EE‑Derived Wnt Hotspots Drive Crypt Niche Collapse During Aging

Aging remodels the intestinal crypt by increasing enteroendocrine (EE) cell density while reducing progenitor proliferation, yet the functional consequence of this shift on Wnt gradient shape remains unclear. We hypothesize that the age‑associated rise in EE cells creates ectopic Wnt‑signaling hotspots that locally elevate β‑catenin activity, disrupting the smooth Wnt gradient required for balanced stem‑cell maintenance and differentiation. These hotspots arise because EE‑derived Kramer protein increasingly antagonizes Kelch, reducing Cullin‑3–mediated degradation of Dishevelled and stabilizing Wnt signaling in a spatially restricted manner. Simultaneously, age‑related shifts in the microbiota diminish production of short‑chain fatty acids that normally suppress EE differentiation, removing a brake on EE expansion. The resulting hotspot‑driven Wnt noise interferes with the positional information that guides stem‑cell fate decisions, leading to premature differentiation and niche erosion.

Key predictions: (1) Spatial transcriptomics of young versus aged crypts will reveal focal zones of heightened Wnt‑target gene expression (e.g., Axin2, Lgr5) colocalized with EE markers (Chga, Tph1) in aged tissue but not in young tissue. (2) Pharmacological inhibition of Kramer‑Kelch interaction in aged mice will flatten ectopic Wnt peaks without globally reducing Wnt signaling, rescuing ISC proliferation. (3) Microbiota transplantation from young donors into aged hosts will lower EE density, diminish Wnt hotspots, and restore a monotonic Wnt gradient along the crypt‑villus axis. (4) Live imaging of a Wnt‑activity reporter (TCF/Lef::H2B‑Venus) combined with EE‑specific calcium flux sensors will show stochastic, high‑amplitude Wnt spikes in EE cells of aged crypts that correlate with nearby ISC exit from the cell cycle.

To test these hypotheses we propose a multidisciplinary approach: (a) Perform 10x Visium spatial transcriptomics on crypt sections from 3‑month and 24‑month mice, quantifying Wnt‑target expression relative to EE markers; (b) Treat aged mice with a peptide disruptor of Kramer‑Kelch binding (validated in vitro) and measure ISC Ki67 index and organoid‑forming capacity; (c) Conduct fecal microbiota transplantation from young to aged recipients, followed by EE immunostaining and Wnt‑reporter imaging; (d) Use intravital microscopy with a FRET‑based β‑catenin sensor and EE‑specific GCaMP to capture real‑time Wnt dynamics in situ. Falsification would occur if aged crypts show no ectopic Wnt hotspots, if Kramer‑Kelch disruption fails to normalize Wnt gradients, or if microbiota rescue does not alter EE density or Wnt patterning despite reversing other age‑related phenotypes. This framework shifts focus from cell‑autonomous ISC decline to an emergent property of spatially distorted morphogen fields driven by niche‑cell composition.