Francesca Pignoni - US grants

DESCRIPTION (Verbatim from applicant's abstract): The long-term objective of this proposal is to gain a better understanding of the genetic control of eye-tissue determination in Drosophila. Several genes that play important roles in this process have been identified in recent years. These are the genes eyeless (ey), sine oculis (so), eyes absent (eya) and dachshund (dac). These genes are required for eye development, can induce other tissues to form eyes, and are linked in a gene network by interactions at the level of protein-protein contacts and transcriptional regulation. In order to generate a more complete picture of thegenetic circuitry involved in establishing eye identity in the fruit fly, we propose to identify additional genes involved in this process and study their relationship to the genes already known. We are pursuing four different approaches to identify novel factors involved in this process. In the first two approaches, we make use of previously identified components of the eye-specification gene network to identify novel factors through protein-DNA and protein-protein interactions. The other twoarebased on genetic screens to identify gene activities required or sufficient for eye induction within theeye disc (normal eyes) or in other tissues (ectopic eyes). A detailed molecular and functional characterization of the genes identified using these approaches will be carried out. Through a combination of forward genetic and reverse genetic approaches, molecular genetic analysis and ectopic expression studies, we will dissect the function of these novel factors in eye-tissue specification and define their role in the context of the already identified genetic pathways, Interestingly, despite the significant differences in structure and function between the fly and human eyes, the components of this genetic network appear to be evolutionarily conserved. Several related genes have been identified in mouse and humans including Pax6 and multiple Six, Eya and Dac genes and some of these genes have been found to regulate eye development in vertebrates. Thus, mutations in eyeless Pax6 cause Aniridia inhumans, the Small eye phenotype in mice, and the eyeless phenotype in Drosophila. Moreover, Pax6 and Six3 have been shown to induce eye structures (lens and retina) when ectopically expressed in Xenopus and Medaka fish, indicating that the homology at the molecular level may extend to a conservation of function in eye development. The extensive conservation of molecular factors involved in the early steps of eye development indicates that work in Drosophila will provide insights into the genetic mechanisms controlling mammalian eye development.

DESCRIPTION (provided by applicant): In the mouse, the Microphthalmia-related Transcription Factor (Mitf) plays a critical role in the development of the retinal pigmented epithelium (RPE) of the eye. Mitf is one of the determinants of RPE identity and contributes to the proliferation, specification and differentiation of this tissue. In the fly, dMitf \s expressed within the eye disc (the progenitor tissue that gives rise to the retina and surrounding head structures). Interestingly, it is expressed in the peripodial membrane (PM) a region of the disc epithelium that, similarly to the presumptive RPE region of the optic vesicle/cup, (a) does not itself form retina, (b) is juxtaposed to and continuous with the retinal epithelium, and (c) transiently expresses the fly homologues of two other transcription factors (Pax6 and Otx2) known to function in RPE formation. As shown here, dMitf controls proliferation in the eye disc can suppress retinal identity and may contribute to the specification of the non-neural portion of the epithelium. These striking similarities strongly suggest that RPE and PM cells are evolutionarily related and that a conserved Mitf genetic pathway functions during RPE and PM development. For these reasons, we propose to investigate the function(s) of dMitf during eye development in the Drosophila model system. The specific aims proposed in this grant focus on two main areas: The analysis of dMitf function in eye disc development and the identification of additional components of the pathway. Through the detailed loss-of-function and gain-of-function studies proposed in the first two aims, we will establish how dMitf contributes to specification/differentiation of the PM and normal proliferation of the eye disc. In the last two aims and as a first step towards characterizing the dMitf pathway, we propose to identify additional components through yeast 2-hybrid and genetic-interaction screens. We believe that these approaches will lead to the identification of additional conserved factors that function with Mitf during eye formation in fly and vertebrates. Mutations in the mouse Mitf locus cause microphthalmia, pigmentation, bone and hearing defects and the RPE promotes proper survival and function of photoreceptor cells throughout life as well as influences retinal neurogenesis and lamination during development. These critical roles of the RPE in eye formation and function are highlighted by the significant number of diseases associated with genes specifically expressed or enriched in the RPE as well as by the link between RPE dysfunction and several eye diseases including age-related macular degeneration (AMD).

DESCRIPTION (provided by applicant): The Drosophila female germline stem cells (GSCs) is among the best-studied stem cell models in any organism permitting analysis at single cell resolution in vivo. Our laboratory has discovered that an uncharacterized, but highly conserved, transmembrane protein is necessary and sufficient for germline stem cell self-renewal in the fly ovary. Interestingly, this factor appears to function as an endocytic receptor in the context of BMP signaling. This proposal investigates how it functions at the molecular level and particularly whether it serves as a more central component of the BMP pathway required for Mad phosphorylation, or as a modulator of the core cascade through known regulatory pathways or another, more general biological process. We also begin the characterization of a putative ligand. Since the BMP pathway is universally important for development and adult homeostasis of metazoa, and pathway components are evolutionarily conserved in animals as different as nematodes and humans, the proposed work has broad implications for areas of stem cell research, developmental biology, and the study of human diseases.

? DESCRIPTION (provided by applicant): The BMP pathway is universally important in multicellular organisms and is known to regulate proliferation, patterning, cell fate and other fundamental processes in model systems. Its components are evolutionarily conserved, and pathway dysfunction leads to human diseases of the skeletal, vascular, and gastrointestinal systems as well as predisposition to colorectal cancer. Work in Drosophila has contributed greatly to our understanding of the molecular mechanisms for signal transduction and its regulation, particularly in the study of the pathway in vivo. We have discovered that Lilipod, an uncharacterized but evolutionarily conserved transmembrane protein of the Lipocalin-receptor family, plays a role in BMP signaling in different contexts and we provide extensive evidence of its contribution to germline stem cell (GSC) self-renewal in the ovarian niche. We have also identified fly Fabp, a protein of the fatty-acid-binding family, as a potential ligand and modulato of Lilipod. Vertebrates have two Lilipod orthologs and at least three Fabp orthologs. Given a high degree of aa sequence conservation (similar to other pathway components), Lilipod/Fabp's role in BMP signaling is likely to be conserved. The multiple Lilipod and Fabp orthologs are broadly expressed in vertebrates and may function sometime redundantly, as well as show pleiotropy given the many roles of BMP; thus, complicating studies in vivo. Drosophila is an ideal model to study Lilipod and Fabp function because it has i) only one lilipod and fabp gene, ii) powerful molecular genetic techniques for loss/gain-of-function analysis in vivo, and iii) a wealth of BMP-pathway (Dpp) reagents for both in vivo and insect cell culture models. We show that lilipod is expressed in GSCs; it is necessary and sufficient for their self-renewal; and it regulates BMP signaling in these cells. For this proposal, we will elucidate the molecular mechanism of Lilipod action by first using an in vitro system (S2 cells) and then testing emerging models in vivo (in the ovarian stem cell niche). In addition, we will also dissect the function of its putative ligand Fabp in vivo and in vitro. Based on our preliminary data, Lilipod's input into the signaling cascade occurs between the receptor Tkv and the activated R-SMAD pMad. Preliminary experiments suggest a direct interaction between Lilipod and Tkv in vivo and a requirement for lilipod in the transduction of the BMP signal in S2 cells. A detailed biochemical analysis of the effect of loss/gain of lilipod in S2 cells will assess the stability, binding and/o activation properties of various BMP signaling components in the presence and absence of Lilipod (Aim 1). The emerging molecular mechanism will then be tested in vivo (Aim 2). In Aim 3, we will investigate the function of Fabp in the germline niche and its role as a putative Lilipod ligand. Preliminary experiments suggest that fabp is required and sufficient in the germline niche to promote the stem cell state; an Fabp isoform directly binds to Lilipod; and a reduction in Fabp level (using a deficiency) impairs Lilipod's function. We will dissect fabp function in vivo (germline niche) and in vitro (in S2 cells) using advanced genetic approaches and biochemical techniques (Aim 3).

Abstract The Microphthalmia-associated Transcription Factor Mitf, the Wnt-pathway effector Arm/?-Catenin, and the Hippo-pathway-regulated transcriptional co-activator Yki control the binary choice between neural epithelium (retina) and non-neural support tissue (peripodial epithelium or PE) in the developing eye disc of Drosophila. Specifically, these factors function as promoters of PE fate and suppressors of retina fate. Using the tools of the Drosophila model, we propose to carry out detailed genetic and molecular analyses of these factors' role in PE determination (Specific Aim 1) and investigate how control of their phosphorylation state (Specific Aim 2) and mRNA stability/translation (Specific Aim 3) regulate their function. Our work will define for the first time the genetic network for eye peripodial specification, the eye-Peripodial Determination Network or ePDN, and lead to the discovery of novel network components. Strikingly, homologous factors play an analogous role during development of the mammalian eye, where MITF, ?-CATENIN, YAP and TAZ, and TEAD specify the Retinal Pigmented Epithelium (RPE) of the optic vesicle. These findings point to an ancient evolutionary relationship between the genetic networks that define accessory support cells of photoreceptor neurons in both flies and vertebrates. Based on this conservation, we believe that our studies will also generate insights into RPE formation.

Branchiootorenal spectrum disorders (BOS) are characterized by craniofacial defects that include malformation of branchial arches (BAs), external ear, middle ear, and inner ear; a subset of patients also has kidney defects. Two causative genes are associated with BOS diagnoses, but these genes account for fewer than half of patient cases: the SIX1 transcription factor and a co-factor protein, EYA1, which binds to SIX1 and modifies its transcriptional activity. Thus, the causative genes for over half of BOS patients are yet to be identified. We hypothesize that there are other key co-factor proteins that bind to SIX1 to regulate its activity, and that mutations in these co-factors contribute to the unknown causes of BOS. The goal of this research program is to identify, in tractable model systems, additional genes whose altered functions contribute to the craniofacial malformations of BOS so that these genes can ultimately be included in human genetic screening. Using the Drosophila interactome data for the fly homologue of Six1, we identified 11 novel putative co-factors in Xenopus and showed that most of these are expressed in the developing BAs, ear and kidney, and therefore are potentially relevant to BOS. These proteins are highly conserved in humans, and our preliminary data show that five of them (Sobp, Zmym2, Zmym4, 2G4, Mcrs1) are required for development of the embryonic precursors of the branchial arches (neural crest [NC]), middle ear (NC) and inner ear (otic placode). In Aim 1, we will use gain- and loss-of-function approaches to determine whether these candidate cofactors play a role NC formation or migration, branchial arch cartilages or inner ear gene expression and formation. In Aim 2, we will evaluate the biochemical interactions of these gene products with Six1 and whether they affect Six1 transcriptional function. In Aim 3, we will determine whether the known BOS mutations in SIX1 affect candidate co-factor binding or function, and map what regions of the protein-protein interaction domains of Six1 and of each co-factor mediate binding and transcriptional activity. Our previous work and established model systems uniquely position us to validate whether these candidates are bone fide Six1 co-factors, and elucidate how they contribute to normal and dysmorphic craniofacial development. These analyses will provide important information that cannot be obtained from the limited patient material available. They also have the future potential to explain the phenotypic variability in BOS patients and provide a rationale for including new causative genes in BOS gene panels.

Project Summary/Abstract The sine oculis (SO) gene belongs to the evolutionarily conserved ?SIX? family of homeobox transcription factors. Family members control cell fate, morphology, proliferation and survival in multiple tissues and organs of metazoans, including humans. In Drosophila, SO functions during development of the visual system, specific neuroblasts and glia of the central and peripheral nervous systems, endocrine glands, and in specialized somatic cells of the testis and the ovary of the adult fly. Several vertebrate SIX genes, including the SO orthologue SIX1, are required for the normal development of the brain, cranial sensory organs and the kidney; thus mutations in human homologues of SO lead to birth defects, including BOS/BOR (OMIM 601205, 600963), Holoprosencephaly 2 and Schizencephaly (OMIM 603714). SO/SIX1 functions as a transcriptional regulator together with a number of protein cofactors. We and others have shown that these cofactors modify the transcriptional activity of SO in vitro; moreover, in vivo evidence suggests that SO-cofactor complexes contribute to development in specific ways. Thus, in order to understand SO function in specific developmental contexts, we need to define the function of the various complexes. Our preliminary findings provide fundamental evidence that targeted mutagenesis of SO can be used to separately and specifically disrupt two well-known SO-cofactor interactions, one with the transcriptional activator Eya/EYA and the other with the transcriptional repressor Gro/TLE/GRG. In addition, we show that it is possible to assess the function of these SO variants in the organism. We propose here to dissect the role of these complexes in vivo as well as initiate an analysis of all known transcriptional cofactors of SO. The goal of this grant is to demonstrate that we can generate ?designer SO proteins? that either lack or retain only one or a few protein-protein interactions, and that we can develop assays for their functional assessment both in vitro and in vivo - in preparation for an R01 submission. Since SO is highly conserved from fly to human and the mouse orthologues, SIX1 and SIX2, can substitute for SO in the fly; most changes that affect partner binding in SO will similarly impact the vertebrate proteins. Thus, the proposed work is directly relevant to the human proteins and their linked human disorders.