ABNORMAL DEVELOPMENT IN FEMALE GONADS OF FUSED TOES MUTANT MICE

Abstract

The goal of the present study is to investigate the functional significance of Iroquois (Irx) genes in developing gonads. The Iroquois homeobox family has been shown to be critical for axis and pattern formation during the development of several organisms. Our experiment utilizes a mutant mouse model called Fused toes (Ft) which lacks 6 genes including the entire IrxB cluster, Irx3, 5 and 6, and 3 other genes of unknown function, Fto, Fts, and Ftm. Homozygote mutants (Ft–/–) die in development (E10–E12) because of severe malformation of the developing brain and loss of left-right asymmetry. All of these genes are present in developing gonads of both sexes except Irx3, which is female specific. Affymetrix GeneChip and quantitative real-time PCR analysis have demonstrated an 8-fold increase in Irx3 expression that is specific to somatic cells of the female gonad during sex determination. Based on these findings, we hypothesize that the IrxB cluster, and specially Irx3, will play an important role in female gonad development. Fused toes embryos were dissected at embryonic day 11.5 and gonads were cultured in vitro for 3 days or in vivo for 2 weeks under the kidney capsule of a castrated wild type male recipient mouse. Embryonic sex and genotype were distinguished by PCR on genomic DNA isolated from embryo tails (wild type Ft/Ft, heterozygote Ft/–, and Ft null −/−). The cultured gonads were assessed for morphology by histological sections and for apoptosis (Tunel) or proliferation (PCNA staining). Morphology and cell proliferation were similar among gonads of both sexes and of all genotypes after 3 days of in vitro explant culture. However, in female gonads from Ft −/− embryos, we observed a significant decrease in cells positive for Tunel staining compared to wild type or heterozygote samples after 3 days of culture. Embryonic gonads subject to 2 weeks of culture under the kidney capsule of recipient mice approximates development at postnatal day 5. In the male gonads, significant development of testicular cords was noted in both wild type and mutant samples. In contrast, we observed differences between the wild type and mutant female gonads. Preliminary data indicates that the wild type ovary can produce primordial follicles and structures consistent with maturation of a single oocyte. Ovaries derived from the Ft–/– embryo, however, do not appear to form primordial follicles and instead, maintain immature structures. Together, these results suggest that the absence of the IrxB cluster, Fto, Fts, and Ftm induces abnormal gonad development in the female. We are currently pursuing additional studies to address the impact of the Ft mutant on gonad development. Supported by the University of Illinois Campus Research Board and the University of Illinois Veterinary Medical Research Fund. (poster)