Fig. 5.
Lama5-deficient luminal MECs are unable to support basal MECs due to defective Wnt signaling. (A) qRT-PCR analysis of Wnt4 expression compared with GAPDH in basal (CD29hi/CD24+) and luminal (CD29low/CD24+) MECs FACS sorted from 8-10-week-old Lama5+/+;K8-CreERT2 and Lama5fl/fl;K8-CreERT2 glands treated with tamoxifen starting from 3 weeks of age (+/+ luminal and basal, fl/fl basal, n=3; fl/fl luminal, n=4 samples analyzed). (B) qRT-PCR analysis of Sox9, Ctnnb1 and Lgr5 expression compared with GAPDH in basal MECs from 10-week-old Lama5fl/fl;K8-CreERT2 and control mice (Sox9 and Ctnnb1 +/+, n=8; fl/fl, n=10; Lgr5 +/+, n=7; fl/fl, n=7 samples analyzed). In A and B, data points indicate RNA samples analyzed from individual mice. (C) Primary MMECs carrying either LAMA5 or SCRA CRISPR guides (gLAMA5 and gSCRA) grown in Matrigel in the presence of 2.5 nM FGF-2 for 4 or 10 days, and quantification of the percentage of branching organoids at day 10 (gSCRA, n=3; gLAMA, n=3). (D) gLAMA5 or gSCRA expressing primary MMECs grown in Matrigel in the presence of 2.5 nM FGF-2 and either Wnt4, Rspo1 or both ligands for 7 days. (E) Quantification of the percentage of branching organoids at day 7 grown as in D (all treatments n=3). (F) Schematic showing the outline of tamoxifen induction and representative images. MECs were isolated from 10-14-week-old Lama5+/+;K8-CreERT2 and Lama5fl/fl;K8-CreERT2 mice treated with tamoxifen 3 days earlier, and grown in Matrigel in the presence of 2.5 nM FGF-2 and either Wnt4 or IWP-2 for 7 days. (G) Quantification of the percentage of branching organoids at day 7 grown as in F (all treatments n=4). In C, E and G, data points indicate RNA samples analysed from individual mice. (H) Representative keratin 8 and E-cadherin immunofluorescence images of primary MMECs organoids treated as in F and grown for 7 days. (I) Representative keratin 14 immunofluorescence images of primary MMECs organoids expressing either gLAMA5 or gSCRA treated as in D and grown for 7 days. (J) Schematic showing the outline of organoid treatment with lactogenic medium. Quantitative PCR analysis of Csn2 gene expression in MECs isolated from 10-14-week-old Lama5+/+;K8-CreERT2 and Lama5fl/fl;K8-CreERT2 mice treated with tamoxifen 3 days earlier, and grown as described in the schematic with or without Wnt4 (all treatments n=3). Data are normalized to lactogenic medium-treated Lama5+/+;K8-CreERT2 for each experiment. Data points indicate RNA samples analysed from independent organoid experiments. Data are mean±s.d. *P<0.05; **P<0.01; ns, not significant [unpaired one-tailed (J) or two-tailed (A,B,C,E,G) Student's t-test]. Scale bars: 100 µm (C,D,H); 50 µm (I).