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The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

Published on: November 20, 2017

Rag GTPases and mTORC1 regulate intestinal stem cell activity in response to nutrient availability.

Weikang Fan^1,2, Churui Ge^1,2, Chunmei Niu^1,2

¹Laboratory for Animal Behavioral Physiology, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China.

The Journal of Cell Biology

|July 13, 2026

Summary

Dietary sensing by gut cells regulates stem cell activity. Nutrient sensing via Rag GTPases and mTORC1 controls intestinal stem cell (ISC) proliferation and differentiation in Drosophila.

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Area of Science:

Cell Biology
Developmental Biology
Gastroenterology

Background:

The intestinal epithelium maintains homeostasis by regulating intestinal stem cell (ISC) activity.
Dietary cues are integrated by enterocytes (ECs) to control ISC function, but mechanisms are unclear.
Nutrient-sensing pathways, including Rag GTPases and mTORC1, are crucial for cellular function.

Purpose of the Study:

To investigate the roles of Rag GTPases and mTORC1 in regulating Drosophila ISC activity.
To elucidate the signaling pathways by which nutrient sensing in ECs impacts ISC proliferation and differentiation.

Main Methods:

Genetic manipulation of Rag GTPases and mTORC1 in Drosophila enterocytes and ISCs.
Analysis of gene expression (Mitf, Upd3) and signaling pathways (JAK-STAT).
Assessment of ISC proliferation and differentiation under varying nutrient conditions.

Main Results:

Inhibition of Rag GTPases in ECs activates Mitf, inducing Upd3 expression and non-cell autonomous ISC proliferation via JAK-STAT.
mTORC1 inactivation in ECs also increases ISC activity through Upd3-JAK-STAT, but independently of Mitf.
Nutrient starvation enhances ISC activity and Upd3 expression, supporting a role for nutrient sensing in ISC regulation.

Conclusions:

A nutrient-sensing regulatory network involving Rag GTPases-Mitf axis and mTORC1 activity controls ISC proliferation.
Enterocyte-specific nutrient sensing pathways modulate ISC behavior through paracrine signaling.
These findings reveal novel mechanisms linking diet to intestinal homeostasis.