The timing of cell fate decisions is crucial for initiating pattern formation in the Drosophila eye.

The eye-antennal disc of Drosophila is composed of three cell layers: a columnar epithelium called the disc proper (DP); an overlying sheet of squamous cells called the peripodial epithelium (PE); and a strip of cuboidal cells that joins the other two cellular sheets to each other and comprises the outer margin (M) of the disc. The M cells play an important role in patterning the eye because it is here that the Hedgehog (Hh), Decapentaplegic (Dpp) and JAK/STAT pathways function to initiate pattern formation. Dpp signaling is lost from the margin of eyes absent (eya) mutant discs and, as a result, the initiation of retinal patterning is blocked. Based on these observations, Eya has been proposed to control the initiation of the morphogenetic furrow via regulation of Dpp signaling within the M. We show that the failure in pattern formation surprisingly results from M cells prematurely adopting a head epidermis fate. This switch in fate normally takes place during pupal development after the eye has been patterned. Our results suggest that the timing of cell fate decisions is essential for correct eye development.

Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system.

Damage in the nervous system induces a stereotypical response that is mediated by glial cells. Here, we use the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. We demonstrate that these cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. We found that this glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. We also present evidence that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that our results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS).

Hexagonal patterning of the Drosophila eye.

A complex network of transcription factor interactions propagates across the larval eye disc to establish columns of evenly-spaced R8 precursor cells, the founding cells of Drosophila ommatidia. After the recruitment of additional photoreceptors to each ommatidium, the surrounding cells are organized into their stereotypical pattern during pupal development. These support cells - comprised of pigment and cone cells - are patterned to encapsulate the photoreceptors and separate ommatidia with an hexagonal honeycomb lattice. Since the proteins and processes essential for correct eye patterning are conserved, elucidating how these function and change during Drosophila eye patterning can substantially advance our understanding of transcription factor and signaling networks, cytoskeletal structures, adhesion complexes, and the biophysical properties of complex tissues during their morphogenesis. Our understanding of many of these aspects of Drosophila eye patterning is largely descriptive. Many important questions, especially relating to the regulation and integration of cellular events, remain.

The Krüppel-like factor Cabut has cell cycle regulatory properties similar to E2F1.

Using a gain-of-function screen in Drosophila, we identified the Krüppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and our data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells.

Inhibition of kinase IKKß suppresses cellular abnormalities induced by the human papillomavirus oncoprotein HPV 18E6.

Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a Drosophila 'HPV 18 E6' model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKK[Formula: see text]-a regulator of NF-κB-as an enhancer of E6-induced cellular defects. We demonstrate that inhibition of IKK[Formula: see text] reduces Magi degradation and that this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKK[Formula: see text] suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKK[Formula: see text] and E6 is conserved in human cells: inhibition of IKK[Formula: see text] blocked the growth of cervical cancer cells, suggesting that IKK[Formula: see text] may serve as a novel therapeutic target for HPV-mediated cancers.

Evolution of Insect Color Vision: From Spectral Sensitivity to Visual Ecology.

Color vision is widespread among insects but varies among species, depending on the spectral sensitivities and interplay of the participating photoreceptors. The spectral sensitivity of a photoreceptor is principally determined by the absorption spectrum of the expressed visual pigment, but it can be modified by various optical and electrophysiological factors. For example, screening and filtering pigments, rhabdom waveguide properties, retinal structure, and neural processing all influence the perceived color signal. We review the diversity in compound eye structure, visual pigments, photoreceptor physiology, and visual ecology of insects. Based on an overview of the current information about the spectral sensitivities of insect photoreceptors, covering 221 species in 13 insect orders, we discuss the evolution of color vision and highlight present knowledge gaps and promising future research directions in the field.

An E3 ubiquitin ligase, cullin-4 regulates retinal differentiation in Drosophila eye.

During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.

Color vision in insects: insights from Drosophila.

Color vision is an important sensory capability that enhances the detection of contrast in retinal images. Monochromatic animals exclusively detect temporal and spatial changes in luminance, whereas two or more types of photoreceptors and neuronal circuitries for the comparison of their responses enable animals to differentiate spectral information independent of intensity. Much of what we know about the cellular and physiological mechanisms underlying color vision comes from research on vertebrates including primates. In insects, many important discoveries have been made, but direct insights into the physiology and circuit implementation of color vision are still limited. Recent advances in Drosophila systems neuroscience suggest that a complete insect color vision circuitry, from photoreceptors to behavior, including all elements and computations, can be revealed in future. Here, we review fundamental concepts in color vision alongside our current understanding of the neuronal basis of color vision in Drosophila, including side views to selected other insects.

Novel type of sub-retinal pigment shield in the miniaturized compound eye of Trichogramma evanescens.

Pigment granules, found in different cell types of the retina in insect compound eyes, fulfill important functions. They isolate the individual ommatidia from stray light, regulate the angular sensitivity, and restrict the light that reaches the photoreceptor according to ambient light intensities. Descriptions of pigment cells within the retina are included in ultrastructural eye descriptions, but knowledge of pigment cell types beneath the retina and basal matrix (BM) are relatively limited in insects. In the miniaturized parasitoid wasp Trichogramma evanescens Westwood 1833, a sub-retinal pigment shield is formed by pigment-bearing cells, which appear in two-dimensional TEM sections to form a separate population beneath the BM. By using three-dimensional reconstructions of serial-section transmission electron microscopy, it was possible to reveal that the sub-retinal pigment shield of T. evanescens is not formed by a separate cell type, but by extensions of the lateral rim pigment cells that penetrate gaps in the BM. The reconstruction is supported by evidence from a statistical analysis of pigment granule volumes of all pigment bearing cell types in the retina and rim region. The study reveals the first known case of the participation of lateral rim cells in a sub-retinal pigment shield in an insect eye. As neither pigmented extensions of secondary pigment cells, nor pigment granules in the extensions of the cone cell projections are present above the BM in T. evanescens, the sub-retinal extensions of the lateral rim cells can be seen as a functional adaptation to miniaturization in order to maintain a proximal shielding function.

Optic lobe organization in stomatopod crustacean species possessing different degrees of retinal complexity.

Stomatopod crustaceans possess tripartite compound eyes; upper and lower hemispheres are separated by an equatorial midband of several ommatidial rows. The organization of stomatopod retinas is well established, but their optic lobes have been studied less. We used histological staining, immunolabeling, and fluorescent tracer injections to compare optic lobes in two 6-row midband species, Neogonodactylus oerstedii and Pseudosquilla ciliata, to those in two 2-row midband species, Squilla empusa and Alima pacifica. Compared to the 6-row species, we found structural differences in all optic neuropils in both 2-row species. Photoreceptor axons from 2-row midband ommatidia supply two sets of lamina cartridges; however, conspicuous spaces lacking lamina cartridges are observed in locations corresponding to where the cartridges of the upper four ommatidial rows of 6-row species would exist. The tripartite arrangement and enlarged projections containing fibers associated with the two rows of midband ommatidia can be traced throughout the entire optic lobe. However, 2-row species lack some features of medullar and lobular neuropils in 6-row species. Our results support the hypothesis that 2-row midband species are derived from a 6-row ancestor, and suggest specializations in the medulla and lobula found solely in 6-row species are important for color and polarization analysis.

A Genetic Screen in Drosophila To Identify Novel Regulation of Cell Growth by Phosphoinositide Signaling.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

Light input pathways to the circadian clock of insects with an emphasis on the fruit fly Drosophila melanogaster.

Light is the most important Zeitgeber for entraining animal activity rhythms to the 24-h day. In all animals, the eyes are the main visual organs that are not only responsible for motion and colour (image) vision, but also transfer light information to the circadian clock in the brain. The way in which light entrains the circadian clock appears, however, variable in different species. As do vertebrates, insects possess extraretinal photoreceptors in addition to their eyes (and ocelli) that are sometimes located close to (underneath) the eyes, but sometimes even in the central brain. These extraretinal photoreceptors contribute to entrainment of their circadian clocks to different degrees. The fruit fly Drosophila melanogaster is special, because it expresses the blue light-sensitive cryptochrome (CRY) directly in its circadian clock neurons, and CRY is usually regarded as the fly's main circadian photoreceptor. Nevertheless, recent studies show that the retinal and extraretinal eyes transfer light information to almost every clock neuron and that the eyes are similarly important for entraining the fly's activity rhythm as in other insects, or more generally spoken in other animals. Here, I compare the light input pathways between selected insect species with a focus on Drosophila's special case.

Identification of CR43467 encoding a long non-coding RNA as a novel genetic interactant with dFIG4, a CMT-causing gene.

The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. We previously performed modifier screening on the dFIG4 knockdown-induced rough eye phenotype and identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA) as genetic interactants with dFIG4. In the present study, in more extensive genetic screening, we found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, we established a CR43467-specific knockdown line using the CRISPR-dCas9 system. By using this system, we demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. The knockdown of CR43467 also suppressed the rough eye phenotype induced by other peripheral neuropathy-related genes, such as dCOA7, dHADHB, and dPDHB. We herein identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.

The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles.

In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. Here we show that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. Our results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina.

A New Rhodopsin Influences Light-dependent Daily Activity Patterns of Fruit Flies.

Rhodopsin 7 ( Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000, but its function has remained elusive. We generated an Rh7 null mutant ( Rh70) by P element-mediated mutagenesis and found that an absence of Rh7 had significant effects on fly activity patterns during light-dark (LD) cycles: Rh70 mutants exhibited less morning activity and a longer siesta than wild-type controls. Consistent with these results, we found that Rh7 appears to be expressed in a few dorsal clock neurons that have been previously implicated in the control of the siesta. We also found putative Rh7 expression in R8 photoreceptor cells of the compound eyes and in the Hofbauer-Buchner eyelets, which have been shown to control the precise timing of locomotor activity. The absence of Rh7 alone impaired neither the flies' responses to constant white light nor the ability to follow phase shifts of white LD cycles. However, in blue light (470 nm), Rh70 mutants needed significantly longer to synchronize than wild-type controls, suggesting that Rh7 is a blue light-sensitive photopigment with a minor contribution to circadian clock synchronization. In combination with mutants that lacked additionally cryptochrome-based and/or eye-based light input to the circadian clock, the absence of Rh7 provoked slightly stronger effects.

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.

Characterization of the first-order visual interneurons in the visual system of the bumblebee (Bombus terrestris).

The bumblebee (Bombus terrestris) has become a common model animal in the study of various aspects of vision and visually guided behavior. Although the bumblebee visual system has been studied to some extent, little is known about the functional role of the first visual neuropil, the lamina. In this work, we provide an anatomical and electrophysiological description of the first-order visual interneurons, lamina monopolar cells (LMCs), of the bumblebee. Using intracellular recording coupled with dye injection, we found that bumblebee LMCs morphologically resemble those found in the honeybee, although only the LMC type L1 cells could be morphologically matched directly between the species. LMCs could also be classified on the basis of their light response properties as spiking or non-spiking. We also show that some bumblebee LMCs can produce spikes during responses to stimulation with naturalistic light contrasts, a property unusual for these neurons.

Neural organization of afferent pathways from the stomatopod compound eye.

Crustaceans and insects share many similarities of brain organization suggesting that their common ancestor possessed some components of those shared features. Stomatopods (mantis shrimps) are basal eumalacostracan crustaceans famous for their elaborate visual system, the most complex of which possesses 12 types of color photoreceptors and the ability to detect both linearly and circularly polarized light. Here, using a palette of histological methods we describe neurons and their neuropils most immediately associated with the stomatopod retina. We first provide a general overview of the major neuropil structures in the eyestalks lateral protocerebrum, with respect to the optical pathways originating from the six rows of specialized ommatidia in the stomatopod's eye, termed the midband. We then focus on the structure and neuronal types of the lamina, the first optic neuropil in the stomatopod visual system. Using Golgi impregnations to resolve single neurons we identify cells in different parts of the lamina corresponding to the three different regions of the stomatopod eye (midband and the upper and lower eye halves). While the optic cartridges relating to the spectral and polarization sensitive midband ommatidia show some specializations not found in the lamina serving the upper and lower eye halves, the general morphology of the midband lamina reflects cell types elsewhere in the lamina and cell types described for other species of Eumalacostraca.

A population of G2-arrested cells are selected as sensory organ precursors for the interommatidial bristles of the Drosophila eye.

Cell cycle progression and differentiation are highly coordinated during the development of multicellular organisms. The mechanisms by which these processes are coordinated and how their coordination contributes to normal development are not fully understood. Here, we determine the developmental fate of a population of precursor cells in the developing Drosophila melanogaster retina that arrest in G2 phase of the cell cycle and investigate whether cell cycle phase-specific arrest influences the fate of these cells. We demonstrate that retinal precursor cells that arrest in G2 during larval development are selected as sensory organ precursors (SOPs) during pupal development and undergo two cell divisions to generate the four-cell interommatidial mechanosensory bristles. While G2 arrest is not required for bristle development, preventing G2 arrest results in incorrect bristle positioning in the adult eye. We conclude that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. Our results suggest that the control of cell cycle progression refines cell fate decisions and that the relationship between these two processes is not necessarily deterministic.

Not flying blind: a comparative study of photoreceptor function in flying and non-flying cockroaches.

Flying is often associated with superior visual performance, as good vision is crucial for detection and implementation of rapid visually guided aerial movements. To understand the evolution of insect visual systems it is therefore important to compare phylogenetically related species with different investments in flight capability. Here, we describe and compare morphological and electrophysiological properties of photoreceptors from the habitually flying green cockroach Panchlora nivea and the American cockroach Periplaneta americana, which flies only at high ambient temperatures. In contrast to Periplaneta, ommatidia in Panchlora were characterized by two-tiered rhabdom, which might facilitate detection of polarized light while flying in the dark. In patch-clamp experiments, we assessed the absolute sensitivity to light, elementary and macroscopic light-activated current and voltage responses, voltage-activated potassium (Kv) conductances, and information transfer. Both species are nocturnal, and their photoreceptors were similarly sensitive to light. However, a number of important differences were found, including the presence in Panchlora of a prominent transient Kv current and a generally low variability in photoreceptor properties. The maximal information rate in Panchlora was one-third higher than in Periplaneta, owing to a substantially higher gain and membrane corner frequency. The differences in performance could not be completely explained by dissimilarities in the light-activated or Kv conductances; instead, we suggest that the superior performance of Panchlora photoreceptors mainly originates from better synchronization of elementary responses. These findings raise the issue of whether the evolutionary tuning of photoreceptor properties to visual demands proceeded differently in Blattodea than in Diptera.