John Flanagan - US grants

The long-term goal of this project is to understand the principles by which polypeptide growth factors their receptors control complex developmental and physiological processes in mammals. The particular focus will be on the class of receptors with tyrosine kinase or tyrosine phosphatase activity. The molecules and cell types that will be studied are directly relevant to many important diseases, including cancer, deafness, malformations of the face and heart, and blood deficiency disorders. Also, much of the work is geared toward using recent techniques to identify new growth factors, which in general have great potential as therapeutic agents. We will extend our studies of a recently identified growth factor, the ligand (KL) for the receptor tyrosine kinase encoded by the c-kit proto-oncogene. Both KL and its receptor are encoded at mouse genetic loci with known mutant alleles, showing that KL is required for the development of some especially interesting cell lineages: cells derived from the neural crest (including melanocytes and probably neurons), germ cells, and hematopoietic cells (including mast cells, red cells and multipotent progenitor cells). Now that KL polypeptides and cDNAs are identified, we are in a position to determine at the molecular level how KL functions. This will be done by three complementary approaches. (1) Analysis of the effects of two different transmembrane forms and the soluble form of KL on cellular processes such as proliferation, differentiation, and migration. (2) Determination of the expression patterns of these different forms of KL in mice. (3) Investigation of the interactions of transmembrane KL with other molecules within the cells that express KL. Much of this work on KL will focus on its control of neural crest cells. This population of multipotent stem cells gives rise to a remarkable range of cell types and tissues, including most of the peripheral nervous system, melanocytes, much of the mesenchyme of the face, and parts of the thymus and heart. These progenitor cells are little characterized at the molecular level, and in order to understand how c-kit and other receptors cooperate to control this complex system it will initially be necessary to know which receptors are present. We propose to use a PCR-based approach to determine the known and possibly unknown receptor-like tyrosine kinases and tyrosine phosphatases that are expressed in neural crest progenitor cells. As another means to understand the biology of the kit family of receptors and ligands, we will study two closely related receptors, flt and flk-2, with unknown ligands. Our approaches will be: (1) to determine where these receptors are expressed, by in situ hybridization; (2) to identify their ligands, using and extending the soluble receptor affinity approach we developed to identify KL; and (3) to study their function by genetic mutant analysis, initially by testing known candidate mouse mutants.

Polypeptide factors that bind to cell surface receptors are powerful mediators of cell-cell communication and have important roles in physiology and disease. The existence of a large number of unidentified polypeptide factors is implied by the identification of "orphan receptors" which appear to be cell surface receptors but for which the ligands are unknown. Identification of these ligands is a critical step in understanding the biology of the orphan receptors, and will also provide important advances in our understanding of cell-cell interaction in general. Moreover, many of the ligands will be good candidates for therapeutic use. We recently identified the ligand (KL) of the orphan receptor tyrosine kinase encoded by the c-kit proto-oncogene by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easily and sensitively traced. The major objective of the present proposal is to develop our soluble receptor affinity approach further, and apply it to identify the ligand of an orphan receptor protein tyrosine phosphatase. Although orphan receptor tyrosine phosphatases are being identified at a rapid rate, little information is available on any of their ligands. Nonetheless, the involvement of the receptors in the control of tyrosine phosphorylation indicates that the ligands are likely to have potent biological effects. In addition to our work on phosphatases, we will perform experiments on the interaction of the c-kit receptor and its recently identified ligand KL. These experiments will have two goals. The first will be to develop methods that can be applied to receptor tyrosine phosphatases and other orphan receptors. The second goal will be to concurrently derive useful information on the biology of c-kit and KL. Studies with the soluble receptor fusion protein are designed to provide important new information on the distribution and the isoforms of the KL polypeptide that are present in tissues. We will also carry out a structure / function analysis of the interaction of c-kit and KL. This will allow us to map functional domains and also to investigate the functional interrelationships between ligand-receptor binding, cell-cell adhesion, proliferation and migration, all of which may be mediated by KL.

Polypeptide factors that bind to cell surface receptors are powerful mediators of cell-cell communication and have important roles in physiology and disease. The existence of a large number of unidentified polypeptide factors is implied by the identification of "orphan receptors" which appear to be cell surface receptors but for which the ligands are unknown. Identification of these ligands is a critical step in understanding the biology of the orphan receptors, and will also provide important advances in our understanding of cell-cell interaction in general. Moreover, many of the ligands will be good candidates for therapeutic use. We recently identified the ligand (KL) of the orphan receptor tyrosine kinase encoded by the c-kit proto-oncogene by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easily and sensitively traced. The major objective of the present proposal is to develop our soluble receptor affinity approach further, and apply it to identify the ligand of an orphan receptor protein tyrosine phosphatase. Although orphan receptor tyrosine phosphatases are being identified at a rapid rate, little information is available on any of their ligands. Nonetheless, the involvement of the receptors in the control of tyrosine phosphorylation indicates that the ligands are likely to have potent biological effects. In addition to our work on phosphatases, we will perform experiments on the interaction of the c-kit receptor and its recently identified ligand KL. These experiments will have two goals. The first will be to develop methods that can be applied to receptor tyrosine phosphatases and other orphan receptors. The second goal will be to concurrently derive useful information on the biology of c-kit and KL. Studies with the soluble receptor fusion protein are designed to provide important new information on the distribution and the isoforms of the KL polypeptide that are present in tissues. We will also carry out a structure / function analysis of the interaction of c-kit and KL. This will allow us to map functional domains and also to investigate the functional interrelationships between ligand-receptor binding, cell-cell adhesion, proliferation and migration, all of which may be mediated by KL.

DESCRIPTION (adapted from investigator's abstract): The long-term goal of this project is to understand the principles by which cell-cell signaling factors control vertebrate development. The investigator's focus is on receptor tyrosine kinases, receptor tyrosine phosphatases, and their ligands, a set of molecules known to have important roles in development, physiology and disease. The Eph receptors are the largest known subfamily of receptor tyrosine kinases, with more than 12 members. Remarkably, all were initially identified as orphan receptors without known ligands, and their specific functions were unknown. Recently, the first members of a corresponding Eph ligand family were identified, opening the way for new studies into function. Novel roles in neural development have already been identified, particularly in axon guidance. The main goal of this proposal is to further characterize functions of the Eph family in neural development. The investigator will focus on roles in axon guidance, but also plans to study related developmental processes such as guidance of cell migration. His studies will involve a combination of molecular-genetic, cellular and embryological approaches, including: 1) characterization of in situ expression and binding patterns of Eph ligands and receptors in embryos, 2) effects of Eph ligands and receptors on assays of cell behavior, in vitro and in vivo, and 3) molecular interactions and signaling properties of Eph receptors and ligands. PTP-NP is a new receptor-type protein tyrosine phosphatase, and is notable as a particularly early and specific marker for developing pancreatic endocrine cells. Despite the medical importance of these cells, no pancreas-specific extracellular control factors have been found. Using a soluble version of the receptor, a candidate ligand for PTP-NP has been identified, which could be a regulator of pancreatic endocrine cells. The investigator's aim is to clone the ligand, and to further characterize the biology of this ligand-receptor system. The investigator expects his work on the basic biology of developmental control could help to understand abnormalities that lead to disease. Because the focus is on the identification and characterization of novel molecules that control cell behavior, the work could lead to the development of new therapeutic agents. For the Eph family, potential applications could be in the repair of damage to neural connections in the brain and spinal cord. For PTP-NP, applications could be in the growth or regeneration of pancreatic endocrine cells to treat diabetes.

DESCRIPTION (provided by applicant): The broad long-term goal of this application is to identify novel ligands for cell surface receptors, and to characterize functions in vertebrate development, particularly development of neural connections. Aim 1 focuses on identification of novel ligands. The first part of aim 1 deals with receptor-like protein tyrosine phosphatases of the mammalian LAR family. Previous genetic studies show functions for these molecules in neural development, but their actions and putative ligands are still not well understood. In preliminary studies, a novel family of three ligands has been molecularly identified, and will be characterized further. The second part of aim 1 deals with the Amyloid Precursor Protein (APP), which is cleaved to form the beta-amyloid peptide implicated in Alzheimer's disease. Despite its pathological relevance, the natural function of APP is not well understood. It has long been suggested that APP may function as a ligand or receptor, and in preliminary studies we have shown a novel-binding pattern to developing axons. Further studies are proposed to identify and characterize the ligand(s) responsible for this binding. Aim 2 proposes continued studies on developmental functions of the ephrins, a family of ligands identified during earlier cycles of the project. The focus is on experiments to elucidate novel principles of ephrin function, such as roles in coordinating cortical cell migration, and roles at the synapse. Aim 3 is to develop new small-molecule approaches to study axon outgrowth and guidance. A "chemical genetic" screen is proposed, to identify chemical compounds that modulate the inhibitory effect of ephrins, as well as other molecular cues that inhibit axon growth during development and regeneration. While this work focuses on basic biology, studies to identify, characterize, and modulate novel cell-cell signaling molecules may ultimately lead to therapeutic agents for maintenance, repair or regeneration of neural connections. Also, our studies on APP ligands may lead to new strategies to regulate proteolytic processing, with potential implications for Alzheimer's disease. Aim 3, involving a screen for chemical compounds that can promote axon growth and regeneration, focuses on basic cell biology but could produce compounds or lead-compounds for further therapeutic development for treatment of nerve injury.

The central long-term goal of the project is to understand molecular signaling mechanisms that underly the development of neural connectivity. Many extracellular guidance cues have been identified in the last decade, but the specific molecular pathways involved in their actions are not yet well understood, and are currently an interesting and important area of research. Our main focus is on ephrins and their Eph receptors, which have important functions in axon guidance, dendrite development, and synapse formation. Aim 1 is to investigate downstream signaling mechanisms. A key feature of the ephrins is that they signal bi-directionally, with a 'forward'signal through the receptor, and a 'reverse'signal transduced through the ligand. Reverse signaling pathways have been identified for the transmembrane ephrin-Bs, but signaling by the lipid-anchored ephrin-As is less well understood, and will be a focus of this Aim. Aim 2 is to study upstream regulation of Eph receptors. The focus will be on RNA-based mechanisms that have recently been identified in axons;RNA-based regulation is currently an exciting area of biology generally, and these studies will delineate novel mechanisms in neural development. Aim 3 is to characterize interactions among receptor-like protein tyrosine phosphatases, Eph receptor kinases, and their common theme of regulatory interactions with heparan sulfate proteoglycans. While our primary goal is to elucidate basic molecular mechanisms, the work has broader implications for understanding and treatment of disease. Ephrins and receptor-like protein tyrosine phosphatases have been identified as regulators of CNS axon regeneration. Beyond axonal connectivity, ephrins have important roles in other aspects of biology, including vascular development, cancer, and stem cell biology.

DESCRIPTION (provided by applicant): The brain relies for its function on a complex pattern of axonal connections that are initially set up during development. The broad long-term goal of the project is to understand molecular signaling mechanisms that underly the process of pathfinding required for axons to grow toward their correct targets. The current proposal focuses particularly on RNA-based mechanisms, which have not been characterized extensively in the axon. Aim 1 builds on our recent work showing that the transmembrane axon guidance receptor DCC physically associates with translation initiation machinery, including eIFs and ribosomal subunits. This finding of functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for extracellular regulation and localization of translation, based on a transmembrane translation regulation complex. Here we propose further studies to survey how general this phenomenon may be for different classes of receptor, focusing on receptors involved in neural development. Identification of this novel regulatory mechanism also raises interesting questions we will address regarding molecular components and interactions involved in the complex. Aim 2 extends our work which previously identified RNA-based mechanisms that can regulate protein expression within spinal commissural axons as they navigate past their well characterized intermediate guidance target, the floor plate of the spinal cord. We have identified RNA-binding proteins in the CPEB family that are involved in spinal commissural neuron pathfinding. We propose further studies of the functions of these RNA-binding proteins in axon guidance, using both in vitro and in vivo functional systems. We also propose studies of the downstream target mRNAs bound by these proteins, which will yield insight into their network of regulatory interactions. While our work focuses primarily on the basic biology of neuron development, it has broad implications for health research. Correct axon pathfinding is required for normal neural development, and RNA-based mechanisms are known to contribute to diseases such as mental retardation. Also, a major health problem is created by inability of adult neurons to regenerate, and ultimately the study of developmental pathfinding is likely to contribute to strategies for axon regeneration. More broadly, our work on the neuron provides a model to uncover fundamental principles with very general implications for biomedical research.

Correct functioning of the visual system requires a precise set of axonal connections from the eye to the brain. If these connections develop abnormally, or are later damaged by injury or degeneration, vision may be impaired or lost. The broad long-term objective of the project is to identify and characterize cell-cell signaling molecules involved in the development and regeneration of visual neuronal connections. In the adult visual system, like other parts of the CNS, axons show very limited capacity for regeneration. This is due, at least in part, to endogenous regeneration inhibitors, leading to great interest in strategies that might overcome the effect of these inhibitors, and thus promote plasticity and regeneration. Chondroitin sulfate proteoglycans (CSPGs) have long been known as an important class of inhibitors, but no corresponding receptor had been identified, limiting further molecular progress in this area. In recent work, we identified Protein Tyrosine Phosphatase sigma (PTPsigma) as a receptor for CSPGs, opening up new opportunities to study mechanisms in regeneration and potential therapeutic strategies. The role of this receptor in regeneration is confirmed by the effects of PTPsigma gene deficiency, which enhances regeneration, including of retinal axons in the optic nerve. In other recent work, we have shown that PTPsigma acts as a ligand- specific molecular switch, mediating not only CSPG inhibition but also heparan sulfate proteogylcan (HSPG) promotion of axon extension, providing a paradigm to understand opposing effects of CSPGs and HSPGs. Aim 1 has two inter-related goals: first, to better understand the basic biology of the interaction between PTPsigma and its proteoglycan ligands; and second, to explore compounds that can promote axon growth and optic nerve regeneration. Whereas Aim 1 focuses on extracellular interactions of PTPsigma, Aim 2 explores downstream transmembrane and intracellular signaling mechanisms. The prior work identifying HSPG and CSPG ligands for PTPsigma opens up new opportunities to understand the downstream mechanistic basis for the contrasting effects of these proteoglycans, and simultaneously to make new progress in understanding basic mechanisms of signaling by the receptor PTP family. Finally, Aim 3 proposes to continue studies of the Amyloid Precursor Protein (APP) and its binding partners expressed in the developing retinotectal system. While APP processing to beta-amyloid is known to have important roles in pathology, neither the normal developmental functions of APP nor the mechanisms that regulate its processing are yet well understood. Binding partners for APP have been identified which are prominently expressed in retinotectal development, and can affect retinal axon growth and APP processing. Further studies will provide improved understanding of these molecular interactions, with potential implications for development, degeneration and regeneration.

The brain relies for its function on a precise and complex pattern of axonal connections. The broad long-term goal of this project is to understand how this pattern of axon connections is set up during development. When such connections fail to form properly, or are subsequently lost, this can lead to a broad range of neurodevelopmental, psychiatric and neurodegenerative disorders. This proposal focuses particularly on RNA-based regulatory mechanisms. A key advantage of regulating gene expression at the mRNA level is that protein expression can be directed to specific subcellular regions with temporal and spatial specificity ? an important advantage in neurons, which have a high degree of spatial organization. Accordingly, RNA-based regulation plays key roles in axon guidance, neuron migration and synapse plasticity, although the specific mechanisms remain poorly understood. Here, RNA-based mechanisms will be studied in regulation of the microtubule cytoskeleton (Aim 1), and in axon pathway selection at a complex choice point (Aim 2). Aim 1 focuses on the microtubule cytoskeleton, which has crucial roles in neuron structure and motility. Our recent work has now identified a mechanism for RNA-based regulation of microtubules. Specifically, microtubule plus-end protein APC binds tubulin Tubb2b mRNA, at a site required for Tubb2b translation in axons, formation of dynamic microtubules in the growth cone, and neuron migration in vivo. This opens up a new field of investigation into RNA-based regulation of the microtubule cytoskeleton. One goal will be to investigate coordinated regulation of specific tubulin mRNAs which have APC binding sites in their 3'UTR and cause most human tubulinopathies. Another objective will use time-lapse imaging to understand specifically how RNA-based regulation controls microtubule dynamics, including fundamental new models for both microtubule initiation at the minus end, and assembly at the plus- end. In addition to axons, these mechanisms will be characterized in formation of synaptic spines. Aim 2 will continue studies of commissural axon guidance at the spinal cord midline, a well-characterized model of developmental axon pathfinding. RNA-based regulation is known to occur within commissural axons, including upregulated translation of mRNAs in distal axon segments that have crossed the midline intermediate target. However, little has been known of the mechanisms, including the RNA-binding proteins involved, their downstream mRNA targets, or upstream regulatory pathways. This Aim will characterize specific RNA-binding proteins that display highly selective expression on axon segments, strong and distinct phenotypes in midline guidance, and interactions with mRNAs regulated in axons at the midline; as well as upstream ligands and receptors that interact physically and functionally with these RNA based regulatory mechanisms. These studies will provide novel information on fundamental mechanisms of axon development, and RNA-based regulation.