Dominant Activation Of Hedgehog Signaling Alters Development Of The Female Reproductive Tract
The hedgehog (HH) signaling pathway regulates the development of multiple organs in the embryo as well as remodeling in adult tissues. Signaling occurs through binding of one of three secreted HH ligands (Indian, desert and sonic) to the membrane receptor patched (PTCH); this relieves inhibition of the signal transducer smoothened (SMO), and downstream signaling occurs through GLI transcription factors. The objective of this study was to determine the role of HH signaling in development of the female reproductive tract. Mice were created in which expression of a dominant active allele of Smo, known as SmoM2, and a fusion protein, yellow fluorescent protein (YFP), are induced in the Müllerian duct by CRE-mediated recombination using the Amhr2cre/+ allele. The Amhr2cre/+ allele is known to be expressed during embryonic development of the Müllerian duct and the ovary. In Amhr2cre/+SmoM2 mutant mice, YFP was detected in the reproductive tract on the day of birth, confirming that expression of the SmoM2/Yfp fusion gene had been induced. At 24 days of age, levels of mRNA for genes that are known transcriptional targets of HH signaling, Gli1, Ptch1 and Hhip, were increased in uteri and oviducts of Amhr2cre/+SmoM2 mutant mice compared to genotype-matched Amhr2+/+SmoM2 controls. Thus, over-activation of HH signaling continued in prepubertal mutant mice. The reproductive tract of mutant mice failed to develop normally. Unlike the uterus in control mice, in which the luminal epithelium consists of a single layer of columnar epithelial cells, the luminal epithelium in mutant mice had stratified squamous cells, resembling those of a normal cervix. In addition, the uterus of mutant mice lacked glands, had a disorganized myometrium and the oviduct lacked coiling. These characteristics suggested partial homeotic transformation of the uterus in mutant mice to resemble the more posterior cervix and failure of the oviduct to develop normally. The anterior-posterior axis of the reproductive tract is known to be regulated by differential expression of genes in the Hox family. Wnt7a and Wnt5a null mice have defects in patterning of the reproductive tract. Furthermore, neonatal treatment with DES was reported to alter development of the reproductive tract and the expression of Hox and Wnt genes. Therefore, steady state levels of mRNA for Hoxa9, Hoxa10, Hoxa11, Hoxa13, Wnt5a and Wnt7a were determined in oviduct, uterus and vagina of 24-day old Amhr2cre/+SmoM2 mutant and control mice. In control mice, expression of Hoxa9 was most prevalent in the oviduct, Hoxa10 and Hoxa11 in the uterus and Hoxa13 in the vagina. mRNA levels of Hoxa9, Hoxa10 and Hoxa11 in the oviduct and uterus of mutant mice were similar to that in control mice. In contrast, mRNA of Hoxa13 in uterus of mutants was 12-fold higher than in controls. Steady state levels Wnt7a mRNA were was similar in the reproductive tract of mutants and controls. In contrast, Wnt5a expression was increased 1.7-fold in the oviducts and 1.8-fold in the uterus of mutant mice compared to control mice, but it did not differ in the vagina. Mutant mice were infertile, in large part due to an ovarian defect that prevented ovulation. In addition, mating with either fertile or vasectomized wild-type males induced a severe inflammatory response in the reproductive tract by a mechanism that remains to be determined. In summary, over-activation of HH signaling results in a defect in patterning of the reproductive tract that is associated with an altered pattern of expression of Hoxa13 in the uterus and elevated expression of Wnt5a in the oviduct and uterus. These results show that development of the reproductive tract can be influenced by HH signaling.
Quirk, Susan Mary
Schaffer, Chris; Fortune, Joanne Elizabeth; Schimenti, John C.
M.S., Animal Science
Master of Science
dissertation or thesis