Microbiota promote secretory cell determination in the intestinal epithelium by modulating host Notch signaling.

Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer.

Publisher Correction: Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.

Lymphocytes cross vascular boundaries via either disrupted tight junctions (TJs) or caveolae to induce tissue inflammation. In the CNS, Th17 lymphocytes cross the blood-brain barrier (BBB) before Th1 cells; yet this differential crossing is poorly understood. We have used intravital two-photon imaging of the spinal cord in wild-type and caveolae-deficient mice with fluorescently labeled endothelial tight junctions to determine how tight junction remodeling and caveolae regulate CNS entry of lymphocytes during the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. We find that dynamic tight junction remodeling occurs early in EAE but does not depend upon caveolar transport. Moreover, Th1, but not Th17, lymphocytes are significantly reduced in the inflamed CNS of mice lacking caveolae. Therefore, tight junction remodeling facilitates Th17 migration across the BBB, whereas caveolae promote Th1 entry into the CNS. Moreover, therapies that target both tight junction degradation and caveolar transcytosis may limit lymphocyte infiltration during inflammation.

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota.

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.

Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation.

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development and tissue physiology. In vertebrates, the most populous and complex community of microbes resides in the digestive tract. Here, we describe the establishment of the gut microbiota and its role in digestive tract differentiation in the zebrafish model vertebrate, Danio rerio. We find that in the absence of the microbiota, the gut epithelium is arrested in aspects of its differentiation, as revealed by the lack of brush border intestinal alkaline phosphatase activity, the maintenance of immature patterns of glycan expression and a paucity of goblet and enteroendocrine cells. In addition, germ-free intestines fail to take up protein macromolecules in the distal intestine and exhibit faster motility. Reintroduction of a complex microbiota at later stages of development or mono-association of germ-free larvae with individual constituents of the microbiota reverses all of these germ-free phenotypes. Exposure of germ-free zebrafish to heat-killed preparations of the microbiota or bacterial lipopolysaccharide is sufficient to restore alkaline phosphatase activity but not mature patterns of Gal alpha1,3Gal containing glycans, indicating that the host perceives and responds to its associated microbiota by at least two distinct pathways.