Philip J. Gage - US grants

DESCRIPTION (provided by applicant): Glaucoma is the second leading cause of blindness overall in the United States and first among African Americans. Elevated intraocular pressure from defects in the iris and aqueous humor drainage system in the anterior segment frequently accompanies glaucoma. Retinal ganglion cell death leading to glaucoma apparently results from the insult of chronically high IOP but the underlying molecular mechanisms are not understood. The genetic regulatory networks governing normal development of anterior segment structures are also poorly understood. Mice provide ideal models for determining the basic mechanisms contributing to normal eye development and for molecularly analyzing genetic eye disease. Mutations in the bicoid-related homeobox gene PITX2 result in Axenfeld-Rieger Syndrome (ARS), an autosomal dominant disease resulting in congenital anterior segment defects and glaucoma. I have cloned Pitx2 from mice and used gene targeting to generate an allelic series (null, hypomorphic, and conditional) for Pitx2 in mice. Initial analysis of the null allele established a more widespread requirement for Pitx2 in eye development than predicted from the human phenotype. Homzygotes mutants have defects in the optic nerve and posterior parts of the eye. The goal of this proposal is to use these mice to analyze the basic mechanisms of Pitx2 function in normal eye development and to identify the molecular consequences of partial or complete loss of Pitx2 activity. We will assess the hypothesis that Pitx2 has distinct functions in the ocular neural crest and mesoderm lineages. Pitx2 expression in each lineage will be determined after using binary transgenic systems to mark each cell lineage. These marking systems will also be used to compare the fates of each lineage in wild type and Pitx2-/- eyes, and determine the genetic mechanisms that require Pitx2. ARS results from altered Pitx2 dosage, indicating certain steps in eye development are highly sensitive to varied PITX2 protein levels. We will vary Pitx2 gene dosage using the null and hypomorphic alleles to identify the specific developmental functions that are most to variations in PITX2 levels and determine the underlying molecular mechanisms. Finally, we will use chimeric mice to rescue the lethality of Pitx2-/- mice in order to analyze Pitx2 functions later in eye development. This multifaceted approach should provide specific mechanistic details about the multiple functions of Pitx2 in eye development and also promises to provide insight into more general fundamental mechanisms of periocular mesenchyme in development. This basic information is essential for understanding eye disease.

DESCRIPTION: Genetic networks regulating normal development of anterior segment structures are poorly understood. Mice are ideal models for determining the basic mechanisms contributing to normal eye development and for analyzing genetic eye disease. Mutations in the homeobox gene PITX2 result in Axenfeld-Rieger Syndrome (ARS), an autosomal dominant disease causing congenital anterior segment defects and glaucoma. I cloned Pitx2 from mouse and used gene targeting in mice to generate a series of Pitx2 alleles that allow for global or conditional ablation of gene function, and the ability to vary gene dose. Based on published data and our own preliminary results, we hypothesize a central role for Pitx2 in periocular mesenchyme during eye development for differentiation of ocular cell types derived from mesenchyme and for mesenchyme expression of extrinsic factors required for normal development of surface and neural ectoderm in the eye. In the previous grant cycle, we demonstrated early Pitx2 expression in ocular neural crest and mesoderm, established that Pitx2 function in neural crest is required for multiple steps in eye development, and identified a role for PITX2 in regulating Wnt signaling in neural crest that could account for Pitx2 mutant phenotypes. The overall goals of this proposal are to use our series of murine Pitx2 alleles to determine the role(s) of Pitx2 in mesoderm during eye development and to establish the mechanistic and functional relationships between Pitx2 and components of the Wnt signaling pathway. In Aim 1, we will test the hypothesis that Pitx2 has distinct functions in ocular mesoderm using a conditional targeting strategy. In Aim 2, we will test a specific Wnt signaling pathway gene as a direct PITX2 target and identify its morphological and molecular roles in eye development to see if this gene accounts for components of the Pitx2 phenotype. We will also screen human patients with specific anterior segment defects and glaucoma for mutations in this gene. In Aim 3, we will directly test for genetic interactions between Pitx2 and the Wnt pathway gene in mice as a mechanism for modification of the Pitx2 mutant phenotype since phenotypic variability is a key feature of ARS. This multifaceted approach will provide specific mechanistic details about the functions of Pitx2 in eye development and new knowledge into more general fundamental mechanisms of periocular mesenchyme in this process. This basic information is essential for understanding eye disease, including glaucoma.

DESCRIPTION (provided by applicant): Our goal is to identify the genetic networks governing ocular anterior segment development and to understand how disruption of these networks leads to defects in anterior segment structure and function. Our objective is to test the functions of the homeodomain transcription factor PITX2 in anterior segment tissues that are most commonly affected in human patients with PITX2 mutations. Our central hypothesis is that PITX2 regulates genetic networks that are required to specify normal corneal cell fates, exclude blood vessels from the developing and mature cornea, and regulate cell proliferation and lineage specification within the iris and structures of the iridocorneal angle. Our hypothesis was formulated on the basis of preliminary data generated by analysis of temporal knockout mice. The rationale for the proposed research is that knowledge of the genetic networks regulated by PITX2 in normal development of the cornea, iris, and iridocorneal angle will advance our understanding of anterior segment dysgenesis and associated pathologies, including elevated intraocular pressure. We will test our hypothesis by pursuing three specific aims: 1) Test the hypothesis that Pitx2 is required for specification and maintenance of corneal cell fates, 2) Test the hypothesis that Pitx2 is required to prevent vascular growth into the developing and mature cornea, and 3) Test the hypothesis that Pitx2 is required for normal development of the iris and structures of the iridocorneal angle. Under the first two aims, a temporal knockout strategy, which is already feasible in the applicant's hands, will be used to ablate Pitx2 at the beginning of corneal development, after which corneal lineages and vascular growth will be assessed using well-established approaches. Under the third aim, an analogous temporal knockout approach, also established as feasible in the applicant's hands, will be used to ablate Pitx2 at the beginning of iris and iridocorneal angle development and the consequences on development of the structures will be determined. The expected outcome is that essential functions of PITX2 in the development of the cornea, iris, and iridocorneal angle will be identified. The approach is innovative because it utilizes a temporal gene knockout strategy to overcome the limitations of global and tissue-specific Pitx2 knockout animals, thereby permitting us to study important later-forming structures in the anterior segment. The proposed research is significant because it will vertically advance and expand understanding of how anterior segment structures are formed during development. Ultimately, such knowledge will provide insights into how anterior segment dysgenesis contributes to vision loss.

Module Use and Impact The Morphology and Imaging Module has two primary functions. First, it processes ocular and brain tissues for microscopy. This is a particularly valuable service for the physiologists and molecular biologists that do not have experience in histological techniques, but are very interested in imaging the tissues and cells that express genes or gene products they are investigating. Tissues are routinely processed for immunocytochemistry and in situ hybridization. Mr. Gillett devised a technique for plastic imbedding zebrafish embryos so the eyes would be stabilized and could be sectioned in the correct orientation. Second, this Module provides expertise for and access to equipment used for tissue processing, microscopy, and image analysis. An additional function of this Module is to provide training in the use of Module microscope and imaging equipment, tissue processing, in situ hybridization, immunohistological techniques, and image analysis software to participating investigators and their staff when requested. The Module will also assist with the planning and execution of immunohistochemistry experiments for investigators who lack the necessary expertise.

Our goal is to identify the regulatory networks that govern corneal development during embryogenesis and corneal healing post-natally, and to determine how disruption of these networks leads to sight-limiting defects in cornea function. As a key step towards this goal, our objective is to identify essential functions of PITX2 in corneal development and wound healing. Our central hypotheses are that activation of Cited2 and repression of Bmp2 and Bmp3 expression are essential mechanisms by which PITX2 regulates corneal development, and that reductions in Pitx2 gene dose adversely affect wound healing in the mature cornea. Our hypothesis was formulated on the basis of data showing that Cited2 expression is lost, and Bmp2 and Bmp3 expression is elevated in the developing cornea in the absence of PITX2, and that corneas of adult Pitx2+/- mice are cloudy and neovascularization is present following subtle insults. The significance of the proposed research is that knowledge of the genetic networks regulated by PITX2 in normal development and healing of the cornea will advance our understanding of these processes in general, an outcome that may in the future contribute to rationale design of improved therapies to prevent and treat vision loss due to corneal disease or injury. We will test our hypotheses via three specific aims: 1) Test the hypothesis that Cited2 is an essential downstream effector of Pitx2 during cornea development, 2) Test the hypothesis that PITX2- mediated suppression of Bmp2 and Bmp3 expression are additional essential mechanisms required for corneal development, and 3) Test the prediction that wound healing in the mature cornea is sensitive to Pitx2 gene dose. Under aim 1, a conditional knockout strategy, which is already available in the applicant?s laboratory, will be used to specifically ablate Cited2 in ocular neural crest or surface ectoderm during corneal development, and assess corneal lineages and vascular growth using well-established approaches. Under aim 2, a conditional strategy will be used to genetically activate Bmp2 or Bmp3 expression in ocular neural crest during corneal development using mice that we have generated, and the resulting mutants will be analyzed by the same criteria as in Aim 1. Under aim 3, the defective Pitx2- allele that the applicant developed will be used to determine the effect(s) of Pitx2 gene dose on wound healing and neovascularization in the mature cornea. The expected outcome is that essential functions of PITX2 in the developing and mature cornea will be identified. The proposed research is innovative because essential functions of PITX2 in the developing and mature cornea will be determined, including the first demonstration that Cited2 expression and BMP signaling activity suppression are essential requirements for corneal development and wound healing. Ultimately, such knowledge may provide insights into productive new therapies for the prevention and treatment of vision loss due to corneal infection and injury. More broadly, the results may also have applicability to understanding mechanisms of neovascularization in certain cancers.

SUMMARY/ABSTRACT - MORPHOLOGY AND IMAGING MODULE The primary function of the Morphology and Imaging Module is to provide access to expert personnel and equipment for microscopic studies of both fixed and live ocular tissues. Housed in a suite of rooms totaling 921 sq. ft. of dedicated space, this Module enhances the research environment by making available state-of-the-art equipment for those investigators who use anatomical and imaging techniques, and, because of the technical expertise provided, it enables others to add anatomical techniques into their repertoire of research tools. Access and training are provided for instrumentation used in 1) the processing of ocular tissue for light and confocal microscopy, immunohistochemistry, and in situ hybridization, and 2) the capturing and processing of images of living and fixed tissue by light and confocal (conventional as well as multiphoton) microscopy. Equipment available in this module includes two cryostats, a paraffin processing and embedding station and paraffin microtome, a Leica laser microdissection microscope, two wide-field fluorescence microscopes (an Olympus EX-51 microscope and a Leica DM6000 microscope), a conventional Leica TCS SP5 confocal microscope, and a multiphoton Leica TCS SP5 confocal microscope. In addition, there is a computer workstation dedicated to image analysis with a stand-alone version of Leica AF, MetaMorph, Imaris and other imaging software. The Module is staffed by a full-time technician who provides assistance with the processing and sectioning of tissue, the design of experiments, training in the use of light and epifluorescence microscopes and associated imaging software, and maintenance of instruments. Also staffing the module (20% effort, paid by the Department of Ophthalmology) is the manager of the Morphology and Image Analysis Core of the Michigan Diabetes and Research Training Center, who provides training in the use of the Leica SP5 confocal microscopes and Leica AF, MetaMorph, and Imaris imaging software. The Morphology and Imaging Module was moderately or extensively utilized by 17 participating investigators during the past 5 years and 16 anticipate using the Module to a moderate or extensive degree in the next funding period.