Figure 4.
Analysis of MEC Lineage Using the SMACreERT2:R26-tdTomato Mouse
(A) Experimental design diagram. TM was administered during embryonic (E15) development, postnatal (P10/11) development, and adulthood (P27/28 and P60/61) and analyzed at variable times.
(B and C) Labeled MECs were found in the external layer of cells of the LG buds (white arrow in C). They did not have processes and presented epithelial morphology.
(D–G) (D) Labeling efficiency in the SMACreERT2:R26-tdTomato mice, as determined by co-immunostaining with the SMA antibody at E19 (E–G) (n = 27; p < 0.05, where n = number of glands).
(H–J) At E19 MECs still remain to be associated with distal parts of LG epithelial tree (H) and mainly found within the LG buds (I), where they form small processes (J; white arrows). All epithelial cells, including MECs, express Pax6 (green) (J, red arrow: Pax6-expressing MEC).
(K–M) (K) By P30 the labeled cells acquired a mature MEC phenotype, i.e., small cell body with several long processes. During postnatal development all MECs maintained close association with the maturating acinar component of the LG, as shown by immunostaining with the ductal marker Panx1 (L, green) and acinar marker Claudin 1 (M, green).
(N and O) MECs and other epithelial cells proliferate during postnatal development, as shown by immunostaining with the anti phospho-histone H3 antibody (green; proliferating MECs, yellow arrowheads; proliferating acinar cells, white arrowheads). Insert in (N) shows proliferating MEC at higher magnification. (O) shows two proliferating MECs (yellow arrowheads) in one acinus.
(P) Quantification of proliferating cells within different LG compartments (MEC, acinar, ductal, and mesenchymal [mes]). Results show mean ± SD, n = 56, ∗ p < 0.05, two-tailed t test.
(Q–S) MECs were labeled at P10/11 and quantified at P14 (Q) and P40 (S); quantification was performed using IMARIS software. Example is shown in (R).
(T) Plot showing results of MEC quantification (n = 35, ∗ p < 0.05, results show mean ± SD; two-tailed t test).
(U) Similarly, MECs were labeled at P27/28 and quantified at P60 and P120. Number of MECs in the LG significantly increases by P60 but remains almost unchanged between P60 and P120 (n = 28; ∗p < 0.05; results show mean ± SD; two-tailed t test).