Johnson, Amy Elizabeth; (2002) Strategies to study sex-determining genes in the mouse. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sox genes encode transcription factors, which possess an HMG box DNA binding domain with a high level of homology to that of SRY, the product of the Y-linked mammalian testes determining gene. The Sox gene family comprises about 26 genes in the mouse and, like Sry, many are involved in cell fate decisions during embryogenesis, where they act in a range of different systems. At least one other member of the family is important for sex determination. Heterozygous mutations in SOX9 lead to the human dwarfism syndrome Campomelic Dysplasia. 75% of XY patients with this condition are sex reversed. In mice, high levels of Sox9 expression are correlated with testis development where it seems to be required for Sertoli cell differentiation. Gene targeting experiments in mice have shown that heterozygous Sox9 null mutations result in death shortly after birth, however no male to female sex reversal was observed. To overcome the issue with lethality, and to generate mice homozygous for a Sox9 null mutation within any tissue of choice, a conditional Sox9 targeting construct was engineered. From the first round of gene targeting in CCE ES cells, chimeras were generated; however no germ line transmission was achieved. Karyotype analysis revealed that a translocation event had occurred within the wild-type CCE ES cells. When gene targeting was repeated using the AB1 ES cell line there was concurrently both random multiple copy integration and homologous recombination. Fortunately, random integration occurred at an additional site in the genome, distinct from the endogenous gene on chromosome 11. Chimeras were generated using these ES cell clones, with the intention of segregating the targeted Sox9 allele away from the random integrants. To permit the conditional deletion of Sox9 in the developing gonads, transgenic mouse lines have also been generated using either Sry or Dax1 regulatory sequences to drive the expression of Cre recombinase. Dax1 was found to be expressed in blastocysts as well as in the gonads; therefore the Dax1 promoter was also used to drive expression of a tamoxifen-inducible Cre (Dax-CreERT2 ). Cre activity within the transgenic mice carrying Sry-Cre, Dax-Cre or Dax-CreERT2 , has been tested after mating them to R26R indicator mice. The latter express the -galactosidase reporter gene from the ubiquitous Rosa26 regulatory region, but only after Cre-mediated excision. In addition to understanding the role Sox9 plays in testis development, the availability of R26R mice made it possible to address a second question concerning cell fate; Male sex determination is triggered by expression of Sry in the undifferentiated, bipotential XY genital ridge where it is hypothesised to act by directing supporting cell precursors to develop as Sertoli cells rather than as follicle cells. However, the evidence that follicle cells are related by lineage to Sertoli cells is only circumstantial. To test this assumption, R26R mice were crossed with mice expressing either the Dax-CreERT2 or Sry-Cre transgenes. Cre expression in the supporting cell precursors would cause these cells and all their descendants, to permanently express [beta]-galactosidase. In this way it should be possible to trace all the fates of the supporting cell precursors in both sexes and to provide definitive proof of the origin of follicle cells and their lineage relationship to Sertoli cells.

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