Gonads from 12.0 dpc Wnt4 -/- embryos were separated from the adjacent mesonephroi, submerged in RNAlater Stabilization Reagent (Qiagen, Cat No. 76104) and stored at -80°C. Gonads were collected from more than 100 embryos for each sex/genotype of interest, and pooled in multiple groups prior to RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (Qiagen Cat No. 74104), following manufacturer’s instructions, and given to the Duke University Microarray Facility.
Gonads from 12.0 dpc XX +/+ embryos were separated from the adjacent mesonephroi, submerged in RNAlater Stabilization Reagent (Qiagen, Cat No. 76104) and stored at -80°C. Gonads were collected from more than 100 embryos for each sex/genotype of interest, and pooled in multiple groups prior to RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (Qiagen Cat No. 74104), following manufacturer’s instructions, and given to the Duke University Microarray Facility.
Scanning was performed by the Duke University Microarray facility using the Axon GenePix Pro 4000A
Description
One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development is inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify genes similarly upregulated in wildtype XY gonads and XX mutant gonads. Expression profiling was performed on gonads collected from XY Wnt4+/+, XX Wnt4+/+ and Wnt4-/- embryos at 12.0 days post coitum, using arrays spotted with the Operon Mouse Genome Oligo Set, version 2.0. Competitive dual-color hybridizations with dye-swap controls were performed in duplicate for each of two comparisons: XY Wnt4+/+, versus XX Wnt4+/+, and XX Wnt4-/- versus XX Wnt4+/+. For both comparisons, the wild-type XX sample is used as the reference.
Data processing
Per spot and per chip intensity dependent Lowess normalizations were performed on median intensity values. Normalized values were used to calculated the log base 2 of the ratio (F635/F532), for all spots with a signal above background.
