Probing the conserved roles of cut in the development and function of optically different insect compound eyes.

Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. Here, we perform RNAi-based knockdowns of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, we investigate two optically different compound eyes: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, we find that multiple aspects of ocular formation are disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, our findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role.

Semper's cells in the insect compound eye: Insights into ocular form and function.

The arthropod compound eye represents one of two major eye types in the animal kingdom and has served as an essential experimental paradigm for defining fundamental mechanisms underlying sensory organ formation, function, and maintenance. One of the most distinguishing features of the compound eye is the highly regular array of lens facets that define individual eye (ommatidial) units. These lens facets are produced by a deeply conserved quartet of cuticle-secreting cells, called Semper cells (SCs). Also widely known as cone cells, SCs were originally identified for their secretion of the dioptric system, i.e. the corneal lens and underlying crystalline cones. Additionally, SCs are now known to execute a diversity of patterning and glial functions in compound eye development and maintenance. Here, we present an integrated account of our current knowledge of SC multifunctionality in the Drosophila compound eye, highlighting emerging gene regulatory modules that may drive the diverse roles for these cells. Drawing comparisons with other deeply conserved retinal glia in the vertebrate single lens eye, this discussion speaks to glial cell origins and opens new avenues for understanding sensory system support programs.

Genetic control of cellular morphogenesis in Müller glia.

Cell shape is critical for the proper function of every cell in every tissue in the body. This is especially true for the highly morphologically diverse neural and glia cells of the central nervous system. The molecular processes by which these, or indeed any, cells gain their particular cell-specific morphology remain largely unexplored. To identify the genes involved in the morphogenesis of the principal glial cell type in the vertebrate retina, the Müller glia (MG), we used genomic and CRISPR based strategies in zebrafish (Danio rerio). We identified 41 genes involved in various aspects of MG cell morphogenesis and revealed a striking concordance between the sequential steps of anatomical feature addition and the expression of cohorts of functionally related genes that regulate these steps. We noted that the many of the genes preferentially expressed in zebrafish MG showed conservation in glia across species suggesting evolutionarily conserved glial developmental pathways.

Mechanisms of Müller glial cell morphogenesis.

Müller Glia (MG), the radial glia cells of the retina, have spectacular morphologies subserving their enormous functional complexity. As early as 1892, the great neuroanatomist Santiago Ramon y Cajal studied the morphological development of MG, defining several steps in their morphogenesis [1,2]. However, the molecular cues controlling these developmental steps remain poorly understood. As MG have roles to play in every cellular and plexiform layer, this review discusses our current understanding on how MG morphology may be linked to their function, including the developmental mechanisms involved in MG patterning and morphogenesis. Uncovering the mechanisms governing glial morphogenesis, using transcriptomics and imaging, may provide shed new light on the pathophysiology and treatment of human neurological disorders.

Multifunctional glial support by Semper cells in the Drosophila retina.

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates.

The cuticular nature of corneal lenses in Drosophila melanogaster.

The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, we used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, we conducted in situ hybridization at 50% pupal development, a key age for lens secretion. Our results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes.

Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

Drosophila Dyrk2 plays a role in the development of the visual system.

The DYRKs (dual-specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that are associated with a number of neurological disorders, but whose biological targets are poorly understood. Drosophila encodes three Dyrks: minibrain/Dyrk1A, DmDyrk2, and DmDyrk3. Here we describe the creation and characterization of a DmDyrk2 null allele, DmDyrk2(1w17) . We provide evidence that the smell impaired allele smi35A(1) , is likely to encode DmDyrk2. We also demonstrate that DmDyrk2 is expressed late in the developing third antennal segment, an anatomical structure associated with smell. In addition, we find that DmDyrk2 is expressed in the morphogenetic furrow of the developing eye, that loss of DmDyrk2 in the eye produced a subtle but measurable defect, and that ectopic DmDyrk2 expression in the eye produced a strong rough eye phenotype characterized by increased secondary, tertiary and bristle interommatidial cells. This phenotype was dependent on DmDyrk2 kinase activity and was only manifest when expressed in post-mitotic non-neuronal progenitors. Together, these data indicate that DmDyrk2 is expressed in developing sensory systems, that it is required for the development of the visual system, and that the eye is a good model to identify DmDyrk2 targets.

Opposite feedbacks in the Hippo pathway for growth control and neural fate.

Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network-including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module-that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.

The lens in focus: a comparison of lens development in Drosophila and vertebrates.

The evolution of the eye has been a major subject of study dating back centuries. The advent of molecular genetics offered the surprising finding that morphologically distinct eyes rely on conserved regulatory gene networks for their formation. While many of these advances often stemmed from studies of the compound eye of the fruit fly, Drosophila melanogaster, and later translated to discoveries in vertebrate systems, studies on vertebrate lens development far outnumber those in Drosophila. This may be largely historical, since Spemann and Mangold's paradigm of tissue induction was discovered in the amphibian lens. Recent studies on lens development in Drosophila have begun to define molecular commonalities with the vertebrate lens. Here, we provide an overview of Drosophila lens development, discussing intrinsic and extrinsic factors controlling lens cell specification and differentiation. We then summarize key morphological and molecular events in vertebrate lens development, emphasizing regulatory factors and networks strongly associated with both systems. Finally, we provide a comparative analysis that highlights areas of research that would help further clarify the degree of conservation between the formation of dioptric systems in invertebrates and vertebrates.

Prospero and Pax2 combinatorially control neural cell fate decisions by modulating Ras- and Notch-dependent signaling.

BACKGROUND: The concept of an equivalence group, a cluster of cells with equal potential to adopt the same specific fate, has served as a useful paradigm to understand neural cell type specification. In the Drosophila eye, a set of five cells, called the 'R7 equivalence group', generates a single photoreceptor neuron and four lens-secreting epithelial cells. This choice between neuronal versus non-neuronal cell fates rests on differential requirements for, and cross-talk between, Notch/Delta- and Ras/mitogen-activated protein kinase (MAPK)-dependent signaling pathways. However, many questions remain unanswered related to how downstream events of these two signaling pathways mediate distinct cell fate decisions. RESULTS: Here, we demonstrate that two direct downstream targets of Ras and Notch signaling, the transcription factors Prospero and dPax2, are essential regulators of neuronal versus non-neuronal cell fate decisions in the R7 equivalence group. Prospero controls high activated MAPK levels required for neuronal fate, whereas dPax2 represses Delta expression to prevent neuronal fate. Importantly, activity from both factors is required for proper cell fate decisions to occur. CONCLUSIONS: These data demonstrate that Ras and Notch signaling are integrated during cell fate decisions within the R7 equivalence group through the combinatorial and opposing activities of Pros and dPax2. Our study provides one of the first examples of how the differential expression and synergistic roles of two independent transcription factors determine cell fate within an equivalence group. Since the integration of Ras and Notch signaling is associated with many developmental and cancer models, these findings should provide new insights into how cell specificity is achieved by ubiquitously used signaling pathways in diverse biological contexts.

Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia.

In the past, vast differences in ocular structure, development, and physiology throughout the animal kingdom led to the widely accepted notion that eyes are polyphyletic, that is, they have independently arisen multiple times during evolution. Despite the dissimilarity between vertebrate and invertebrate eyes, it is becoming increasingly evident that the development of the eye in both groups shares more similarity at the genetic level than was previously assumed, forcing a reexamination of eye evolution. Understanding the molecular underpinnings of cell type specification during Drosophila eye development has been a focus of research for many labs over the past 25 years, and many of these findings are nicely reviewed in Chapters 1 and 4. A somewhat less explored area of research, however, considers how these cells, once specified, develop into functional ocular structures. This review aims to summarize the current knowledge related to the terminal differentiation events of the retina, corneal lens, and pigmented epithelia in the fly eye. In addition, we discuss emerging evidence that the different functional components of the fly eye share developmental pathways and functions with the vertebrate eye.

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.

Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila.

A major question in development is how different specialized cell types arise from a common progenitor. In the adult Drosophila compound eye, color discrimination is achieved by UV-, blue- and green-sensitive photoreceptors (PRs). These different PR subsets arise from neuronal precursors called R7 and R8 cells. Recent studies have demonstrated that R7-based UV-sensitive PRs require the repression of R8-based blue/green-sensitive PR characteristics to properly develop. This repression is mediated by the transcription factor Prospero (Pros). Here, we report that Senseless (Sens), a Drosophila ortholog of the vertebrate Gfi1 transcription factor, plays an opposing role to Pros by both negatively regulating R7-based features and positively enforcing R8-based features during terminal differentiation. In addition, we demonstrate that Pros and Sens function together with the transcription factor Orthodenticle (Otd) to oppositely regulate R7 and R8 PR Rhodopsin gene expression in vitro. These data show that sens, previously shown to be essential for neuronal specification, also controls differentiation of specific neuronal subtypes in the retina. Interestingly, Pros has recently been shown to function as a tumor suppressor, whereas Gfi1 is a well-characterized oncogene. Thus, we propose that sens/pros antagonism is important for regulating many biological processes.