Justin P. Kumar

A Novel System for the Launch of Alphavirus RNA Synthesis Reveals a Role for the Imd Pathway in Arthropod Antiviral Response

Alphaviruses are RNA viruses transmitted between vertebrate hosts by arthropod vectors, primarily mosquitoes. How arthropods counteract alphaviruses or viruses per se is not very well understood. Drosophila melanogaster is a powerful model system for studying innate immunity against bacterial and fungal infections. In this study we report the use of a novel system to analyze replication of Sindbis virus (type species of the alphavirus genus) RNA following expression of a Sindbis virus replicon RNA from the fly genome. We demonstrate deficits in the immune deficiency (Imd) pathway enhance viral replication while mutations in the Toll pathway fail to affect replication. Similar results were observed with intrathoracic injections of whole virus and confirmed in cultured mosquito cells. These findings show that the Imd pathway mediates an antiviral response to Sindbis virus replication. To our knowledge, this is the first demonstration of an antiviral role for the Imd pathway in insects.

Dual transcriptional activities of SIX proteins define their roles in normal and ectopic eye development

The SIX family of homeodomain-containing DNA-binding proteins play crucial roles in both Drosophila and vertebrate retinal specification. In flies, three such family members exist, but only two, Sine oculis (So) and Optix, are expressed and function within the eye. In vertebrates, the homologs of Optix (Six3 and Six6) and probably So (Six1 and Six2) are also required for proper eye formation. Depending upon the individual SIX protein and the specific developmental context, transcription of target genes can either be activated or repressed. These activities are thought to occur through physical interactions with the Eyes absent (Eya) co-activator and the Groucho (Gro) co-repressor, but the relative contribution that each complex makes to overall eye development is not well understood. Here, we attempt to address this issue by investigating the role that each complex plays in the induction of ectopic eyes in Drosophila. We fused the VP16 activation and Engrailed repressor domains to both So and Optix, and attempted to generate ectopic eyes with these chimeric proteins. Surprisingly, we find that So and Optix must initially function as transcriptional repressors to trigger the formation of ectopic eyes. Both factors appear to be required to repress the expression of non-retinal selector genes. We propose that during early phases of eye development, SIX proteins function, in part, to repress the transcription of non-retinal selector genes, thereby allowing induction of the retina to proceed. This model of repression-mediated induction of developmental programs could have implications beyond the eye and might be applicable to other systems.

Building an ommatidium one cell at a time.

Since the discovery of a single white‐eyed male in a population of red eyed flies over 100 years ago (Morgan, 1910), the compound eye of the fruit fly, Drosophila melanogaster, has been a favorite experimental system for identifying genes that regulate various aspects of development. For example, a fair amount of what we know today about enzymatic pathways and vesicular transport is due to the discovery and subsequent characterization of eye color mutants such as white. Likewise, our present day understanding of organogenesis has been aided considerably by studies of mutations, such as eyeless, that either reduce or eliminate the compound eyes. But by far the phenotype that has provided levers into the greatest number of experimental fields has been the humble “rough” eye. The fly eye is composed of several hundred unit‐eyes that are also called ommatidia. These unit eyes are packed into a hexagonal array of remarkable precision. The structure of the eye is so precise that it has been compared with that of a crystal (Ready et al., 1976). Even the slightest perturbations to the structure of the ommatidium can be visually detected by light or electron microscopy. The cause for this is two‐fold: (1) any defect that affects the hexagonal geometry of a single ommatidium can and will disrupt the positioning of surrounding unit eyes thereby propagating structural flaws and (2) disruptions in genes that govern the development of even a single cell within an ommatidium will affect all unit eyes. In both cases, the effect is the visual magnification of even the smallest imperfection. Studies of rough eye mutants have provided key insights into the areas of cell fate specification, lateral inhibition, signal transduction, transcription factor networks, planar cell polarity, cell proliferation, and programmed cell death just to name a few. This review will attempt to summarize the key steps that are required to assemble each ommatidium. Developmental Dynamics 241:136–149, 2012.

Retinal determination the beginning of eye development.

The road to producing an eye begins with the decision to commit a population of cells to adopting an eye tissue fate, the process of retinal determination. Over the past decade and a half, a network of transcription factors has been found to mediate this process in all seeing animals. This retinal determination network is known to regulate not only tissue fate but also cell proliferation, pattern formation, compartment boundary establishment, and even retinal cell specification. The compound eye of the fruit fly, Drosophila melanogaster, has proven to be an excellent experimental system to study the mechanisms by which this network regulates organogenesis and tissue patterning. In fact the founding members of most of the gene families that make up this network were first isolated in Drosophila based on loss-of-function phenotypes that affect the eye. This chapter will highlight the history of discovery of the retinal determination network and will draw attention to the molecular and biochemical mechanisms that underlie our understanding of how the fate of the retina is determined.

My what big eyes you have: how the Drosophila retina grows.

The compound eye of the fruit fly, Drosophila melanogaster, has for decades been used extensively to study a number of critical developmental processes including tissue development, pattern formation, cell fate specification, and planar cell polarity. To a lesser degree it has been used to examine the cell cycle and tissue proliferation. Discovering the mechanisms that balance tissue growth and cell death in developing epithelia has traditionally been the realm of those using the wing disc. However, over the last decade a series of observations has demonstrated that the eye is a suitable and maybe even preferable tissue for studying tissue growth. This review will focus on how growth of the retina is controlled by the genes and pathways that govern the specification of tissue fate, the division of the epithelium into dorsal‐ventral compartments, the initiation, and progression of the morphogenetic furrow and the second mitotic wave.

Differential requirements for the Pax6(5a) genes eyegone and twin of eyegone during eye development in Drosophila

In eye development the tasks of tissue specification and cell proliferation are regulated, in part, by the Pax6 and Pax6(5a) proteins respectively. In vertebrates, Pax6(5a) is generated as an alternately spliced isoform of Pax6. This stands in contrast to the fruit fly, Drosophila melanogaster, which has two Pax6(5a) homologs that are encoded by the eyegone and twin of eyegone genes. In this report we set out to determine the respective contributions that each gene makes to the development of the fly retina. Here we demonstrate that both eyg and toe encode transcriptional repressors, are expressed in identical patterns but at significantly different levels. We further show, through a molecular dissection of both proteins, that Eyg makes differential use of several domains when compared to Toe and that the number of repressor domains also differs between the two Pax6(5a) homologs. We predict that these results will have implications for elucidating the functional differences between closely related members of other Pax subclasses.

Identification of Retinal Transformation Hot Spots in Developing Drosophila Epithelia

Background The retinal determination (RD) network is an evolutionarily conserved regulatory circuit that governs early events in the development of eyes throughout the animal kingdom. Ectopic expression of many members of this network leads to the transformation of non-retinal epithelia into eye tissue. An often-overlooked observation is that only particular cell-populations within a handful of tissues are capable of having their primary developmental instructions superseded and overruled. Methodology/Preliminary Findings Here we confirm that indeed, only a discrete number of cell populations within the imaginal discs that give rise to the head, antenna, legs, wings and halteres have the cellular plasticity to have their developmental fates altered. In contrast to previous reports, we find that all transformable cell populations do not lie within the TGFβ or Hedgehog signaling domains. Additionally neither signaling cascade alone is sufficient for non-retinal cell types to be converted into retinal tissue. The transformation “hot spots” that we have identified appear to coincide with several previously defined transdetermination “weak spots”, suggesting that ectopic eye formation is less the result of one network overriding the orders of another, as previously thought, but rather is the physical manifestation of redirecting cell populations of enormous cellular plasticity. We also demonstrate that the initiation of eye formation in non-retinal tissues occurs asynchronously compared to that of the normal eye suggesting that retinal development is not under the control of a global developmental clock. Conclusions/Significance We conclude that the subregions of non-retinal tissues that are capable of supporting eye formation represent specialized cell-populations that have a different level of plasticity than other cells within these tissues and may be the founder cells of each tissue.

Transcriptional activities of the Pax6 gene eyeless regulate tissue specificity of ectopic eye formation in Drosophila

Pax genes encode DNA binding proteins that play pivotal roles in the determination of complex tissues. Members of one subclass, Pax6, function as selector genes and play key roles in the retinal development of all seeing animals. Mutations within the Pax6 homologs including fly eyeless, mouse Small eye and human Pax6 lead to severe retinal defects in their respective systems. In Drosophila eyeless and twin of eyeless, play non-redundant roles in the developing retina. One particularly interesting characteristic of these genes is that, although expression of either gene can induce ectopic eye formation in non-retinal tissues, there are differences in the location and frequencies at which the eyes develop. eyeless induces much larger ectopic eyes, at higher frequencies, and in a broader range of tissues than twin of eyeless. In this report we describe a series of experiments conducted in both yeast and flies that has identified protein modules that are responsible for the differences in tissue transformation. These domains appear to contain transcriptional activator and repressor activity of distinct strengths. We propose a model in which the selective presence of these activities and their relative strengths accounts, in part, for the disparity to which ectopic eyes are induced in response to the forced expression of eyeless and twin of eyeless. The identification of both transcriptional activator and repressor activity within the Pax6 protein furthers our understanding of how this gene family regulates tissue determination.

Restriction of ectopic eye formation by Drosophila teashirt and tiptop to the developing antenna

In Drosophila, the retinal determination network comprises a set of nuclear factors whose loss‐of‐function phenotypes often include the complete or near total elimination of the developing eye. These genes also share the ability of being able to induce ectopic eye formation when forcibly expressed in nonretinal tissues such as the antennae, legs, halteres, wings, and genitals. However, it appears that the ability to redirect and transform tissue fates is limited; not all tissues and cell populations can be forced into adopting an eye fate. In this report, we demonstrate that ectopic eye formation by teashirt and its paralog tiptop, a potential new eye specification gene, is restricted to the developing antennae. Of interest, tiptop appears to be a more effective inducer of retinal formation than teashirt. A genetic screen for interacting proteins failed to identify paralog‐specific relationships suggesting that the differences between these two genes may be attributed instead to structural differences between the duplicates. We also demonstrate that in addition to being expressed in coincident patterns within the developing eye, both paralogs are transcribed at very similar levels. Developmental Dynamics 238:2202–2210, 2009.

Position dependent responses to discontinuities in the retinal determination network

The development of any cell and/or tissue is dependent upon interconnections between several signaling pathways and myriad transcription factors. It is becoming more apparent that these inputs are best studied, not as individual components, but rather as elements of a gene regulatory network. Over the last decade several networks governing the specification of single cells, individual organs and entire stages of development have been described. The current incarnations of these networks are the products of the continual addition of newly discovered genetic, molecular and biochemical interactions. However, as currently envisaged, network diagrams may not sufficiently describe the spatial and temporal dynamics that underlie developmental processes. We have conducted a developmental analysis of a sub circuit of the Drosophila retinal determination network. This sub circuit is comprised of three genes, two (sine oculis and dachshund) of which code for DNA binding proteins and one (eyes absent) that encodes a transcriptional co-activator. We demonstrate here that the nature of the regulatory relationships that exist between these three genes changes as retinal development progresses. We also demonstrate that the response of the tissue to the loss of any of these three RD genes is dependent upon the position of the mutant cells within the eye field. Depending upon its location, mutant tissue will either overproliferate itself or will signal to surrounding cells instructing them to propagate and compensate for the eventual loss through apoptosis of the mutant clone. Taken together these results suggest that the complexities of development are best appreciated when spatial and temporal information is incorporated when describing gene regulatory networks.