In this year, we analyzed the role of LGR4, which contributed in the generation and maintenance of mammary gland ductal structure, by using epithelial cell-specific lgr4 gene deficient female mice (lgr4^{K5 KO}), and lgr4-EGFP-CreERT2 mutants mice in our project. Resultantly, we’ve found that Lgr4 gene defect caused the decrease in the number of branching structures and impairment in milk production with hypoplasia of alveolar in pregnancy, indicating that LGR4 had an essential role in the structural and functional properties of mammary duct. The flow cytometry analysis using cells prepared from mammary glands of the tissue-specific lgr4 gene deficient mice, showed decreased expression of the marker genes related to undifferentiated state of the mammary epithelial cells. This result indicates that LGR4 contributes to the maintenance of undifferentiated state of mammary epithelial cells. In addition, we tried the 3D culture of mammary epithelial cells. By this in vitro experiment, we obtained interesting results that suggested that several soluble factors involved in Wnt signaling might regulate the tubular structural formation in the mammary-like organoids.

On the other hand, we found that lgr4^{K5 KO} female showed the subfertily with the impaired formation of the uterine gland, which is the micro-tubular tissue in uterine epithelium. This study implies that Wnt/Rspo/LGR4 signaling is also playing important roles in the development of glandular tissue in uterine epithelium, and we published these data in FASEB Journal in 2013.