A transgenic bacterial artificial chromosome approach to identify regulatory regions that direct Amhr2 and Osterix expression in Mullerian duct mesenchyme

A transgenic mouse approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that direct Mullerian duct expression for Amhr2 and Osterix (Osx, also known as Sp7). Amhr2 encodes the receptor that mediates anti-Mullerian hormone (AMH) signaling for Mullerian duct regression in male embryos. Amhr2 is expressed in the Mullerian duct mesenchyme of both male and female embryos. A similar to 147-kb BAC clone containing the Amhr2 locus was used to generate transgenic mice. The transgene was able to rescue the block in Mullerian duct regression of Amhr2-null males, suggesting that the BAC clone contains regulatory sequences active in male embryos. Osx is expressed in the developing skeleton of male and female embryos but is also an AMH-induced gene that is expressed in the Mullerian duct mesenchyme exclusively in male embryos. Osx-Cre transgenic mice were previously generated using a similar to 204-kb BAC clone. Crosses of Osx-Cre mice to Cre-dependent lacZ reporter mice resulted in reporter expression in the developing skeleton and in the Mullerian duct mesenchyme of male but not female embryos. Osx-Cherry transgenic mice were previously generated using a 39-kb genomic region surrounding the Osx locus. Osx-Cherry embryos expressed red fluorescence in the developing skeleton and Mullerian duct mesenchyme of male but not female embryos. In addition, female Osx-Cherry embryos ectopically expressing human AMH from an Mt1-AMH transgene activated red fluorescence in the Mullerian duct mesenchyme. These results suggest that the 39-kb region used to generate Osx-Cherry contains male-specific Mullerian duct mesenchyme regulatory sequences that are responsive to AMH signaling. These BAC transgenic mouse approaches identify two distinct regions that direct Mullerian duct mesenchyme expression and contribute fundamental knowledge to define a gene regulatory network for sex differentiation.

Automated summary

A transgenic approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that control the expression of Amhr2 and Osterix genes. This study provides important insights into the gene regulatory network for sex differentiation.