Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization

Abstract

The intestinal epithelium maintains a remarkable balance between proliferation and differentiation despite rapid cellular turnover. A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogen signals in the tissue. Here, to dissect epithelial-autonomous morphogenic signaling circuits, we developed an enteroid monolayer culture system that recapitulates four key properties of the intestinal epithelium, namely the ability to maintain proliferative and differentiated zones, self-renew, polarize, and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization, and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen-mediated feedback.

Keywords: BMP; Wnt; crypt; epithelium; feedback; homeostasis; intestine; organoid; proliferation.

Figure 1

Enteroid monolayers establish proliferative focal patterning and contain differentiated epithelial cell types. (A) Bright field images of 2D intestinal sheet 7 days after seeding. Arrowheads mark putative crypt foci. Scale bar = 25μM. (B) 2D intestinal cultures treated with EdU for 2 hours, then fixed and stained for EdU and DAPI. EdU clusters and dense nuclei foci co-localize (merged), indicating that the dense nuclear regions are proliferative crypt foci. Scale bar = 25μM. (C) Confocal image of crypt foci showing proliferative cells (EdU, green) clustered with Paneth cells (UEA-1, red) surrounded by non-proliferative cells (DAPI, blue). Scale bar = 25μM. (D) Confocal image of enteroid monolayers stained for actin (green) and DNA (blue). Arrowheads mark apical actin bundles. Right and bottom panels in c.–d. indicate horizontal (H) and vertical (V) projections. Scale bar = 25μM. (E) Enteroid monolayer cultures from Lgr^5eGFP-DTR mice showing Lgr5⁺ stem cells (green) juxtaposed with Paneth cells (lysozyme, red) and surrounded by proliferative TA cells (EdU, blue). Scale bar = 25μM. (F) Enteroid culture images co-stained for cell-fate marker (red: Ki-67 – proliferative; UEA-1 – Paneth; Muc2 – Goblet; villin – enterocyte; DCLK-1 – Tuft; ChgA – enteroendocrine; Olfm4 – stem), EdU (green) and DAPI (blue). Scale bar = 25μM. Crypts in B–F were seeded 7 days before staining and imaging. See also Figures S1, S2, and S3.

Figure 2

Enteroid monolayers are dynamically established and maintained. (A) Time course of crypt development after seeding on ECM. Cells labeled with EdU two hours before fixation at indicated time. Scale bar = 25 μM. (B) Timeline of experiments. (C, D), Pulse-chase time-course experiment shows tissue turnover time of ~4 days. Enteroids were pulsed with EdU two hours prior to time 0 and then chased with fresh media lacking EdU. (C) Representative images of enteroid monolayer cultures, fixed and stained for DNA (blue), EdU (green) and Villin (red) at indicated times post EdU pulse. Scale bar = 50 μM. (D) Quantification of pulse-chase experiment showing colocalization of EdU with the proliferation marker Ki67 (grey, Ki-67⁺ and EdU⁺) and colocalization of EdU with non-proliferative cells (red, Ki-67⁻ and EdU⁺); Error bars represent mean ± SEM of triplicate wells, each containing ≥10000 cells. (E) Quantification of total cell number, fraction of EdU⁺, fraction of stem cells (OLFM4⁺), and fraction of Paneth cells (lysozyme⁺) in enteroid cultures over a two-week time course. Error bars represent mean ± SEM of triplicate wells. (F) Cleaved caspase staining shows apoptosis occurs in the non-proliferative regions as opposed to the proliferative crypt regions (EdU+). Scale bar = 50 μM. (G) Annexin V staining (magenta) overlay on phase contrast image shows cell death occurs on the margins of tissues. Asterisks denote clusters of Paneth cells. Scale bar = 50 μM. See also Figure S3.

Figure 3

The maintenance of growth and pattern formation for crypt foci is regulated by WNT and BMP. (A) Crypts were maintained for 2 days under a matrix of WNT3a vs. BMP4 concentrations in ENR media (see Experimental Procedures). Cells were labeled with EdU for two hours before fixation and staining for EdU and DNA. (B,C) As in (A), but crypt number (B) and average number of cells per crypt (C) were quantified. (D, E, F) As in (A–C) but with 5 days of growth factor treatment. Scale bars = 25 μM. See also Figure S3.

Figure 4

The establishment of crypt foci requires intrinsic WNT and BMP signaling to regulate growth and patterning. (A–F) Enteroid monolayers respond to WNT or BMP after initial seeding. Four hours after seeding, cells were treated with ENR basal media and the indicated perturbation, WNT3a (8 nM), IWP-2 (10 uM), BMP4 (9 nM), or LDN (100 nM), throughout the time course. Cells were labeled with EdU two hours before fixation at the indicated time. (A) Staining of nuclei (DAPI) and proliferative cells (EdU). Scale bar = 50 μM. (B,C) Quantification of fraction EdU⁺ cells (B), and ratio of crypt to villus cell numbers (C) of experiment in (A) throughout the time course. Error bars represent mean ± SEM of triplicate wells. (D, E, F) WNT3a treatment rescues proliferation but not patterning of enteroid monolayers. 48 hour treatment. (D) Quantification of fraction EdU⁺ shows rescue of proliferation. Error bars represent mean ± SEM of triplicate wells. (E) Representative images of crypts under treatment at 48 hours. Scale bar = 50 μM. (F) Density plot of dispersion, as measured by distance between nearest neighbor EdU+ cells. Co-treatment with IWP-2 and WNT3a significantly shifts distribution compared to control. (G) Suppression of WNT3a-mediated growth collapse at 72 hours by LDN treatment. Error bars represent mean ± SEM of triplicate wells. (H) Quantification of fraction of Paneth cells out of total cells under 48 hours of WNT3a/BMP4 treatment. For morphogen concentrations, see Experimental Procedures. (I) Model of tissue-autonomous growth and patterning self-regulation via WNT/BMP signaling. See also Figure S4.