Supplementary Figure 3: Basal and luminal Lgr6 + progenitors contribute to the alveolar network during pregnancy.

(a, b) Confocal images of K14/K8 immunostained whole mount mammary glands from LT female at 17.5 dpc in first pregnancy (17.5 dpc P1). tdTomato⁺ clones dispersed over newly formed alveolar structures contain either basal K14⁺ cells (a) or K8⁺ alveolar cells (b). Scale bars, 40 μm (a), 20 μm (b). (c) Confocal overview image of mammary gland on day 1 of the first lactation (1d L1). LN: lymph node. Scale bar, 2 mm. (d) 3D reconstruction of 1d L1 mammary gland showing an alveolar network of myoepithelial tdTomato⁺ cells. Scale bar, 50 μm. (e) Quantification of alveoli containing basal and luminal tdTomato + cells in the first lactation after tamoxifen administration to LT females at 4w of age. (1,115 alveoli pooled from 3 mice were analysed). (f) Confocal z-stack image of tdTomato⁺ cells lining ducts and alveolar remnants (arrows), and adjacent to apoptotic cells (arrowheads) at the end of involution (21d Inv1). Autofluorescent signal (arrowheads) comes from dying cells. Scale bar, 50 μm. (g) Confocal z-stack image of K14/K8 immunostained mammary duct at the end of involution. Scale bar, 25 μm. (h) 3D reconstruction of K8 stained mammary duct showing luminal clones remaining after involution. Scale bar, 50 μm.