Stratification of enterochromaffin cells by single-cell expression analysis

Reviewer #1 (Public Review):

The authors have performed extensive work generating reporter mice and performing single-cell analysis combined with in situ hybridization to arrive at 14 clusters of enterochromaffin (EC) cells. Then, they focus on Piezo channel expression in distal EC cells and find that these channels might play a role in regulating colonic motility. Overall, this is an informative study that comprehensively classifies EC cells in different regions of the small and large intestine. From a functional point of view, however, the authors seem to ignore the fact that the expression of Piezo-2-IRES-Cre is broad, which would raise concerns regarding their physiological conclusions.

The authors may wish to consider the following specific points:

It is surprising that the number of ileal EC cells is less than that of the distal colon, and it would be interesting to know whether the authors can comment about ileal EC cells. It is unclear why ileal ECs were not included in the study, even though they are mentioned in the diagram (Fig. 2c).

Based on their analysis, there are 10 EC cell clusters in SI while there are only 4 clusters in the colon. The authors should comment on whether this is reflective of lesser diversity among colonic ECs or due to the smaller number of colonic ECs collected.

The authors previously described that distal colonic EC cells exhibit various morphologies (Kuramoto et al., 2021). Do Ascl1(+) EC cells particularly co-localize with EC cells with long basal processes? Also, to validate the RNA seq data, the authors might show co-localization between Piezo2/Ascl1/Tph1 in distal EC cells. It would be interesting to see whether Ascl1-CreER (which is available in Jax) specifically labels distal colonic EC cells as this could provide a good genetic tool to specifically manipulate distal colonic EC cells.

The authors used Piezo2-IRES-Cre mice, whose expression is rather broad. They might examine the distribution of Chrm3-mCitrine in the intestine (IF/IHC would be straightforward). And if the expression is in other cell types (which is most likely the case), they should justify that the observed phenotype derives from Piezo2-expressing EC cells. Alternatively, they could use Piezo2-Cre;ePetFlp (or Vil-Flp);Chrm3 to specifically express DREADD receptors in distal colonic EC cells. Also, what does 5HT release look like in jejunal EC cells in Piezo-CHRM3 mice?

For the same reasons as above, DTR experiments may also be non-specific. For example, based on the IF staining (Fig. 6b,d), there seems to be a loss of Tph1+ cells in the proximal colon of Piezo2-DTR mice, so the effects of the Piezo2-DTR likely extend beyond the distal colon.

It is unclear why the localized loss of Piezo2 in Piezo2-DTR mice alters small intestinal transit (Fig. 6g,h). The authors should discuss the functional differences observed between Piezo2-DTR (intraluminal app) and Vil1-Piezo2 KO mice i.e., small intestinal transit, 5HT release, etc. Are these differences due to the residual Piezo2 expression in Piezo2 KO mice? In this context, the authors may want to discuss their findings in the context of recent papers, such as those from the Patapoutian and Ginty groups.

Reviewer #2 (Public Review):

Summary:
The authors investigated the expression profile of enterochromaffin (EC) cells after creating a new tryptophan hydroxylase 1 (Tph1) GFP-reporter mouse using scRNAseq and confirmative RNAscope analysis. They distinguish 14 clusters of Tph1+ cells found along the gut axis. The manuscript focuses on two of these, (i) a multihormonal cell type shown to express markers of pathogen/toxin and nutrient detection in the proximal small intestine, and (ii) on a EC-cluster in the distal colon, which expresses Piezo2, rendering these cells mechanosensitive. In- and ex- vivo data explore the role of the mechanosensitive EC population for intestinal/colonic transit, using chemogenetic activation, diptheria-toxin receptor dependent cell ablation and conditional gut epithelial specific Piezo2 knock-out. Whilst some of these data are confirmative of previous reports - Piezo2 has been implicated in mechanosensitive serotonin release previously, as referred to by the authors - the data are solid and emphasize the importance of mechanosensitive serotonin release for colonic propulsion. The transcriptomic data will guide future research.

Strengths:
The transcriptomic data, whilst confirmative, is more granular than previous data sets. Employing new tools to establish a role of mechanosensitive EC cells for colonic and thus total intestinal transit.

Weaknesses:

1. The proposed villus/crypt distribution of the 14 cell types is not verified adequately. The RNAscope and immunohistochemistry samples presented do not allow assessment of whether this interpretation is correct - spatial transcriptomics, now approaching single-cell resolution, would be likely to help verify this claim.

2. The physiological function and/or functionality of most of the transcriptomically enriched gene products has not been assessed. Whilst a role for Piezo2 expressing cells for colonic transit is convincingly demonstrated, the nature of the mechanical stimulus or the stimulus-secretion coupling downstream of Piezo2 activation is not clear.