Peristaltic contractions drive gut anisotropic growth through collective cell rearrangements

Reviewer #1 (Public review):

Kawamura et al. investigated the role of circumferential smooth muscle contractions in chick gut tube elongation, addressing the hypothesis that "peristaltic activity generated by the gut promotes its own elongation during embryogenesis". Although not acknowledged in the current manuscript, this interesting premise was, in fact, previously demonstrated.

Indeed, the experiments in the present manuscript closely parallel a previous study (Khalipina et al, 2019: "Smooth muscle contractility causes the gut to grow anisotropically") that also cultured chick gut tissue and performed time-lapse analyses to quantify peristalsis. Both studies showed that inhibiting peristalsis with Ca-channel blockers induces a switch from elongational to radial growth in the gut.

However, one of the main strengths of the current study is the innovative use of optogenetic manipulation to rescue gut lengthening in drug-inhibited gut tissue by re-stimulating peristaltic contractions. In addition, the authors use aphidicolin to show that peristalsis-mediated gut elongation is independent of cell division. They also track individual smooth muscle cells and show that they divide circumferentially, but become redistributed along the length of the gut tube with peristalsis.

While these data are solidly quantitative, they do not provide mechanistic insight into how peristaltic contractions cause smooth muscle cells to be redistributed.

The evidence presented in this manuscript supports the main conclusion that peristalsis plays a critical role in embryonic gut elongation, but this conclusion itself is not novel. In addition to corroborating previous work, this manuscript provides some useful additions to our existing knowledge of the role of mechanical forces in embryonic gut morphogenesis and illustrates the utility of a previously published optogenetic manipulation technique.

Reviewer #2 (Public review):

Summary:

This study uses the chicken caecum ex vivo culture to show that embryonic peristaltic activity is a key mechanical factor for gut elongation. It is shown that pharmacological inhibition arrests intestinal growth, while optogenetic restoration rescues longitudinal elongation. The authors propose a two-step mechanism in which circular smooth muscle cells proliferate circumferentially, but peristalsis pushes them toward longitudinal rearrangement, which explains the anisotropic growth of the gut.

Strengths:

The experiments combine loss-of-function (peristalsis inhibition) with gain-of-function (optogenetic rescue) experiments and quantifiable readouts in an embryonic gut culture model. The work is clearly presented with nice microscopy videos and offers a potentially valuable conceptual framework linking tissue-scale mechanics to smooth muscle cell behaviors during development.

Weaknesses:

Some results appear conceptually inconsistent with the claim of peristalsis-essential rearrangement (e.g., longitudinal separation of daughter cells even without peristalsis), and the mechanistic link would benefit from clearer quantification and reconciliation. The study largely overlooks contributions from other gut layers and the ECM (and aphidicolin affects all proliferating cells), limiting interpretation of how smooth muscle rearrangement translates into whole-wall elongation.

Reviewer #3 (Public review):

Summary:

The authors noted a steep increase in the rate of growth with the onset of more frequent peristaltic-like movements and hypothesized that peristaltic activity rearranges the orientation of cell growth from circumferential to longitudinal. This study sought to alter peristalsis and then (1) carefully examine the growth of the chick cecum relative to the frequency of peristaltic-like movements and (2) examine the orientation of cells relative to the circumferential and longitudinal axes to determine whether peristalsis is required for cecum lengthening. To alter peristaltic-like movements, contraction was inhibited through treatment with nifedipine (a calcium channel blocker that acts to relax smooth muscle) or Ani9 (inhibits Ca-activated chloride channels), and contractions were induced through activation of a blue light-activatable channel rhodopsin 2 (introduced through electroporation).

Strengths:

(1) Use of multiple methods to alter peristalsis in initial studies.

(2) Live imaging.

(3) Careful measurements.

(4) Nicely presented figures.

Weaknesses:

(1) Only Nifedipine inhibition was examined for cell positional changes.

(2) Ki67 was not carefully analysed, and apoptosis was not shown at all.

(3) The results shown are suggestive of a role for peristalsis in the lengthening of the cecum. Demonstration that increased peristalsis could further increase lengthening would be helpful.

(4) The novelty of this work is incremental for the field in that the reagents used and the model of smooth muscle driving gut lengthening in mouse and chick small intestines have both previously been published. This manuscript does suggest that the role of smooth muscle in longitudinal growth may extend to other tubular organs (chick cecum).

Author response:

We sincerely appreciate the efforts of the Senior and Reviewing Editors, as well as the three reviewers, for their careful evaluation of our manuscript and their insightful comments. Previous studies have suggested that smooth muscle activity contributes to gut elongation; however, these studies do not directly demonstrate that peristaltic movements per se drive elongation. For example, studies in mouse have primarily focused on residual stress of smooth muscle (Yang et al., 2021), rather than the dynamic spatiotemporal nature of peristalsis. In chickens, inhibition of peristalsis by nifedipine has been interpreted as evidence for a role of peristalsis in gut elongation (Khalipina et al., 2019). However, because nifedipine broadly affects calcium-dependent cellular processes, these experiments cannot distinguish whether the observed effects arise specifically from loss of peristalsis or from other cellular perturbations. In our current study, we aimed to challenge this limitation by combining pharmacological inhibition with optogenetic reactivation. This approach allows us to selectively restore peristaltic movements under conditions in which endogenous peristalsis are suppressed. Based on these experiments, we provide evidence supporting a causal contribution of peristalsis to the anisotropic gut growth. We agree with the reviewers that the positioning of our study relative to previous work should be clarified. In a revised manuscript, we will more clearly distinguish between static mechanical tension and endogenous peristaltic movements, and better define the conceptual advance of our study. In addition to macroscopic growth analysis, we identified cellular dynamics associated with elongation, including circumferentially oriented cell division and peristalsis-dependent longitudinal cell rearrangement. We agree that the mechanistic link between peristalsis and downstream cellular behaviors remains incompletely understood. In the revised manuscript, we will clarify this limitation and outline future directions, including experiments to test the role of mechanical cues (e.g., mechanical perturbation and pharmacological manipulation of mechanotransduction pathways).

Public Reviews:

Reviewer #1 (Public review):

The mechanism by which peristalsis and the cell rearrangement are mediated

We appreciate this important point. As suggested, the possibility that mechanical aspects of peristalsis contribute to the gut elongation is highly plausible. To address this, we plan to perform additional experiments aimed at isolating the mechanical component of peristalsis. Furthermore, we will investigate the involvement of mechanotransduction pathways, including Piezo-mediated pathway, using pharmacological approaches. We will revise the manuscript to better discuss these possibilities and clarify the current limitations of our study.

The novelty and positioning of our study

We appreciate this comment and have addressed this point in the General response above. In the revised manuscript, we will more clearly position our study relative to the previous studies.

Reviewer #2 (Public review):

Longitudinal separation of daughter cells even without peristalsis

We appreciate this insightful and important comment. As noted, daughter cells can exhibit longitudinal separation even under nifedipine treatment, whereas the divergence index (DI) shows a clear increase only in the control (with peristalsis) condition. We interpret this as follows; immediately after cell division, two daughter cells occupy nearly identical positions along the longitudinal axis, and stochastic fluctuations may cause them to separate each other. Such local separation does not necessarily reflect population-level cell rearrangement. In contrast, DI captures collective dispersion of a cell population, which reflects organized tissue-level rearrangement associated with elongation. We will revise the manuscript to clarify this distinction between local cell behavior and population-level dynamics, and to better explain how DI reflects elongation-related processes.

Contributions from other gut layers and ECMs

We agree that contributions from other tissue layers and extracellular matrix (ECM) components might be important. To address this, we plan additional experiments including targeted ablation of specific tissue layers and pharmacological manipulation of ECM remodeling (e.g., using MMP modulators). We will also expand the Discussion to better acknowledge these factors.

Reviewer #3 (Public review):

(1) We agree that experiments based solely on nifedipine treatment cannot fully exclude potential off-target effects. To address this limitation, we plan to perform additional experiments that rescue the mis-rearrangement of cells by applying mechanical forces.

(2) We agree that more elaborate analyses of cell proliferation and apoptosis are needed. In the revised manuscript, we will incorporate additional analyses using appropriate markers and methods suitable for developing gut tissue.

(3) In Figure 2, we had already shown an increased the frequency of peristaltic contractions (30 s intervals, Fig. 2i, j, k, n). This did not result in a significant increase in elongation or widening compared to the control condition (120 s intervals). This suggests that the effect of peristalsis on elongation may reach a plateau at a certain frequency. We will revise the manuscript to clarify this interpretation and discuss its implications.

(4) We appreciate this important comment and have addressed the issue of novelty and positioning in the General response shown above.

Reference

Yang, Y. et al. Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation. Nat. Commun. 12, 7186 (2021).

Khalipina, D., Kaga, Y., Dacher, N. & Chevalier, N. R. Smooth muscle contractility causes the gut to grow anisotropically. J. R. Soc. Interface 16, 20190484 (2019).