Muscarinic receptor M3 contributes to intestinal stem cell maintenance via EphB/ephrin-B signaling

A signaling cascade composing M3, EphB/ephrin-B, and MEK synergizes to guide the homeostasis of intestinal epithelial cell growth and differentiation following modifications of the cholinergic intestinal niche.

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Thank you very much for your positive comments concerning our manuscript. I would like to answer your minor comments as follows.
Paneth cell quantification (Answer): Concerning Paneth cell quantification, we carried out quantitative RT-PCR to evaluate the functional consequence of deletion of M3. mRNA level of Paneth cell marker gene (Lysozyme) was significantly enhanced in M3 −/− crypts (1.5 folds over WT crypts) (Fig. 3E). Furthermore, mRNA level of EphB3 gene which is mainly expressed in Paneth cells was also enhanced compared with that of WT crypts (2 folds) (Fig. 5D). The data support increased numbers of ISCs with a proportionate increase in Paneth cells.
To have a fruitful discussion in our manuscript, I added a new paragraph as follows.
(New paragraph): The enlarged crypt morphology can be explained by a specific increase in the transit-amplifying compartment and by increased numbers of ISCs with a proportionate increase in transit-amplifying and Paneth cells. We identified the major intestinal epithelial cell populations according to their differential expression of EphB2 and marker gene expression. Loss of M3 caused profound increase in the population of the EphB2 high stem cell and EphB2 med progenitor compartment. In addition to their increased populations, we observed increased expression of ISC markers within EphB2 high and of progenitor markers within EphB2 med populations, supporting an expansion of each compartment upon loss of M3. We also observed increased expression of the marker genes for Paneth cells (Lysozyme and EphB3) as well as for enterocytes (villin) and goblet cells (mucin-2) in M3 −/− crypts, quantitatively. Paneth cells are the source of multiple stem cell growth factors (for example, Wnt3, EGF, and TGF-α) which are essential signals for stem cell maintenance (Sato et al, 2011). Indeed, co-culturing of sorted ISCs with Paneth cells dramatically improves organoid formation (Sato et al, 2011). Thus, the number of Paneth cell increased can induce to increase the number of ISCs in M3 −/− crypts. Collectively, the data support a direct effect of M3 on ISCs, which subsequently affects the size of ISC compartment and the crypt.
The role of the ENS I agree with the reviewer's comment. Understanding the role of the ENS in the intestinal crypts is of key importance. According to the reviewer's comment, I added new sentences in the third paragraph in discussion and new references as follows.
(New sentences): In a more recent study, co-culture of a monolayer of organoid-grown differentiated cells with dissociated adult mouse ENS cells shows that the epithelial cell density increases by 40% (Puzan et al, 2018). Especially, chromogranin A-positive epithelial cells increased, suggesting enhancement of enteroendocrine cell population (Puzan et al. 2018). The data imply that epithelial lineage composition may ultimately benefit from the presence of the ENS. As one of the major pathways of excitatory transmission within ENS is mediated by cholinergic transmission (Galligan et al, 2000), neuronal ACh is expected to have the potential functional effects on crypt homeostasis. The new link between M3 and EphB/ephrin-B signaling To have a fruitful discussion in our manuscript, I added a new paragraph as follows.
Additionally, I added new references as follows.
(New paragraph): The evidence that EphB and ephrin-B associate and interact with other cell surface receptors such as channel type receptors and G protein-coupled receptors suggests integration of different signaling routes or modulation of signal transmission. The N-methyl-Daspartate (NMDA) receptor channel, which is a receptor for glutamate in the central nervous system, interacts with EphB at the cell surface; this interaction is mediated by the extracellular regions of the two receptors (Dalva et al, 2000). Bruno and coworkers (Bruno et al, 2005) have provided evidence for a novel type of interaction between ephrin-B2, NMDA receptors, and metabotropic glutamate 1 receptors, a new partner in the network in the developing brain. M3 and metabotropic glutamate 1 receptors are both coupled to Gq proteins, and their activation stimulates phosphatidylinositol hydrolysis with ensuing intracellular Ca 2+ release and activation of protein kinase C (De Blasi et al, 2001). This is the first time that M3 signaling has controlled the EphB/ephrin-B system in the small intestine. However, the molecular details of the new link between M3 and EphB/ephrin-B signaling remain unclear.
(New references) 1) Dalva MB, Takasu MA, Lin MZ, Shamah SM, Hu L, Gale NW, Greenberg ME (2000)  If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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