R-Spondin1 Regulates Fate of Enteric Neural Progenitors via Differential LGR4/5/6 Expression in Mice and Humans.

BACKGROUND & AIMS: Regeneration and cytodifferentiation of various adult epithelial stem cell compartments are controlled by the WNT agonist R-Spondin1 (RSPO1) and the Leucin-rich-repeat-containing G-protein-coupled receptors (LGR4/5/6). We hypothesized that RSPO1-LGR signaling is also involved in regulating neuroregeneration and homeostasis of the postnatal enteric nervous system (ENS). METHODS: We isolated neural crest-derived ENS cells from wnt1-tomato mice and patient samples, which were evaluated using pharmacological in vitro studies under RSPO1 stimulation. We use proliferation assays (BrdU incorporation, Ki67 staining), as well as neuronal differentiation screenings. We performed fluorescence-activated cell sorting-based in vitro assays to stratify human ENS cells for LGR receptor expression, and to characterize them by immunofluorescence colabeling studies in vivo. RESULTS: If applied to murine and human ENS progenitors, RSPO1 led to an increased proliferation (P = .002), followed by enhanced enteric neurogenesis (P < .001). This coincided with an upregulation of LGR4 expression during ENS progenitor proliferation (P ≤ .001) in vitro. In contrast, we observed a reduced proliferation in ENS progenitors expressing LGR5 (P ≤ .001), whereas LGR6 was not expressed by proliferative ENS progenitors (P ≤ .05). Instead, LGR5 and LGR6 expression increased over the course of induced neuronal differentiation (LGR5: P ≤ .001 and LGR6: P ≤ .05), consistent with the in vivo expression. CONCLUSIONS: LGR receptor expression therefore might represent a previously unknown mechanism influencing the fate decision of ENS progenitor cells between proliferation and neuronal differentiation. Thus, our study is essential for our understanding of regenerative aspects of the postnatal ENS in health and disease.