The nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist muramyl dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cell (ISC) survival through NOD2 activation upon an otherwise lethal oxidative stress-mediated signal. However, the underlying protective mechanisms remain unknown. Here, using irradiation as stressor and primarily murine-derived intestinal organoids as a model system, we show that MDP induced a significant reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 knockout (KO) and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We confirmed the MDP-dependent induction of ISC mitophagy upon stress in vivo. These findings elucidate the NOD2-mediated mechanism of cytoprotection involving the clearance of the lethal excess of ROS molecules through mitophagy, triggered by the coordinated activation of NOD2 and ATG16L1 by a nuclear factor κB (NF-κB)-independent pathway.

Conditional deletion of Notchless leads to rapid deletion and exhaustion of HSCs and early progenitor cells, whereas committed progenitor cells survive as a result of differences in ribosomal biogenesis.

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Our knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. To investigate this issue, we studied one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. We show that element C contains multiple binding sites for Meis and Hox/Pbx factors and that these proteins synergize to activate the enhancer. Mutation of these binding sites allowed us to establish that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, our data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, we show that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, our data clarify the molecular mechanisms of an essential step in hindbrain patterning. We propose a model for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops.