Brandon P. Weasner

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.

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.

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.

Conservation of an Inhibitor of the Epidermal Growth Factor Receptor, Kekkon1, in Dipterans

Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1s function and evolution, we identified kek1 orthologs within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, we find that the cytoplasmic domain contains a region with the highest degree of conservation, which we have implicated in EGFR inhibition and dubbed the Kek tail (KT) box.

A dissection of the teashirt and tiptop genes reveals a novel mechanism for regulating transcription factor activity.

In the Drosophila eye the retinal determination (RD) network controls both tissue specification and cell proliferation. Mutations in network members result in severe reductions in the size of the eye primordium and the transformation of the eye field into head cuticle. The zinc-finger transcription factor Teashirt (Tsh) plays a role in promoting cell proliferation in the anterior most portions of the eye field as well as in inducing ectopic eye formation in forced expression assays. Tiptop (Tio) is a recently discovered paralog of Tsh. It is distributed in an identical pattern to Tsh within the retina and can also promote ectopic eye development. In a previous study we demonstrated that Tio can induce ectopic eye formation in a broader range of cell populations than Tsh and is also a more potent inducer of cell proliferation. Here we have focused on understanding the molecular and biochemical basis that underlies these differences. The two paralogs are structurally similar but differ in one significant aspect: Tsh contains three zinc finger motifs while Tio has four such domains. We used a series of deletion and chimeric proteins to identify the zinc finger domains that are selectively used for either promoting cell proliferation or inducing eye formation. Our results indicate that for both proteins the second zinc finger is essential to the proper functioning of the protein while the remaining zinc finger domains appear to contribute but are not absolutely required. Interestingly, these domains antagonize each other to balance the overall activity of the protein. This appears to be a novel internal mechanism for regulating the activity of a transcription factor. We also demonstrate that both Tsh and Tio bind to C-terminal Binding Protein (CtBP) and that this interaction is important for promoting both cell proliferation and eye development. And finally we report that the physical interaction that has been described for Tsh and Homothorax (Hth) do not occur through the zinc finger domains.

The non-conserved C-terminal segments of Sine Oculis Homeobox (SIX) proteins confer functional specificity.

The Sine Oculis Homeobox (SIX) proteins play critical roles in organogenesis and are defined by the presence of two evolutionarily conserved functional motifs: a homeobox DNA binding domain and the SIX protein–protein interaction domain. Members of this transcription factor family can be divided into three subgroups: Six1/2, Six4/5, and Six3/6. This partitioning is based mainly on protein sequence similarity and genomic architecture, and not on specificities of DNA binding or binding partners. In fact, it is well demonstrated that members of the different subgroups can bind to and activate common transcriptional targets as well as form biochemical complexes with communal binding partners. Here we report that the C‐terminal segment, which is not conserved across different SIX subfamilies, may serve to functionally distinguish individual SIX proteins. In particular, we have dissected the C‐terminal region of Optix, the Drosophila ortholog of mammalian Six3/6, and identified three regions that distinguish Optix from Sine Oculis, the fly homolog of Six1/2. Two of these regions have been preserved in all Six3/6 family members while the third section is present only within Optix proteins in the Drosophilids. The activities of these regions are required, in unison, for Optix function. We suggest that biochemical/functional differences between members of large protein families as well as proteins encoded by duplicate genes can, in part, be attributed to the activities of nonconserved segments. Finally, we demonstrate that a subset of vertebrate SIX proteins has retained the ability to function during normal fly eye development but have lost the ability to induce the formation of ectopic eyes. genesis 47:514–523, 2009.

The Drosophila Wilms’ Tumor 1-Associating Protein (WTAP) Homolog is Required for Eye Development

Sine Oculis (So), the founding member of the SIX family of homeobox transcription factors, binds to sequence specific DNA elements and regulates transcription of downstream target genes. It does so, in part, through the formation of distinct biochemical complexes with Eyes Absent (Eya) and Groucho (Gro). While these complexes play significant roles during development, they do not account for all So-dependent activities in Drosophila. It is thought that additional So-containing complexes make important contributions as well. This contention is supported by the identification of nearly two-dozen additional proteins that complex with So. However, very little is known about the roles that these additional complexes play in development. In this report we have used yeast two-hybrid screens and co-immunoprecipitation assays from Kc167 cells to identify a biochemical complex consisting of So and Fl(2)d, the Drosophila homolog of human Wilms׳ Tumor 1-Associating Protein (WTAP). We show that Fl(2)d protein is distributed throughout the entire eye-antennal imaginal disc and that loss-of-function mutations lead to perturbations in retinal development. The eye defects are manifested behind the morphogenetic furrow and result in part from increased levels of the pan-neuronal RNA binding protein Embryonic Lethal Abnormal Vision (Elav) and the RUNX class transcription factor Lozenge (Lz). We also provide evidence that So and Fl(2)d interact genetically in the developing eye. Wilms׳ tumor-1 (WT1), a binding partner of WTAP, is required for normal eye formation in mammals and loss-of-function mutations are associated with some versions of retinoblastoma. In contrast, WTAP and its homologs have not been implicated in eye development. To our knowledge, the results presented in this report are the first description of a role for WTAP in the retina of any seeing animal.

Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements.

The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. In this report we explore the mechanisms by which eya expression is spatially and temporally governed in the developing eye. We demonstrate that multiple cis-regulatory elements function cooperatively to control eya transcription and that spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, we show that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis.