Innate immune receptor NOD2 mediates LGR5+ intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation.

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.

Decrypting the communication between microbes and the intestinal mucosa-A brief review on Pathogénie Microbienne Moléculaire's latest research.

Over the past 10 years, the "Pathogénie Microbienne Moléculaire" unit of Professor Philippe Sansonetti has studied the molecular cross talk between the intestinal microbiota and the gut epithelium, aiming to better understand how this mutualistic symbiosis delineates homoeostasis and, when perturbed, prompts pathology. To do so, the unit has manipulated both bacterial and epithelial cells, and used cutting-edge technology. More recently, the lab has turned its focus also on studying the intestinal crypt and more specifically the intestinal stem cell for their role in epithelial regeneration and long-term epithelium renewal. Here, we provide a brief review summarising recent results obtained from the lab, with particular focus on the intestinal crypt.

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

A functional interaction between Irx and Meis patterns the anterior hindbrain and activates krox20 expression in rhombomere 3.

Patterning of the vertebrate hindbrain involves a segmentation process leading to the formation of seven rhombomeres along the antero-posterior axis. While recent studies have shed light on the mechanisms underlying progressive subdivision of the posterior hindbrain into individual rhombomeres, the early events involved in anterior hindbrain patterning are still largely unknown. In this paper we demonstrate that two zebrafish Iroquois transcription factors, Irx7 and Irx1b, are required for the proper formation and specification of rhombomeres 1 to 4 and, in particular, for krox20 activation in r3. We also show that Irx7 functionally interacts with Meis factors to activate the expression of anterior hindbrain markers, such as hoxb1a, hoxa2 and krox20, ectopically in the anterior neural plate. Then, focusing on krox20 expression, we show that the effect of Irx7 and Meis1.1 is mediated by element C, a conserved cis-regulatory element involved in krox20 activation in the hindbrain. Together, our data point to an essential function of Iroquois transcription factors in krox20 activation and, more generally, in anterior hindbrain specification.

Compensation between Wnt-driven tumorigenesis and cellular responses to ribosome biogenesis inhibition in the murine intestinal epithelium.

Ribosome biogenesis inhibition causes cell cycle arrest and apoptosis through the activation of tumor suppressor-dependent surveillance pathways. These responses are exacerbated in cancer cells, suggesting that targeting ribosome synthesis may be beneficial to patients. Here, we characterize the effect of the loss-of-function of Notchless (Nle), an essential actor of ribosome biogenesis, on the intestinal epithelium undergoing tumor initiation due to acute Apc loss-of-function. We show that ribosome biogenesis dysfunction strongly alleviates Wnt-driven tumor initiation by restoring cell cycle exit and differentiation in Apc-deficient progenitors. Conversely Wnt hyperactivation attenuates the cellular responses to surveillance pathways activation induced by ribosome biogenesis dysfunction, as proliferation was maintained at control-like levels in the stem cells and progenitors of double mutants. Thus, our data indicate that, while ribosome biogenesis inhibition efficiently reduces cancer cell proliferation in the intestinal epithelium, enhanced resistance of Apc-deficient stem and progenitor cells to ribosome biogenesis defects may be an important concern when using a therapeutic strategy targeting ribosome production for the treatment of Wnt-dependent tumorigenesis.

Pancreatic Ductal Deletion of Hnf1b Disrupts Exocrine Homeostasis, Leads to Pancreatitis, and Facilitates Tumorigenesis.

BACKGROUND & AIMS: The exocrine pancreas consists of acinar cells that produce digestive enzymes transported to the intestine through a branched ductal epithelium. Chronic pancreatitis is characterized by progressive inflammation, fibrosis, and loss of acinar tissue. These changes of the exocrine tissue are risk factors for pancreatic cancer. The cause of chronic pancreatitis cannot be identified in one quarter of patients. Here, we investigated how duct dysfunction could contribute to pancreatitis development. METHODS: The transcription factor Hnf1b, first expressed in pancreatic progenitors, is strictly restricted to ductal cells from late embryogenesis. We previously showed that Hnf1b is crucial for pancreas morphogenesis but its postnatal role still remains unelucidated. To investigate the role of pancreatic ducts in exocrine homeostasis, we inactivated the Hnf1b gene in vivo in mouse ductal cells. RESULTS: We uncovered that postnatal Hnf1b inactivation in pancreatic ducts leads to chronic pancreatitis in adults. Hnf1bΔduct mutants show dilatation of ducts, loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis. We deciphered the early events involved, with down-regulation of cystic disease-associated genes, loss of primary cilia, up-regulation of signaling pathways, especially the Yap pathway, which is involved in acinar-to-ductal metaplasia. Remarkably, Hnf1bΔduct mutants developed pancreatic intraepithelial neoplasia and promote pancreatic intraepithelial neoplasia progression in concert with KRAS. We further showed that adult Hnf1b inactivation in pancreatic ducts is associated with impaired regeneration after injury, with persistent metaplasia and initiation of neoplasia. CONCLUSIONS: Loss of Hnf1b in ductal cells leads to chronic pancreatitis and neoplasia. This study shows that Hnf1b deficiency may contribute to diseases of the exocrine pancreas and gains further insight into the etiology of pancreatitis and tumorigenesis.

[Microbiota-intestinal stem cells dialog: a key element for intestinal regeneration]. / Le dialogue microbiote-cellules souches - Un élément clé pour la régénération intestinale.

The most abundant and well-studied microbiota on the human body resides in the intestinal tract. Its impact extends the limits of the mucosal interface as it plays an essential role in systemic functions such as development of the immune system. At the level of the intestine, commensal microbes play important metabolic functions and promote the integrity of the mucosal barrier. Moreover, a large number of studies points to a role of the microbiota in intestinal regeneration both under homeostatic conditions and after epithelial damage. As intestinal regeneration is sustained by highly proliferative intestinal stem cells (ISCs), these observations raise the question of a direct impact of commensals on the activity of these cells. Key mediators of the dialog between microbes and the epithelium are the immune cells residing in the gut. Consistently, both innate lymphoid cells and macrophages activated by microbial stimuli have been shown to promote ISCs proliferation by secreting cytokines. More direct routes of communication have been described recently, either through the binding of bacterial ligands to Pattern Recognition Receptors expressed in ISCs, or through the sensing by ISCs of bacterial metabolites. In this review, we explore this stem cell-microbiota dialog and its impact on gut homeostasis.

Initiation of cyp26a1 Expression in the Zebrafish Anterior Neural Plate by a Novel Cis-Acting Element.

Early patterning of the vertebrate neural plate involves a complex hierarchy of inductive interactions orchestrated by signalling molecules and their antagonists. The morphogen retinoic acid, together with the Cyp26 enzymes which degrade it, play a central role in this process. The cyp26a1 gene expressed in the anterior neural plate thus contributes to the fine modulation of the rostrocaudal retinoic acid gradient. Despite this important role of cyp26a1 in early brain formation, the mechanisms that control its expression in the anterior neural plate are totally unknown. Here, we present the isolation of a 310-base-pair DNA element adjacent to cyp26a1 promoter, displaying enhancer activity restricted to the anterior neural plate of the zebrafish gastrula. We show that unlike that of cyp26a1, expression driven by this cyp26a1 anterior neural plate element (cANE) is independent of retinoic acid. Through deletion analysis, we identify a 12-nucleotide motif essential for cANE activity. A consensus bipartite binding site for SoxB:Oct transcription factors overlaps with this motif. Mutational analysis suggests that SoxB binding is essential for its activity. We discuss the contribution of this study to the elucidation of the regulatory hierarchy involved in early neural plate patterning.

Notchless-dependent ribosome synthesis is required for the maintenance of adult hematopoietic stem cells.

Blood cell production relies on the coordinated activities of hematopoietic stem cells (HSCs) and multipotent and lineage-restricted progenitors. Here, we identify Notchless (Nle) as a critical factor for HSC maintenance under both homeostatic and cytopenic conditions. Nle deficiency leads to a rapid and drastic exhaustion of HSCs and immature progenitors and failure to maintain quiescence in HSCs. In contrast, Nle is dispensable for cycling-restricted progenitors and differentiated cells. In yeast, Nle/Rsa4 is essential for ribosome biogenesis, and we show that its role in pre-60S subunit maturation has been conserved in the mouse. Despite its implication in this basal cellular process, Nle deletion affects ribosome biogenesis only in HSCs and immature progenitors. Ribosome biogenesis defects are accompanied by p53 activation, which causes their rapid exhaustion. Collectively, our findings establish an essential role for Nle in HSC and immature progenitor functions and uncover previously unsuspected differences in ribosome biogenesis that distinguish stem cells from restricted progenitor populations.

Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors.

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.

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant.

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.