Andrew Tomlinson - US grants

DESCRIPTION: The applicant hopes to increase understanding of the Hh and Wg signaling pathways by elucidating mechanisms about how the known components work and will identify new components in these pathways. Aim 1 will test the proposed model for Hh signaling, will map the domain of Smoothened that confers sensitivity to Ptc, and will test specificity of activating downstream components. Aim 2 will test the specific hypothesis that the serpentine receptors activate heterotrimeric G proteins. Aim 3 will use an elegant genetic screen to identify new genes in the Wg signaling pathway.

DESCRIPTION (Adapted from applicant's abstract): Patterning of the fly retina starts as the morphogenetic furrow sweeps through the eye imaginal disc. Successive waves of activation of the Ras pathway recruit the photoreceptors that form the ommatidia. However, the ommatidia are not all identical and later patterning events add at least two new features to the eye: one is to define the type of inner photoreceptors that are involved in detection of colored or polarized light. Another is to create chirality to the ommatidium that is essential for the correct projection pattern of the photoreceptors to the neural cartridges in the optic lobe. This new application offers to test the mechanisms underlying these two events and in particular the role of wnt's.

P_.ROVIDED. Pdncipal Investigator/Program Director (Last, first, middle): Tomlinson, Andrew A critical question in retinal development is how different photoreceptor types are specified and positioned within a retina. The Drosophila eye is a valuable model system with which to address this question. The fly eye is a compound aggregate of many hundred sub-units called ommatidia. Within each ommatidium there are distinct photoreceptor types and ommatidia occur as different classes containing varying photoreceptor types. The photoreceptor types differ by their opsin expressions, their axonal projections, and their positions within the ommatidia. The major questions we are addressing here are: (i) how the photoreceptors within each ommatidium are uniquely specified? (ii) How the different types of ommatidia are specified? (I) Specification of the R7photoreceptor: The UV sensitive photoreceptor in each ommatidium is the R7 cell. Signals from differentiating photoreceptors that contact the presumptive R7 and activate two distinct intra- cellular signals within the cell - the Ras and Notch (N) pathways. We wish to understand how these two pathways interact to specify the R7 cell. Do they act combinatorially to provide the R7 precursor with a unique developmental cue, or do they act in a different manner? For example could N pathway activation allow the activation of the Ras pathway and have no other function? Once these questions are answered we will examine the nature of the molecular interaction, and or integration of the two pathways. (2) Specification of asymmetry within the ommatidia: The photoreceptors in each ommatidium are arrayed in an asymmetric manner that is critically related to the optical properties of the eye. Ommatidia decode graded information the retina to establish an initial asymmetry that is then communicated to the other cells of the ommatidium. The questions here are how the graded information is established, and how it is decoded and communicated to all cells of the unit. (3) The specification of the dorsal rim: The dorsal rim ommatidia are polarized light detectors cells found in the dorsal extreme of the eye and contain specializations of the R7 and R8 ceils. Signals emanating from the neighboring head tissue induce these ommatidia in only dorsal tissues. W e wish to understand how signals from the head tissue organize the dorsal rim ommatidia and other associated retinal specializations.

DESCRIPTION (provided by applicant): Cell-cell signaling is a primary mechanism by which development is orchestrated. Cells transmit signals that direct the fate of other cells. These signals are usually small polypeptides secreted by the signaling cells or presented on their surfaces. The signals bind and activate receptors on the receiving cells, which results in the initiation of intracellular second messenger cascades. The Wnt signaling pathway is of interest for two major reasons. First, it represents one of the major types of cell-cell signaling that occurs during development. Second, inappropriate activation of the pathway causes oncogenic transformation of cells. Thus from the perspectives of disease and development understanding this signaling pathway is of clear importance. Frizzled (Fz) proteins are a family of serpentine receptors that bind Wnt peptides and transduce the signal across the plasma membrane and activate a second messenger system that transduces the signal to the nucleus to effect transcriptional regulation. Fz proteins also transduce a second distinct class of signal known as the polarity pathway. The ligand(s) for this pathway is unknown and this signal targets the recipient cells' cytoskeletons, leading to the uniform polarization of cells within epithelia, which for example project hairs in the same direction. The mechanism by which the signal is received and interpreted by the cells is seen as equivalent to that which occurs in neutrophil or slime mold amoebae chemotaxis in an extracellular signaling gradient. Thus this signaling pathway is of importance for understanding how cells respond and polarize with respect to gradients and how effectors are able to reorganize the cellular cytoskeleton. In Drosophila there are two well-characterized Wg (the fly Wnt-1) receptors - Fz1 and Fz2. Fz2 only transduces the Wg signal whereas Fzl transduces both the Wg and polaritysignals. This application proposes: 1. A detailed structure/function analysis of the intra-cellular portions of Fz1is proposed to locate the sequences that allow this receptor to engage polarity transduction machinery. 2. We have identified G-alpha-o (part of a trimeric complex that transduce signals from this class of receptors) as a candidate mediator of Fz1 signaling and we have shown that G-alpha-o and Fz1 bind in vitro. Using biochemical techniques we intend to determine which other G-alpha subunits bind Fz1 and which other Fz receptors bind to G-alpha-o. 3. We will use fly genetics to determine whether G-alpha-o also transduces the Wg signal, and also identify its molecular partners in the trimeric complex.

DESCRIPTION (provided by applicant): The human eye is made from many diverse cell types in various tissue layers and structures. In diseased or damaged eyes the ability to replace defective or absent cell types has major therapeutic importance. Here retinal precursor cells need to be guided through various developmental steps in order to generate the required cell types. This grant aims to understand the processes of cell specification in a model retina; the developing Drosophila eye. The study is focused on the Notch and RTK signaling pathways. A series of complex interactions between these two pathways specify a number of distinct cell types, and we aim to use a variety of molecular, genetic and histological techniques to understand how the two signaling pathways are integrated and interpreted in the cells, and how this information then directs the specification of discrete retinal cell types.

Project Summary Receptor Tyrosine Kinases (RTKs) play profound and pervasive roles in biology and are associated with many different cancers, and are currently the focus of intense research. For practical reasons, most RTK studies are carried out in tissue culture which has both advantages and limitations. One limitation is that these studies do not examine RTKs in their native biological setting, and a deep understanding of how they perform their normal functions is often lacking. This application uses the developing Drosophila eye as a setting in which to study two distinctly different RTKs in their normal biological roles. One is the Drosophila EGF receptor (DER), which by structure and behavior is a standard RTK: it is a simple plasma membrane protein expressed ubiquitously at low levels, its ligand is a diffusible peptide, and its activation occurs on the plasma membrane. The other is Sevenless (Sev), an RTK with a number of unusual features. It has a multimeric structure, it is expressed at high levels and transduces a potent RTK signal, its ligand is an integral membrane protein, and its activation is linked to endocytosis which occurs in a cell-type specific manner. These features of Sev reflect its use by the developmental program to generate a high RTK signal in one specific photoreceptor precursor. Here we compare and contrast the structure and regulation of the two RTKs to understand how the unusual features of Sev allow it to perform its specific functions.

Project Summary In highly structured organs, cells are arranged in precise patterns with each cell occupying a defined position and performing a specific function. Thus, there is an intimate linkage between a cell?s position and its fate. The mechanisms that regulate position usually involve changes in cell affinities and movement of cells, whereas the processes of fate assignment involve the execution of specific programs of cellular differentiation. This raises the question of how at the molecular level the mechanisms that determine position are integrated with those that determine fate. This proposal uses the developing Drosophila ommatidium as a model system with which to study the linkage between cell position and fate. Ommatidia grow by recruiting cells to precise positions in their structure, and the cell fate specifications depends on the cells they contact. The Receptor Tyrosine Kinase and Notch signaling pathways play key roles in ommatidial development, and in this work we test whether these two pathways control both the recruitment and cell fate specifications. Specifically, we ask whether the recruitment and fate specification steps represent early and late aspects of the same signaling process.