Research interests

Identifying Genes Involved in Primary (Gonadal) Sex Determination and Other Important Developmental Processes

The primary research focus of our laboratory is to identify and determine the role of genes involved in gonadal sex determination.

Overview of Sex Determination and Sex Reversal

Normally XX individuals develop as females with two ovaries and XY individuals develop as males with two testes. There are exceptions, however, in which XX individuals develop testes and XY individuals develop ovaries. The discovery of sex reversed conditions in mice and humans led to the cloning of Sry (sex-determining region of Chromosome Y) which initiates testicular development in the mammalian bipotential gonad. The gene responsible for initiating ovarian development awaits discovery.

The easiest sex reversal cases to understand are those in which XY females contain a non-functional Sry gene or XX males contain an intact Sry gene. Most human sex reversal cases, however, remain unexplained, including XY females or hermaphrodites who contain an apparently normal Sry gene, and XX males who lack the Sry gene.

We are investigating a number of inherited sex reversal conditions in mice with the premise that unraveling their genetic basis will lead to the identification of additional gonadal sex-determining genes and a better understanding of human sex reversal conditions. Many unexplained human sex reversal cases are reminiscent of our finding in mice that genetic background (i.e., modifier genes) can determine whether a mutation causes ovarian development in an XY individual.

Comparing Sex Reversal Conditions in Humans and Mice

In humans, heterozygosity for a non-functional (null) mutation in the autosomal transcription factor WT1 (Wilms' tumor 1) or SF1 (NR5A1, nuclear receptor subfamily 5, group A, member 1) can cause XY sex reversal. In contrast, previous reports indicate that heterozygosity for a null allele of Wt1 or Sf1 in mice does not cause XY sex reversal. This apparent human versus mouse difference led to the hypothesis that the correct dosage of gonadal sex determining transcription factors is necessary for normal testis development in humans but not in mice. Studies in our laboratory contradict this hypothesis and indicate that testis development in XY mice also depends on presence of the normal dosage of specific transcription factors.

Our experiments use mice of two inbred strains, C57BL/6J (B6) and DBA/2J (D2). We chose B6 because they are exceptionally sensitive to disturbances in the early events of testicular development and thus provide a genetic test system for identifying genes involved in gonadal sex determination. We chose D2 because the same genetic perturbations that cause ovarian tissue development in B6 XY mice allow normal testicular development in D2 mice.

We have investigated gonad development in XY mice containing a null allele of the transcription factors Wt1, Sf1, Gata4 (GATA binding protein 4), and Fog2 (Zfpm2, zinc finger protein, multitype 2). Ovarian tissue was present in B6 XY mice if they are heterozygous for the null allele for one of these transcription factors and contain the AKR-Sry gene, whereas testicular tissue developed if they contained the B6-Sry gene. In contrast, heterozygous (B6 x D2)F1 and D2 mice developed exclusively testicular tissue regardless of which Sry allele was present. We also investigated gonad development in XY mice containing a mutation in the X-linked transcription factor Dax1 (Nr0b1, nuclear receptor subfamily 0, group B, member 1). If the mutant Dax1 gene was present, B6 XY mice developed ovaries. If the Dax1 mutant gene was present in D2 or F1 XY mice, they developed testes.

We conclude that the apparent differences between testis development in humans and mice relating to incorrect dosage of gonadal sex determining transcription factors are best explained by genetic background diversity within a species not differences between species. We predict that mutations within GATA4, FOG2, and DAX1 will be associated with human XY sex reversal.

Identifying Genes Involved in the Sex Reversal of B6 XY mice

We are using two approaches to identify the modifier genes in the B6 genome that, under certain conditions, cause sex reversal in XY mice. The first employs standard genetic crosses in which (B6 x D2)F1 mice carrying a null allele for one of the transcription factors are backcrossed to B6 mice. Gonads of XY offspring are analyzed and a genome scan is conducted to identify chromosomal regions that cause sex reversal when homozygous for B6 derived genes. In collaboration with Gary Churchill, The Jackson Laboratory, we have identified several chromosomal regions involved in sex reversal of Dax1-/Y and Gata4-/+ XY mice. One region, located on distal Chromosome 4, is of special interest because it contains a gene involved in three inherited XY sex reversal models. Experiments to identify this gene are in progress.

The second approach, conducted in collaboration with Jason Affourtit and Carol Bult, The Jackson Laboratory, is based on our hypothesis that the genes involved in B6 XY sex reversal conditions are expressed during fetal Sertoli cell development and critical for normal Sertoli cell differentiation. To identify these genes, we are using a microarray approach that involves applying complementary DNA obtained from fetal Sertoli cells and fetal granulosa cells to Affymetrix Mouse Genome 430 2.0 arrays. We have identified a number of genes that are differentially expressed in Sertoli cells and granulosa cells and these genes form the basis for identifying genes involved in B6 XY sex reversal. In addition, we predict that one of the genes expressed during early differentiation of granulosa cells is the ovarian determining gene.

Analyzing Gene Expression During Normal and Abnormal Gonad Development

As the list of gonadal sex-determining genes expands, understanding the interactions between genes will become increasingly difficult. To aid in identifying potential gene interactions that occur during normal XX and XY gonad development and to reveal abnormal gene interactions that occur in mutant XX and XY gonads, we developed a real time RT-PCR protocol to examine and compare the relative expression levels of genes expressed during fetal gonad development. Some genes are expressed equally in XX and XY gonads, whereas others are expressed at higher levels in XY or XX gonads. Previous to this study, Bmp2 (bone morphogenetic protein 2), Emx2 (empty spiracles homolog 2, Drosophila) and Fgfr2 (fibroblast growth factor receptor 2) were not known to be expressed at higher levels in XX versus XY gonads. The increased expression of these genes in XX embryonic gonads indicate that they play an important role in ovarian development.

Ongoing Future Directions

The inherited XY sex reversal conditions we are investigating share a common feature: One or more B6-derived genes must be homozygous for ovarian tissue to develop in XY mice whereas presence of D2-derived alleles results in normal testis development. To test the hypothesis that these XY sex reversal conditions share common modifier genes, we are fine-mapping the modifier genes involved in several XY sex reversal conditions. This information will identify the genes that make B6 a sensitive strain for studying gonadal sex determination and will identify new gonadal sex determining genes that play a role in human XY sex reversal.

Other Projects

Several other projects are underway. We are collaborating with Richard Chaillet, University of Pittsburgh School of Medicine, to identify genes involved in two inherited ovarian teratoma conditions, and with Lisa Guay-Woodford, University of Alabama at Birmingham, to identify the mutated gene involved in an inherited cystic liver condition and in an inherited diabetes insipidus condition.

Publications

• (From 2002)
• Albrecht KH, Eicher EM. 2002. Sex determination, mouse. In: Encyclopedia of Genetics, Brenner S, Miller JH (eds). Academic Press, New York 1816-1819.
• Albrecht KH, Eicher EM. 2002. The mouse sex-reversed rearrangement (Sxr). In: Encyclopedia of Genetics, Brenner S, Miller JH (eds). Academic Press, New York 1253-1254.
• Tevosian SG, Albrecht KH, Crispino JD, Fujiwara Y, Eicher EM, Orkin SH. 2002. Gonadal differentiation, sex determination and normal Sry expression in mice require direct interaction between transcription partners GATA4 and FOG2. Development 129:4627-4634.
• Albrecht KH, Young M, Washburn LL, Eicher EM. 2003. Sry expression level and protein isoform differences play a role in abnormal testis development in C57BL/6J mice carrying certain Sry alleles. Genetics 164:277-288.
• Bergstrom DE, Bergstrom RA, Munroe RJ, Lee BK, Browning VL, You Y, Eicher EM, Schimenti JC. 2003. Overlapping deletions spanning the proximal two-thirds of the mouse t complex. Mamm Genome 14:817-829.
• Zhang Q, Zhao B, Li W, Oiso N, Novak EK, Rusiniak ME, Gautam R, Chintala S, O’Brien EP, Zhang Y, Roe BA, Elliott RW, Eicher EM, Liang P, Kratz C, Spritz RA, Copeland NG, Jenkins NA, Swank. 2003. Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6. Nat Genet 33:145-153.
• Bouma GJ, Hart GT, Washburn LL, Recknagel AK, Eicher EM. 2004. Using real time RT-PCR analysis to determine multiple gene expression patterns during XX and XY fetal gonad development. Gene Expr Patterns 5:141-149.
• Lorenz-Depiereux B, Guido VE, Johnson KR, Zheng QY, Gagnon LH, Bauschatz JD, Davisson MT, Washburn LL, Donahue LR, Strom TM, Eicher EM. 2004. New intragenic deletions in the Phex gene clarify X-linked hypophosphatemia-related abnormalities in mice. Mamm Genome 15:151-161.
• Bouma GJ, Albrecht KH, Washburn LL, Recknagel AK, Churchill GA, Eicher EM. 2005. Gonadal sex reversal in mutant Dax1 XY mice: A failure to up-regulate Sox9 in pre-Sertoli cells. Development 132:3045-3054.

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