O'Neill, HC; (2014) Transcriptional Regulation of Foxl2 and its role in Ovarian Development. Doctoral thesis , UCL (University College London).

Abstract

If information is power, then the battle of the sexes has been won for many a year by males, led by SRY and an army of male factors. While many key female-specific genes are known to both promote female development and antagonise these male factors, no one-leader stands out as a sole “switch” governess. Foxl2 is a strong candidate for an ovary-determining and/or anti-testis gene in mammals. Mutations in the gene are associated with premature ovarian failure in women (as part of the Blepharophimosis Ptosis Epicanthus Inversus (BPES) syndrome) and XX male sex reversal in goats with the polled intersex syndrome (PIS). However, despite it being expressed from 12.0 dpc specifically in granulosa cells in the early ovary, no gonadal phenotype is seen in XX mice homozygous for null mutations in Foxl2 until after birth. Wnt signalling is also implicated in repressing testis development, but ovary development still occurs with mutations affecting both Foxl2 and Wnt signalling, suggesting that they are part of a redundant system, with one or more critical additional genes or pathways still to be found. Sox9, together with its close homologues within the SoxE subfamily, Sox8 and 10, are required for Sertoli cell differentiation, typical of the testis, and must be repressed to allow granulosa cell and ovary development. Through examining how these SoxE genes are expressed during development of XX gonads mutant in Foxl2 and/or Wnt signalling, additional complex stage-specific requirements for anti-testis gene activity have been revealed. Two approaches have also been taken to begin to define the regulatory regions driving Foxl2 expression in the ovary, with the long-term aim of unravelling a critical enhancer(s), as a female equivalent of the Sertoli cell-specific Sox9 element TESCO, and identifying the factors that bind to this, which may include missing ovary-determinants. First, a strategy was used that makes no prior assumptions, where BAC sequences flanking Foxl2 were used to drive reporter gene expression in transgenic mice. Preliminary data suggests that a 110 kb BAC can replicate aspects of Foxl2 expression, in ovaries and the pituitary. Secondly, an approach based on genomic studies was employed, which assumes that mutations associated with Foxl2 that lay outside the coding region may directly affect tissue-specific regulatory sequences. The caveat with this approach is that these may be associated with levels of expression, or as chromatin organisers, rather than tissue-specific enhancers. D’haene et al (2009) showed a de novo deletion as small as 7.4 kb, located 283 kb 5’ to FOXL2, was capable of causing BPES in humans, while a 11.7 kb DNA element, reported to affect the transcription of Caprine Foxl2, was deleted in PIS from a region about 200 Kb upstream (Pailhoux et al., 2001). These regions contain many Conserved Non-Coding sequences (CNCs), which, in other loci, have sometimes been implicated in the regulation of gene expression. A 6 kb mouse sequence homologous to the 7.4Kb human region was cloned into EYFP, LacZ and Luciferase reporter vectors, and assayed in cell culture and transgenic mice, where it was shown to be able to drive expression in vivo. The results are discussed with reference to the complex genomic architecture of the Foxl2 locus.

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