Compartment specific responses to contractility in the small intestinal epithelium

Tissues are subject to multiple mechanical inputs at the cellular level that influence their overall shape and function. In the small intestine, actomyosin contractility can be induced by many physiological and pathological inputs. However, we have little understanding of how contractility impacts the intestinal epithelium on a cellular and tissue level. In this study, we probed the cell and tissue-level effects of contractility by using mouse models to genetically increase the level of myosin activity in the two distinct morphologic compartments of the intestinal epithelium, the crypts and villi. We found that increased contractility in the villar compartment caused shape changes in the cells that expressed the transgene and their immediate neighbors. While there were no discernable effects on villar architecture or cell polarity, even low levels of transgene induction in the villi caused non-cell autonomous hyperproliferation of the transit amplifying cells in the crypt, driving increased cell flux through the crypt-villar axis. In contrast, induction of increased contractility in the proliferating cells of the crypts resulted in nuclear deformations, DNA damage, and apoptosis. This study reveals the complex and diverse responses of different intestinal epithelial cells to contractility and provides important insight into mechanical regulation of intestinal physiology.

The Arhgef11CA construct has been extensively tested by both us and others in the past to induce contrac@lity in cultured cell lines, primary cells and mice.We have included greater descrip@on of this in the text.Optogene@c ac@va@on of the construct has been shown to result in rapid contrac@le responses (Berlew et al 2021).The construct contains only the GEF domain of Arhgef11 and not addi@onal domains that may mediate other func@ons.Further, when expressed in mouse epidermis (where it is much more uniformly expressed, making these experiments possible), it causes a @ssue wide compac@on that was measured in ex vivo skin explants (Ning et al, 2021).We have been unable to replicate this in organoids as in this system we lost the compartmentaliza@on of expression and organoids died soon aPer Arhgef11CA induc@on.The very similar effects on ac@n, myosin, cell shape, and junc@onal tension between the epidermis and the villar epithelium provide strong support for a contrac@le func@on.
To convincingly demonstrate that the phenotypes observed are due to cell contrac;lity or tension heterogeneity within the epithelium, the authors should 1) test whether these phenotypes (in both crypt and villus domains) are recapitulated in 2D or 3D intes;nal organoids isolated from these mice, and if so whether they can be rescued by ROCK inhibi;on (e.g.Y27632 or Thiazovivin).
As men@oned above, we are unable to perform these experiments in organoids as we see rapid death aPer induc@on of Arhgef11CA in this system (we see widespread expression of the transgene and apoptosis).For this reason, we turned to inhibi@on of contrac@lity in vivo and we chose to assay y-H2aX induc@on as it is a clear and rapid response to contrac@lity.We fed VillinCre;RosartTA;TreArhgef11 mice doxycycline food, and injected them with 10mg/kg Y27632 (Rock inhibitor) approximately 8 hours later.We then measured the percentage of y-H2aX posi@ve cells per crypt and found that Rock inhibi@on significantly rescued the DNA damage phenotype in Arhgef11 Crypt animals.We have included this quan@fica@on in Figure 6 of the manuscript.

They should test whether ac;va;on of contrac;lity in vitro through Calyculin A treatment or other means (e.g. len;viral transduc;on of other contrac;lity inducers) recapitulates the observed phenotypes.
To address this point, we treated wildtype organoids with Calyculin A and assayed for DNA damage (yH2ax expression).We find that there is a marked induc@on of yH2aX expression specifically in the crypt base of these organoids aPer two hours of calyculin a treatment.We have not included this in the manuscript due to the broad effects of Calyculin A.

"The changes in contrac;lity appear to be quite local and we have no evidence that they are transmiTed through mul;ple cells." This claim should be inves;gated in more detail by quan;fying the junc;onal MyoII C etc. as well as phosphorylated myosin light chain in cells adjacent and within the vicinity of the
Arhgef11CA-expressing villus cells.From some of the images (e.g.1I and S2C), it appears that there might be subtle differences that could be more obvious when quan;fied and would help explain the non-cellautonomous effects -perhaps one of the most exci;ng findings of this manuscript.
We included data showing that there is only an increase in α-18 staining in Arhgef11CA-expressing cells (Fig S1C -F).In these quan@fica@ons, cells immediately adjacent to Arhgef11CA-expressing cells have α-18 levels that are not significantly different from control @ssue.

Minor comments:
The ;ming of dox induc;on and ;ssue collec;on is unclear for some of the experiments.The manuscript would benefit from adding the experimental ;melines to all figures.
We apologize for the confusion and have added diagrams highligh@ng experimental @melines to necessary figures.
Where along the crypt-villus axis were the line profiles for quan;fica;on drawn?Was this specifically for cells along the villus sides or at the ;ps?The loca;ons could themselves have heterogeneity between them in control samples, so an important control would be to measure the line profiles of Ac;n/MyoIIC at different posi;ons along the villus or at least clearly state where the measurements are made.
As the primary expression loca@on of Arhgef11-CA expressing cells in the Arhgef11 Villi model was the lower third of the villus, our line scan quan@fica@ons were consistently measured from this region.We have included this informa@on in the methods sec@on of the text.
Are there changes to villus morphology in the Arhgef11-Crypt mice?It would be assumed that the villi would undergo atrophy but this should be more clearly shown.
There are no major morphological differences in the villus aPer 24 hours of induc@on in Arhgef11 Crypt mice.Most of the rapid weight loss seems to be due to defects in the colon leading to mis regula@on of water absorp@on.However, aPer 48 hours of induc@on, Arhgef11 Crypt do begin to exhibit some villus atrophy, consistent with the massive @ssue disorganiza@on at that point.
Are the Arhgef11-crypt phenotypes specific to the small intes;ne, or is the expression/phenotype also observed in the colon?
Quan@fica@on of DNA damage in colon samples of Arhgef11 Crypt animals suggest that there are similar phenotypes in this @ssue as well.However, the induc@on of Arhgef11CA expression and of DNA damage is more variable from animal to animal in the colon resul@ng in an upward trend of γ-H2aX expression that does not reach sta@s@cal significance via paired t-test.We have included the γ-H2aX colon data in Supplemental Figure 5.

Mul;ple misspellings of "Arhgef"
Thank you for bringing this to our agen@on.We have corrected the spelling mistakes in the text.
"Regardless, these data indicate that the crypt hyperplasia, in response to increased actomyosin contrac;lity in villar cells, is driven by transit amplifying cell hyperprolifera;on and not an expansion of stem cells."This is not convincing from the graph of Sox9 expression alone.To make this claim the authors should look at the propor;on of more bona fide ISC markers, such as Lgr5 and Olfm4.Like point #4, the authors need to use a more robust list of markers to support their conclusion that the TA progenitors, not the stem cells, are the cell popula;on driving the hyperprolifera;ve phenotype.Lgr5,Olfm4,Math1,Notch1,etc., markers should be included.
We appreciate this important point.We have now included quan@fica@on of Olfm4 expression in control and Arhgef11 Villi samples.The length of the Olfm4 expression domain, as well as the number of posi@ve cells per crypt is not significantly different between groups, which is consistent with the Sox9 data.We have added representa@ve images and the quan@fica@on to Supplemental Figure 7 and included this data in the text.
"Together these data demonstrate that mechanical deforma;on induces DNA damage leading to apoptosis of these cells" -Not directly tested that this is causal.
Thank you for this comment.Based on our @me course experiments, the events of DNA damage occur before apoptosis (and independently of it), but we did not test whether there is a direct connec@on.We have adjusted our conclusions in the text to beger reflect our observa@ons.
Reviewer 2 comments: Major comments: The authors must elaborate on the reasons for actomyosin gain-of-func@on experiments in the introduc;on.It needs to be clarified what was the hypothesis that the authors were trying to test in this study.For example, the following sentence is vague and needs clarifica;on on what func;on remains unclear: "Despite the importance of actomyosin-mediated contrac;lity in intes;nal development and homeostasis, the effects that contrac;lity has on cell and ;ssue func;on remain unclear." We have expanded on our hypothesis in the text.
"Regulated F-ac@n dynamics are required for ac@ve villar cell migra@on, driving the homeosta@c flux of the @ssue [21].Addi@onally, actomyosin is required for both cell extrusion, which is important for villar cell turnover, and cell contrac@on in response to infec@on [22,23].Despite the importance of actomyosin-mediated contrac@lity in intes@nal development and homeostasis, and its modula@on during pathologic condi@ons, the effects that contrac@lity has on cell and @ssue func@on remain unclear.This is largely due to the fact that very few tools exist to specifically tune a cell's contrac@le state in vivo, in order to study its cell and @ssue level effects.By manipula@ng contrac@lity in vivo, in the absence of addi@onal perturba@ons, we hope to define the cell and @ssue-specific responses that are induced by increased acto-myosin ac@vity." The authors should refrain from statements that are not accurate, such as: "This study highlights dis;nct, compartment specific responses to cell contrac;lity and, for the first ;me, reveals the mechanical sensi;vity of small intes;nal epithelial stem cells."There are published studies tes;ng "mechanical sensi;vity" in the crypt compartment, including stem cells.To name a few: Pen;nmikko et al. 2022;Pérez-González et al. 2021;Yang et al. 2021;Sumigray et al. 2018).
• "for the first ;me, reveals the mechanical sensi;vity of small intes;nal epithelial stem cells" o Mul;ple studies, included some cited here, have shown this (e.g. PMIDs: 31019299, 31019299, 31019299, 31019299) We apologize that this statement was not as clear as intended.We have adjusted it to state that they are highly sensi@ve to internal mechanical forces generated by the acto-myosin cytoskeleton.Previous work focused on external forces.
The authors should explore the expression of NMIIA, which is also expressed in the intes;nal epithelium, and they are strongly encouraged to stain for pMLC, which indicates ac;vated myosin and contrac;ng stress fibers.