Haploinsufficiency of Homeodomain Proteins Six3, Vax1, and Otx2 Causes Subfertility in Mice via Distinct Mechanisms

Abstract

Haploinsufficiency occurs when loss of one copy of a diploid gene (hemizygosity) causes a phenotype. It is relatively rare, in that most genes can produce sufficient mRNA and protein from a single copy to prevent any loss of normal activity and function. Reproduction is a complex process relying on migration of GnRH neurons from the olfactory placode to the hypothalamus during development. We have studied 3 different homeodomain genes Otx2, Vax1, and Six3 and found that the deletion of one allele for any of these genes in mice produces subfertility or infertility in one or both sexes, despite the presence of one intact allele. All 3 heterozygous mice have reduced numbers of GnRH neurons, but the mechanisms of subfertility differ significantly. This review compares the subfertility phenotypes and their mechanisms.

Keywords: Heterozygous; Homeodomain; Infertility.

Fig. 1.

Developmental GnRH neuron migration and homeodomain gene expression. GnRH neuron maturation depends on internal and external factors to the GnRH neurons, allowing developmental migration and maturation. Correct GnRH neuron migration and increased Gnrh1 expression is required for fertility. a Schematic of a sagittal mouse head illustrating developmental GnRH neuron migration. GnRH neurons arise in the olfactory placode around embryonic day 11 (e11) in the mouse. From there they migrate through the cribriform plate into the brain. Once within the brain, GnRH neurons follow a more ventral trajectory to finally localize scattered throughout the anterior hypothalamus. On completion of their migration, GnRH neurons send projections to the median eminence, where GnRH is released in a pulsatile fashion into the hypophyseal portal system. GnRH neurons require ~4 days to complete this migration. Blue dots illustrate the location of GnRH neurons at e13 and e17, and the blue arrows indicate the migration path. b During GnRH neuron development, a complex gene expression pattern is required for increased Gnrh1 expression in parallel with expression of receptors and other factors allowing GnRH neuron pathfinding. We used RNA-Seq comparison of homeodomain transcription factor gene expression in immortalized mature non-migratory GnRH neurons (GT1–7), immature migratory GnRH neurons (GN11), and fibroblasts (NIH-3T3) to identify novel transcription factors potentially involved in GnRH neuron maturation. RNA-Seq data are shown as the average of 2 biological replicates, and expressed as RPKM (reads per kilobase million). These data support quantitative RTPCR analyses previously published for these genes [–26].

Fig. 2.

Reduced number of GnRH-expressing neurons in the hypothalamus of 17 day-old mouse embryos heterozygous for Otx2, Vax1, or Six3. To determine how haploinsufficiency of Otx2, Vax1, and Six3 impacted GnRH neuron development, we performed immunohistochemistry (IHC) for GnRH in mouse embryo heads from control, Otx2^Het, Vax1^Het, and Six3^Het mice at e13 and e17. The red squares on the schematic illustrate the area imaged. GnRH IHC at e13 shows normal location and numbers of GnRH neurons at the cribriform plate in Otx2^Het, Vax1^Het, and Six3^Het embryos as compared to wildtype. In contrast, at e17, Otx2^Het, Vax1^Het, and Six3^Het embryos have fewer detectable hypothalamic GnRH neurons, causing a reduction of GnRH terminals at the median eminence (black arrow). Follow-up studies found that reduced release of GnRH caused subfertility in these heterozygote mouse lines. Scale bar represents 100 μm. pit, pituitary.

Fig. 3.

Summary of role of Otx2, Vax1, Six3, and Six6 in GnRH neuron development and fertility. a Sagittal illustration of GnRH neuron location after termination of migration in the adult brains of control and heterozygote animals and the correlation with fertility. Box indicates normal location of neurons that are most likely to project to the median eminence allowing GnRH release into the hypophysial portal system promoting LH and FSH release from the pituitary to promote gonadal function and fertility. b Summary of findings in the Otx2, Vax1, Six3, and Six6 heterozygote (Het) and knock-out (null) mice. * Studies only performed in males due to inability to generate adult Otx2^Het females. N/A, not applicable.