A. N. Combes

Publications (5)4.54 Total impact

• Article: Expression and functional analysis of Dkk1 during early gonadal development.

  A N Combes, J Bowles, C-W Feng, H S Chiu, P-L Khoo, A Jackson, M H Little, P P L Tam, P Koopman
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  ABSTRACT: WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.
  Sexual Development 01/2011; 5(3):124-30. · 2.27 Impact Factor
• Article: Expression and Functional Analysis of Dkk1 during Early Gonadal Development

  A.N. Combes, J. Bowles, C.-W. Feng, H.S. Chiu, P.-L. Khoo, A. Jackson, M.H. Little, P.P.L. Tam, P. Koopman
  [show abstract] [hide abstract]
  ABSTRACT: WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.
  Sexual Development 08/1970; 5(3):124-130. · 2.27 Impact Factor
• Article: SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

  D Wilhelm, R. Hiramatsu, H. Mizusaki, L. Widjaja, A. N. Combes, Y Kanai, P Koopman
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  ABSTRACT: In mammals, male sex is determined by the Y-chromosomal gene Sry ( sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 ( SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D-2 ( PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase ( Pgds), the enzyme that produces PGD2, is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immuno-precipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.
• Article: Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis

  T Svingen, D Wilhelm, A. N. Combes, B. Hosking, V R Harley, A H Sinclair, Peter Koopman
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  ABSTRACT: Gene function during mouse development is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with microinjection and magnetically induced transfection ( magnetofection) of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Furthermore, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues.
• Article: Gonadal defects in Cited2 -mutant mice indicate a role for SF1 in both testis and ovary differentiation

  A. N. Combes, C. M. Spiller, V R Harley, A H Sinclair, S. L. Dunwoodie, D Wilhelm, P Koopman
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  ABSTRACT: Sex determination is regulated by a molecular antagonism between testis- and ovary-determining pathways in the supporting cell lineage of the gonadal primordia. Genes important for maintaining this lineage play critical roles in early gonadal development, but their influence on testis and ovary differentiation is unclear due to the severity of loss-of-function phenotypes. The transcription factor SF1 (Nr5a1/Ad4BP) is one such factor, required for establishing the supporting cell lineage, and for propagating the male pathway. In the gonad, Sf1 expression is enhanced by the transcriptional co-factor Cited2. We have used the reduced levels of Sf1 expression in Cited2 -/- mice as a hypomorphic model to gain insight into the sex-specific roles of SF1 function in gonadal development. In XY mutant mice, we found that testis development was delayed in Cited2 -/- gonads, and that testis structure was permanently disrupted. In XX Cited2 -/- gonads, ectopic cell migration was observed which correlated with a transient upregulation of Fgf9, and a delay in Wnt4 then Foxl2 expression. These data suggest a novel role for SF1 in promoting ovarian development in addition to its roles in testis differentiation.