Justin Kumar - US grants

Project Summary The early development of a number of insect and mammalian tissues including the eye requires the activity of an evolutionarily conserved regulatory circuit that includes members of the PAX, SIX, EYA and DACH gene families. Of particular interest to this proposal is the role that the SIX and EYA proteins play in the early formation and initial patterning of the retina. The function of these two gene families are highly significant to human health as mutations can lead to holoprosencephaly with associated cyclopia, bilateral anophthalmia and congenital cataracts as well as non-retinal defects such as myotonic dystrophy and branchio-oto-renal syndrome. Furthermore, these genes are also implicated in tumorigenesis and numerous cancers. SIX proteins are homeobox containing transcription factors whereas EYA proteins have transcriptional co-activator and protein tyrosine phosphatase activities. These proteins regulate the transcription of target genes as SIX-EYA heterodimers. Disruption of these complexes, in addition to causing retinal disorders in both mouse models and human patients, leads to a complete block in retinal development in Drosophila melanogaster. The fruit fly eye has become a premier model system for studying the genetic and molecular mechanisms that govern tissue determination. We will take advantage of the no-eye phenotypes caused by mutations in the sine oculis (so) and eyes absent (eya) genes (the founding members of the SIX and Eya gene families) to investigate novel activities of the So-Eya complex. The ability of this complex to also coax non-ocular tissue into adopting an eye fate (ectopic eyes) is another tool that can be utilized to dissect the roles that these proteins play in early retinal determination and patterning. In this application I propose to (1) study a novel mechanism by which the So-Eya complex represses transcription of non-ocular selector genes during early eye formation; (2) identify transcription factors that bind to and activate recently identified novel enhancers of the eya gene; and (3) determine the molecular and functional relationships between the So-Eya complex and the Decapentaplegic (Dpp) signaling pathway. The association of SIX and EYA gene lesions with retinal disorders in both insect and mammalian systems provides us with an exciting opportunity for studies in the Drosophila eye to advance our understanding of mammalian eye formation and human retinal disorders.

The Indiana University REU Site program was established in 1998 and has been funded by NSF since 2000. Ten undergraduate students per year come to the Bloomington campus to participate in research at the frontiers of molecular biology and genetics. Applications from all students are considered, especially those from minority groups traditionally under-represented in science and those from institutions with limited research opportunities. Students conduct research in laboratories that use molecular biology and genetic techniques to investigate basic biological mechanisms, including the regulation of gene expression, the mechanisms of cell differentiation and development in plants, animals, fungi, and bacteria, host-parasite interaction and disease resistance in plants, molecular evolution, signal transduction, DNA repair, cellular mechanisms of mitosis and meiosis, protein structure, and virus infection and replication. This program is designed to develop both the technical and the intellectual abilities of the participants. Each student conducts a research project under close mentor supervision. Students also write research proposals and formal papers, present research talks at an end-of-program symposium, and participate in sessions on research ethics, graduate school, and other topics of interest. Students are also exposed to research conducted in a range of laboratories during weekly lunchtime seminars. More information is available at http://www.bio.indiana.edu/undergrad/opportunities/mbgreu/index.html, or by contacting the REU coordinator at iusummer@bio.indiana.edu.

DESCRIPTION (provided by applicant): The goal of the proposed Genetics, Cellular and Molecular Sciences Training Grant (GCMSTG) is to provide comprehensive, relevant and engaging pre-doctoral training in the molecular sciences across a range of scales from atomic resolution, through macromolecules, to the cell and its structure, and to populations of cells and tissues. Fifty six Training Faculties from the Indiana University Biology, Biochemistry and Chemistry departments and the Medical Sciences Program, cover a broad range of expertise and research interests that encompass diverse disciplines including biochemistry, development, cell biology, microbiology, molecular genetics, structural biology, and molecular evolution. Trainees have the opportunity to engage in the highly collaborative and stimulating intellectual environment facilitated by a large, integrated faculty who span a tremendous breadth of life science research. Trainees enroll in a core curriculum designed to teach molecular science from a primary research perspective. Courses on literature analysis, grant writing, teaching and ethics, plus opportunities to develop presentation skills complement the core curriculum. Trainees cultivate their specific interests with advanced coursework. Throughout their studies, students are extensively mentored by the Training Faculty, the TG Committee and their thesis committees. Specific training grant activities including a yearly symposium, a monthly trainee meeting, travel support, and opportunities to meet with visiting seminar speakers, enhance their experience, and create a rich intellectual environment. Many students supported though the GCMSTG are from the Molecular, Biology and Genetics graduate program in the Biology Department, although a significant number of trainees with a molecular biological focus will also be supported from the Ecology, Evolution and Behavior program in Biology, and the Interdisciplinary Biochemistry and Medical Sciences graduate programs. The lU GCMSTG has a long and outstanding track record of training pre-doctoral students, who go on to develop successful and productive careers in academic and industrial life science sectors. This resubmitted proposal requests continuation of 16 training positions, through which we will contribute to and enhance this tradition. RELEVANCE: The lU GCMS TG enables talented pre-doctoral students to realize their potential and develop into outstanding life scientists, contributing to human health and society at many different levels. Comprehensive coursework, careful mentoring, advanced facilities, intensive scientific discourse and teaching, creates a program in which dedicated students thrive, and propel their careers forward into the biomedical sciences.

? DESCRIPTION (provided by applicant): Specification of organ/tissue identity is a fundamental requirement of animal development as it is imperative that each tissue/organ type be made in the right numbers, placed in the right location and constructed to function properly. The traditional view of this process has been that gene regulatory networks function exclusively to promote the desired fate. However, a growing body of evidence, including several discoveries from my research group, strongly suggests that determination of organ and tissue identity actually has two requirements: 1) specifying the desired tissue and 2) repressing alternate fates. A situation where this paradigm-shifting model is likely to be particularly valuable is in establishing boundaries between distinct tissue types. In this respect eye development provides an ideal opportunity to test our new models of tissue specification. In Drosophila, several non-ocular structures such as the head epidermis, antenna, and maxillary palp border the developing fly retina. We examined sine oculis (so) and eyes absent (eya) mutant retinas and have determined that selector genes normally expressed in the surrounding non-ocular tissues are ectopically activated in the eye field. Activation of these factors within retinal progenitor clls forces a homeotic transformation of the eye field into epidermal tissue. The vertebrate eye similarly arises from a territory that is bordered by non-ocular tissues including the telencephalon, diencephalon, and hypothalamus. Recent studies have shown that several selector genes controlling their development are also ectopically activated in the eyes of mouse Lhx2 and frog rax mutants. The transformations that are seen in both vertebrates and flies indicate that segregation of ocular and non-ocular fates is essential for proper head and eye formation. The objective of this proposal is to determine how Sine Oculis Homeobox (SIX) and Eyes Absent (EYA) proteins promote eye formation by repressing non-ocular fates in the developing retina. The rationale for the proposed research is that the chosen questions are focused on processes that are likely to be highly conserved, thereby allowing studies in Drosophila to uncover general mechanisms of tissue/organ formation. Exciting preliminary data guides the following specific aims: (1) Investigate the role that SIX/EYA proteins play in the novel suppression of non-ocular fates during eye specification; (2) Test the hypothesis that retinal patterning by the morphogenetic furrow requires suppression of non-ocular fates by Dpp signaling and SIX/EYA proteins; and (3) Identify the molecular mechanism by which eya expression is activated in the eye field and repressed in bordering non-ocular tissues. The proposed studies test innovative hypotheses and are significant because our results will uncover new far-reaching principles governing tissue/organ specification and patterning. The work also will further our understanding of how errors in these processes facilitate the induction of congenital disorders.

Project Summary: Induction, the process by which one tissue signals to and influences the development of another, is a central feature of metazoan development. Some of the most famous and best studied examples of inductive interactions include the communication that takes place between the ectoderm, mesoderm, and endoderm during early embryonic development. Other examples include the signaling that leads to proper neural plate, somite, and brain development. Relevant to this proposal are the inductive cues that are sent by the vertebrate lens to ensure proper specification, positioning, and patterning of the adjacent retina. Mutations that either disrupt transcriptional networks within and signaling emanating from the lens lead to catastrophic retinal disorders. As such, there is intense interest in identifying and understanding the mechanisms underlying the induction of retinal development by the adjoining lens. This application is focused on using the eye- antennal disc of the fruit fly, Drosophila melanogaster, as an experimental system for studying inductive events during eye formation. The eye-antennal disc is a sac-like structure that contains three different tissues. The retina develops from a columnar epithelium called the disc proper. Overlying the disc proper is a sheet of squamous cells called the peripodial epithelium. These two layers are stitched together along their edges by a strip of cuboidal cells called the margin (which is itself derived from the peripodial epithelium). As such, the eye-antennal disc resembles a closed pillowcase. Evidence from the published literature indicates that signaling from the peripodial epithelium is important for inducing fate specification, growth, patterning, and cell fate choices within the retina. While the vertebrate lens and fly peripodial epithelium are non-homologous structures it appears that both tissues make use of common regulatory modules to induce developmental changes in the retina. For example, recent studies have shown that Pax6 and BMP4/TGF? signaling are both required in the lens and peripodial epithelium for retinal development. In this proposal we will address a number of exciting questions that go to the heart understanding how development of the retina is induced by neighboring tissues. Using modern molecular, cellular, and genetic methods we will develop a state-of-the-art contemporary perspective on how the peripodial epithelium influences the development of the eye and directly contributes to formation of the head. As part of these studies we will pursue the identification of transcription factors and signaling pathways that are important in the peripodial epithelium for retinal development. These gene regulatory networks will be relevant to understanding how the vertebrate lens communicates to the adjacent retina. We will test the specific hypotheses that Pax6 and the So-Eya complex regulate the production of ligands for the TGF? and Notch signaling pathways. From the aims presented here we will acquire new insights into the mechanisms by which transcriptional networks and signaling pathways are integrated to control inductive events during retinal specification and patterning.