? ? The overall objective of this proposal is to establish a genome-wide, cell type-specific, computer-based searchable map of gene expression associated with critical periods of development of the normal fetal testis. The fetal testis plays an essential role in normal male urogenital tract development and many male urogenital tract disorders are linked to fetal testis perturbations. To understand the impact of genetic and environmental influences on testicular development, it is essential to determine normal gene expression patterns in the fetal testis during development. The goals of this proposal are to 1) define the 3-dimensional (3-D) spatial gene dynamics in relationship to structural development and hormonal balance in the developing testis and 2) identify cell-specific genes that can be used as biomarkers for key differentiation events. The following specific aims will address these goals: 1) Map global gene expression within specific regions in the developing fetal testis at critical developmental stages using laser capture microdissection and microarray analysis; 2) Quantitatively map key gene expression changes to specific regions within the developing fetal testis using laser capture microdissection and high throughput quantitative RT-PCR; 3) Identify testicular biomarkers representing cell-specific differentiation genes using in situ hybridization; 4) Develop a computer-based, 3-D model of the fetal testis incorporating the spatial and temporal expression patterns of key genes. Laser capture microdissection of select regions within the fetal testis during late gestation (gestation days 14-18) will be combined with microarray analysis and high-throughput real-time RT-PCR to characterize global gene expression. In situ hybridization will be used to identify gene expression in specific cell types. These data will be combined with a computerized structural mesh of the fetal testis to create a 3-D spatial and temporal gene expression model. This information will provide researchers with valuable tools for further investigations into normal fetal testicular development as well as the causes, treatment, and prevention of male urogenital tract disorders ? ?
