatin remodeling regulating apoptosis at the epigenetic levels.

Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay

The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3′UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.

Quantitative Relationships Between Growth, Differentiation, and Shape That Control Drosophila Eye Development and Its Variation.

The size of organs is critical for their function and often a defining trait of a species. Still, how organs reach a species-specific size or how this size varies during evolution are problems not yet solved. Here, we have investigated the conditions that ensure growth termination, variation of final size and the stability of the process for developmental systems that grow and differentiate simultaneously. Specifically, we present a theoretical model for the development of the Drosophila eye, a system where a wave of differentiation sweeps across a growing primordium. This model, which describes the system in a simplified form, predicts universal relationships linking final eye size and developmental time to a single parameter which integrates genetically-controlled variables, the rates of cell proliferation and differentiation, with geometrical factors. We find that the predictions of the theoretical model show good agreement with previously published experimental results. We also develop a new computational model that recapitulates the process more realistically and find concordance between this model and theory as well, but only when the primordium is circular. However, when the primordium is elliptical both models show discrepancies. We explain this difference by the mechanical interactions between cells, an aspect that is not included in the theoretical model. Globally, our work defines the quantitative relationships between rates of growth and differentiation and organ primordium size that ensure growth termination (and, thereby, specify final eye size) and determine the duration of the process; identifies geometrical dependencies of both size and developmental time; and uncovers potential instabilities of the system which might constraint developmental strategies to evolve eyes of different size.

Motif 1\xa0Binding Protein suppresses wingless to promote eye fate in Drosophila

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. We found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a “no-eye” phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a “no-eye” phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. We found that upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. Our data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

Histone deacetylase (Rpd3) regulates Drosophila early brain development via regulation of Tailless.

Tailless is a committed transcriptional repressor and principal regulator of the brain and eye development in Drosophila. Rpd3, the histone deacetylase, is an established repressor that interacts with co-repressors like Sin3a, Prospero, Brakeless and Atrophin. This study aims at deciphering the role of Rpd3 in embryonic segmentation and larval brain development in Drosophila. It delineates the mechanism of Tailless regulation by Rpd3, along with its interacting partners. There was a significant reduction in Tailless in Rpd3 heteroallelic mutant embryos, substantiating that Rpd3 is indispensable for the normal Tailless expression. The expression of the primary readout, Tailless was correlative to the expression of the neural cell adhesion molecule homologue, Fascilin2 (Fas2). Rpd3 also aids in the proper development of the mushroom body. Both Tailless and Fas2 expression are reported to be antagonistic to the epidermal growth factor receptor (EGFR) expression. The decrease in Tailless and Fas2 expression highlights that EGFR is upregulated in the larval mutants, hindering brain development. This study outlines the axis comprising Rpd3, dEGFR, Tailless and Fas2, which interact to fine-tune the early segmentation and larval brain development. Therefore, Rpd3 along with Tailless has immense significance in early embryogenesis and development of the larval brain.

Abstract IA11: Hippo signaling Is Intrinsically regulated during cell cycle progression

Abstract The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration, and its dysregulation is widely implicated in various cancers. Hippo signaling has been recently identified as a major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival, and hepatocellular carcinoma (HCC) formation. We have found that Hippo/YAP signaling cell-autonomously inhibits HCC proliferation by interacting with Wnt and Notch signaling. We have also found that Hippo/YAP signaling cell-nonautonomously inhibits HCC by shaping its inflammatory microenvironment. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, LATS kinases and YAP/TAZ transcription factors, are regulated during cell cycle progression by core cell cycle machinery components. Furthermore, we show in Drosophila eye and wing development that cell cycle is required in vivo to regulate Hippo signaling with a conserved mechanism. Cell cycle control of Hippo signaling represents a previously unappreciated and evolutionarily conserved new layer of Hippo signaling regulation. Citation Format: Yingzi Yang, Wantae Kim, Xiaohui Wang. Hippo signaling Is Intrinsically regulated during cell cycle progression [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA11.