Fourth Chromosome Resource Project: a comprehensive resource for genetic analysis in Drosophila that includes humanized stocks.

The fourth chromosome is the final frontier for genetic analysis in Drosophila. Small, heterochromatic, and devoid of recombination the fourth has long been ignored. Nevertheless, its long arm contains 79 protein-coding genes. The Fourth Chromosome Resource Project (FCRP) has a goal of facilitating the investigation of genes on this neglected chromosome. The project has 446 stocks publicly available at the Bloomington and Kyoto stock centers with phenotypic data curated by the FlyBase and FlyPush resources. Four of the five stock sets are nearly complete: (1) UAS.fly cDNAs, (2) UAS.human homolog cDNAs, (3) gene trap mutants and protein traps, and (4) stocks promoting meiotic and mitotic recombination on the fourth. Ongoing is mutagenesis of each fourth gene on a new FRT-bearing chromosome for marked single-cell clones. Beyond flies, FCRP facilitates the creation and analysis of humanized fly stocks. These provide opportunities to apply Drosophila genetics to the analysis of human gene interaction and function. In addition, the FCRP provides investigators with confidence through stock validation and an incentive via phenotyping to tackle genes on the fourth that have never been studied. Taken together, FCRP stocks will facilitate all manner of genetic and molecular studies. The resource is readily available to researchers to enhance our understanding of metazoan biology, including conserved molecular mechanisms underlying health and disease.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are mis-regulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate. Summary Statement: Here we describe a novel mechanism by which Pc promotes an eye fate during normal development and how the eye is reprogrammed into a wing in its absence.

A CUT&RUN protocol to determine patterns of epigenetic marks in imaginal discs of Drosophila.

Cleavage Under Targets & Release Using Nucleases (CUT&RUN) sequencing is a technique used to study gene regulation. The protocol presented here has been used successfully to identify the pattern of histone modifications within the genome of the eye-antennal disc of the fruit fly, Drosophila melanogaster. In its present form, it can be used to analyze genomic features of other imaginal discs. It can be modified for use with other tissues and applications including identifying the pattern of transcription factor occupancy.

The early history of the eye-antennal disc of Drosophila melanogaster.

A pair of eye-antennal imaginal discs give rise to nearly all external structures of the adult Drosophila head including the compound eyes, ocelli, antennae, maxillary palps, head epidermis, and bristles. In the earliest days of Drosophila research, investigators would examine thousands of adult flies in search of viable mutants whose appearance deviated from the norm. The compound eyes are dispensable for viability and perturbations to their structure are easy to detect. As such, the adult compound eye and the developing eye-antennal disc emerged as focal points for studies of genetics and developmental biology. Since few tools were available at the time, early researchers put an enormous amount of thought into models that would explain their experimental observations-many of these hypotheses remain to be tested. However, these "ancient" studies have been lost to time and are no longer read or incorporated into today's literature despite the abundance of field-defining discoveries that are contained therein. In this FlyBook chapter, I will bring these forgotten classics together and draw connections between them and modern studies of tissue specification and patterning. In doing so, I hope to bring a larger appreciation of the contributions that the eye-antennal disc has made to our understanding of development as well as draw the readers' attention to the earliest studies of this important imaginal disc. Armed with the today's toolkit of sophisticated genetic and molecular methods and using the old papers as a guide, we can use the eye-antennal disc to unravel the mysteries of development.

A multi-gene knockdown approach reveals a new role for Pax6 in controlling organ number in Drosophila.

Genetic screens are designed to target individual genes for the practical reason of establishing a clear association between a mutant phenotype and a single genetic locus. This allows for a developmental or physiological role to be assigned to the wild-type gene. We previously observed that the concurrent loss of Pax6 and Polycomb epigenetic repressors in Drosophila leads the eye to transform into a wing. This fate change is not seen when either factor is disrupted separately. An implication of this finding is that standard screens may miss the roles that combinations of genes play in development. Here, we show that this phenomenon is not limited to Pax6 and Polycomb but rather applies more generally. We demonstrate that in the Drosophila eye-antennal disc, the simultaneous downregulation of Pax6 with either the NURF nucleosome remodeling complex or the Pointed transcription factor transforms the head epidermis into an antenna. This is a previously unidentified fate change that is also not observed with the loss of individual genes. We propose that the use of multi-gene knockdowns is an essential tool for unraveling the complexity of development.

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.

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.

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.

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.

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.

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.

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 Kek1's 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.

The Eye Specification Network in Drosophila.

One of the most exciting revelations in retinal biology is the realization that the molecules and mechanisms that regulate eye development have been conserved in all seeing animals including such diverse organisms as the fruit fly, mouse and man. The emerging commonality among mechanisms used in eye development allows for the use of model systems such as the fruit fly, Drosophila melanogaster, to provide key insights into the development and diseases of the mammalian eye. Eye specification in Drosophila is controlled, in part, by the concerted activities of eight nuclear proteins and several signal transduction cascades that together form a tightly woven regulatory network. Loss of function mutations in several components lead to the complete derailment of eye development while ectopic expression of threse genes in non-retinal tissues can direct the fates of these tissues towards eye formation. Here we will describe what is currently known about this remarkable regulatory cassettee highlight some of the outstanding questions that still need to be answered.