Figure 2. Postnatal epithelial BMP signaling coincides with isthmus ISEMF aggregation and young trophocytes provide limited support for organoid growth.

A-B) In Pdgfra^H2B-eGFP mice, GFP^hi ISEMFs present on established and emerging (arrowhead) villi on postnatal day (P) 1 concentrate over the next 3 weeks at the crypt-villus isthmus shelf (white brackets). From representative images of proximal SI at the indicated ages (A, scale bars 50 μm) we quantified GFP^hi ISEMF cell numbers (B) at the isthmus (white brackets, 5 lowest villus epithelial cells) in relation to villus trunks (red brackets, next 5 cells toward the villus tip); graph displays data from >25 villi/sample (n=3 animals at each age). Statistical differences are reported relative to P1 after two-way ANOVA followed by Tukey’s multiple comparisons test. ns: not significant, *P <0.0001.

C) Representative pSMAD1/5/9 (magenta) immunostaining (green: LAMININ/basement membrane, blue: DAPI) in mouse proximal SI on indicated postnatal days. cr, crypts; scale bars 50 μm; boxed regions are magnified in adjoining images; grey scale and extended images are shown in Figure S1D. pSMAD⁺ epithelial cell nuclei (filled arrowheads; pSMAD⁻ cells indicated by empty arrowheads) are present at villus tops from birth, while pSMAD⁺ cells at villus bottoms increase over the ensuing 2 weeks. Graph displays pSMAD⁺ epithelial cell fractions at the bottoms (blue dots) and tops (grey dots) of >25 villi/sample (n=3 mice at each age). Statistical differences are reported relative to P1 villus bottoms after two-way ANOVA followed by Tukey’s multiple comparisons test. ns: not significant, *P <0.0001.

D) Increased epithelial pSMAD1/5 coincides with increasing isthmus ISEMF density.

E) PDGFRA^lo mesenchymal cells include trophocytes (CD81⁺, red) and CD81⁻ stromal cells (CD81⁻PRA^lo, purple). Co-cultures of P14 (left graph) or adult (right graph) SI crypts with isolated adult (black) or P14 (blue) mesenchymal cells in factor-free medium. In contrast to adult trophocytes, which support robust organoid growth, P14 trophocytes are inactive. Graph depicts organoids per sample relative to control wells (- cells + EGF, NOG, and RSPO1 (ENR); 94.5 ±46.8 P14 organoids/well; 64.5 ±20.03 adult organoids/well; n=6 each). Statistical significance determined by Student’s t-test at **P <0.01, ***P <0.001 (n=3–6 biological replicates).

See also Figure S1.