Optogenetic control of gut movements reveals peristaltic wave-mediated induction of cloacal contractions and reactivation of impaired gut motility

Gut peristalsis, recognized as a wave-like progression along the anterior-posterior gut axis, plays a pivotal role in the transportation, digestion, and absorption of ingested materials. The embryonic gut, which has not experienced ingested materials, undergoes peristalsis offering a powerful model for studying the intrinsic mechanisms underlying the gut motility. It has previously been shown in chicken embryos that acute contractions of the cloaca (an anus-like structure) located at the posterior end of the hindgut are tightly coupled with the arrival of hindgut-derived waves. To further scrutinize the interactions between hindgut and cloaca, we here developed an optogenetic method that produced artificial waves in the hindgut. A variant form of channelrhodopsin-2 (ChR2(D156C)), permitting extremely large photocurrents, was expressed in the muscle component of the hindgut of chicken embryos using Tol2-mediated gene transfer and in ovo electroporation techniques. The D156C-expressing hindgut responded efficiently to local pulses of blue light: local contractions emerge at an ectopic site in the hindgut, which were followed by peristaltic waves that reached to the endpoint of the hindgut. Markedly, the arrival of the optogenetically induced waves caused concomitant contractions of the cloaca, revealing that the hindgut-cloaca coordination is mediated by signals triggered by peristaltic waves. Moreover, a cloaca undergoing pharmacologically provoked aberrant contractions could respond to pulsed blue light irradiation. Together, the optogenetic technology developed in this study for inducing gut peristalsis paves the way to study the gut movement and also to explore therapeutic methodology for peristaltic disorders.


Introduction
Gut peristalsis is recognized as a wave-like propagation of a local contraction along the anterior-posterior (A-P; also called oral-aboral) gut axis. The physiology of gut peristalsis has extensively been studied in adults, and the importance of peristaltic movements for the transportation, digestion, and absorption of ingested materials has greatly been appreciated (Huizinga et al., 1998;Huizinga and Lammers, 2009;Spencer et al., 2016). Although it is known that many gut-related disorders are associated with dysfunction of gut motility/peristalsis, efficient therapeutic methods have not been developed. A main reason is that the intrinsic mechanisms underlying the initiation and maintenance of gut peristalsis remain largely unexplored.
It has been shown that the embryonic gut in vertebrates undergoes peristalsis, offering a powerful model to understand the intrinsic mechanisms by which the peristalsis is regulated (Holmberg et al., 2007;Roberts et al., 2010;Chevalier et al., 2017;Chevalier et al., 2019;Chevalier et al., 2020;Okamoto and Hatta, 2022;Shikaya et al., 2022). We have recently shown in chickens that the embryonic cloaca, the posterior orifice of the gut, which is important for excretion of the urine-feces complex (Romanoff, 1960;King and McLelland, 1981), undergoes acute contractions occurring concomitantly with the arrival of hindgut-derived waves (Shikaya et al., 2022). Moreover, when the cloaca is isolated from the hindgut, its contractions are abolished implying intimate coordination between cloaca and its adjacent hindgut (Shikaya et al., 2022). To know whether the cloacal contractions are triggered by the arrival of peristaltic waves from the hindgut, it is necessary to experimentally/ artificially produce peristalsis in the hindgut and observe if this manipulation causes acute contractions in the cloaca at the timepoint of wave arrival.
To test this hypothesis, we sought an optogenetic approach whereby we could induce artificial peristaltic waves. A commonly used protein for the optogenetics is channelrhodopsin-2 (ChR2) discovered originally in microbes. This protein is a non-selective cation channel that opens in response to blue light (470 nm) leading to a cellular excitation (Nagel et al., 2003). The wild type and several variants of ChR2 have been used to control the gut motility. While wild type ChR2 and its variant H134R (a mutation in histidine-134 to arginine) evoked mouse and zebrafish gut peristalsis when expressed in the enteric nervous system (ENS) (Hibberd et al., 2018;Perez-Medina and Galligan, 2019), they induced only a local contraction with no following peristaltic waves when expressed in the muscle layer of the gut (Vogt et al., 2021;Okamoto and Hatta, 2022).
In this study, we aimed at optogenetic control of gut peristalsis by targeting the muscle layer of developing gut in chicken embryos, since it is known that myogenic function precedes neural ones during development (Holmberg et al., 2007;Roberts et al., 2010;Chevalier et al., 2020;Shikaya et al., 2022). Recently, another variant D156C was reported to exhibit an extremely large photocurrent and a prolonged open-state lifetime (Dawydow et al., 2014). By optimizing conditions for Tol2-mediated gene transfer with the D156C-encoding gene and in ovo electroporation targeting the muscle layer of hindgut and cloaca, we found that D156C successfully evoked a local contraction and its following peristaltic movement along the A-P axis in the hindgut. Importantly, when the artificially produced peristaltic waves reached the cloaca, they induced acute contractions in this tissue in accordance with the rhythm of blue light irradiation, demonstrating that the hindgut-derived peristaltic waves mediated the coordination between the hindgut and cloaca. Moreover, cloacae with drug-provoked aberrant contractions were able to respond to blue light pulse to implement rhythmic contractions, suggesting a possible therapeutic methodology for peristaltic disorder.

Chicken embryos
Fertilized chicken eggs were obtained from the Yamagishi poultry farms (Wakayama, Japan). Embryos were staged according to embryonic day (E) or the somite number (somite stage; ss). All animal experiments were conducted with the ethical approval of Kyoto University (#202110).

Monitoring of gut motility and kymograph preparation
A portion of gut posterior to the duodenum was dissected from E12 embryos, and placed on a silicone-coated Petri dish (6 cm diameter) filled with 10 mL of high-glucose DMEM (Wako, 048-33575) warmed at 38.5°C with a heating plate (MSA Factory, PH200-100/PCC100G). These procedures were performed under the red-light condition when ChR2-expressing guts were analyzed. To avoid drifts of the specimen during imaging, gut-attached remnants such as pancreas, vitelline membrane, and the surrounding tissues of the cloaca were pinned to the silicone dish with fine needles. Following 10 min of resting, time-lapse images were captured using the Leica MZ10 F microscope with a DS-Ri1 camera (Nikon). The obtained images were processed by ImageJ software (NIH) to analyze gut peristalsis and converted to kymographs as previously described (Shikaya et al., 2022). For the experiments demonstrated in Figures 5, 6, the medium containing 100 μM carbenoxolone (nacalai tesque, 32775-51) or water (Merck Millipore, Elix essential UV3) was used.

Quantification of cloacal contractions
Our region-of-interest (ROI) was set around the cloaca, and changes in the movement of ROI in captured images were converted to intensity values using Stack Difference of ImageJ. The values of intensity were normalized by the first frame of filmed data. Peaks of contractions were detected using SciPy library (scipy.signal.find_ peaks) in Python (parameters: height = 2, distance = 10 s, prominence = 1). Intervals were calculated by the time between two successive peaks. Peaks appearing within 6.5 s after photostimulation were defined as the light-induced contractions. Responsivity was calculated by the number of light-induced contractions to all photo-stimulation (10 times).

Statistical analysis
The box plots represent the median, upper and lower interquartile. Wilcoxon rank-sum test was conducted using R to compare data statistically. Graphs were made by R or matplotlib and seaborn library in Python.

Genetic labeling of the muscle layer of hindgut and cloaca
To efficiently express the ChR2 gene into the muscle layer of developing hindgut and cloaca, we determined the presumptive regions of hindgut and cloaca in the splanchnopleural mesoderm at E2.5 (23ss-25ss) embryos that participates in forming the gut muscle layer (Romanoff, 1960). We divided an area of splanchnopleural mesoderm spanning from the vitelline artery to the posterior end of neural tube into four different regions (#1 to #4) along the A-P axis, and targeted these regions roughly with CAGGS-mCherry ( Figure 1A). In this study, the gene expression into the splanchnopleural mesoderm was carried out using the Tol2mediated in ovo electroporation ( Figure 1A) (Sato et al., 2007;Takahashi et al., 2008;Sato and Takahashi, 2009). In all cases with regions #1 to #4, mCherry-positive cells at E12 were observed in the muscle layer of developing gut (such as circular muscles), consistent with previous studies ( Figures 1A,B) (Mahadevan et al., 2014;Sanketi and Kurpios, 2022). Transverse views of electroporated hindgut confirmed that mCherry-positive cells were contained in the muscle layer without overlapping with neural population (neural crest derived) revealed by staining with Tuj1 or αSMA antibodies ( Figure 1B, Supplementary Figure S1). We found that the regions #1 to #4 along the A-P axis at E2.5 largely corresponded to labelled regions along the A-P axis at E12 in the gut tube ranging from the ileum to the cloaca ( Figure 1C), with the region #4 contributing to the hindgut-cloaca region ( Figures 1C, D). In the following experiments, we focused on the region #4 to manipulate the muscle layer of hindgut and cloaca.

Optogenetic control of gut motility with ChR2(D156C) variant
The ChR2-expressing gut region was dissected from E12 embryos, and subjected to blue light irradiation ex vivo (see Materials and Methods). Exploiting the observation that the middle site in the hindgut never exhibits an origin of peristaltic waves [OPWs, (Shikaya et al., 2022)], this site was focally irradiated with blue light using a fine optic fiber at 1-min intervals (Figure 2A). We Frontiers in Physiology frontiersin.org 03

FIGURE 1
Genetic labeling of hindgut-cloaca muscle layers by in ovo electroporation into the splanchnopleural mesoderm. (A) The region of splanchnopleural mesoderm in the range from vitelline artery (VA) to the end of the neural tube was divided into 4 regions: region 1, region 2, region 3, and region 4. Each area was roughly targeted by mCherry plasmids by in ovo electroporation at 23-25 ss and re-incubated until E12. Tol2-expression vector and transposase are shown. (B) mCherry + cells were observed in circular smooth muscles in the gut at E12. Lateral view shows that the mCherry + cells (red) are arranged in the circumferential direction. Transverse view shows immunostaining of αSMA and Tuj1 (green in both). mCherry signals were partially detected in αSMA + cells but not in Tuji1 + cells. Nuclei were stained with DAPI (blue). (C) A schematic view of the intact gut from duodenum to cloaca. The gut from the ileum to the cloaca (frame) was analyzed. mCherry + cells (red signals in fluorescent views, red color in the schematic diagrams) in regions 1 to 4 are shown in the ileum, posterior ileum and caecum, posterior ileum to anterior hindgut/caecum, and caecum/hindgut to cloaca, respectively. (D) Table shows the number of mCherry + gut over the total examined. Scale bars: B, 100 μm; C, 5 mm. ss, somite stage; DA, dorsal aorta; AI, anterior ileum; PI, posterior ileum; DC, distal caecum; PC, proximal caecum; AH, anterior hindgut; PH, posterior hindgut; Cl, Cloaca.

FIGURE 2
Optogenetic control of hindgut peristaltic waves followed by cloacal contraction. (A) Specimen was video recorded for 10 min each before and during photo-stimulation. Blue light irradiation was focally delivered by a fine optical fiber to the middle of hindgut expressing ChR2 (red) with the intermittent stimulation cycle (20-ms pulse width, 5 Hz, 2s, every 1 min). (B) DNAs of mCherry, ChR2 (WT, wild type), or ChR2 (D156C) were expressed in the muscle layer of hindgut-cloaca by in ovo electroporation. White dotted lines and rods indicate the outlines of the gut tube and optical fibers, respectively. Only the intrinsic peristalsis (the red slanted lines) and cloaca contractions (black horizontal lines) were shown in mCherry-and wild-type ChR2-expressing guts in both the pre-photo and the photo recordings (n = 6, n = 6, respectively). In the D156C-expressing guts, while sporadic intrinsic waves were observed in pre-photo recordings, upon the photo-stimulation light-induced waves were evoked propagating both anteriorly and posteriorly represented by inverted V-shaped blue lines in kymograph (n = 5). Origins of the peristaltic waves are indicated by light blue arrowheads. used the wild-type and D156C variant of ChR2 ( Figure 2B). Control hindgut (mCherry-expressed) exhibited no additional OPWs or waves in the hindgut upon irradiation, whereas intrinsic waves normally occurred (Figures 2B,C, red slanted lines; n = 6, Supplementary Movie S1).
Wild-type ChR2 also failed to evoke ectopic contraction/peristalsis ( Figures 2B,C, n = 6, Supplementary Movie S2). As previously reported, the frequency of intrinsic waves in the hindgut propagating from the ileum was variable between individuals (Shikaya et al., 2022). The horizontal lines in the kymograph shown in black dotted lines in the trace ( Figure 2B) indicate a secondary effect in the hindgut being pulled by acute contractions of the cloaca (Shikaya et al., 2022), which is also explained below.
In clear contrast, the D156C-expressing hindgut responded efficiently to the light irradiation, in which the irradiated site displayed a local contraction in response to blue light that was followed by wave propagation along the hindgut (Figures 2B,C; n = 5, Supplementary Movie S3). Intriguingly, these induced waves were recognized as inverted v-shaped lines in kymograph (blue lines, D156C in Figure 2B), showing that the waves propagated both anteriorly (to stomach) and posteriorly (to cloaca), contrasting with the normal hindgut in which waves propagate only posteriorly. These observations suggest that the hindgut has a potential to accommodate waves in both directions. In addition, the optogenetic induction of artificial waves reduced the occurrence rate of intrinsic peristaltic movements (Figures 2B,C). These phenomena are accounted for by the observation that blue light-induced waves proceeding anteriorly into the ileum, which are seen in Supplementary Movie S3 and highlighted in Supplementary Figure S2, met intrinsic waves coming posteriorly from the ileum resulting annihilation (Chevalier et al., 2017;Chevalier, 2022;Shikaya et al., 2022). We also tested another variant C128S/D156A (CSDA) with an open-state lifetime and photocurrents longer and smaller than those in D156C, respectively (Dawydow et al., 2014), but this variant failed to evoke artificial contraction/waves (data not shown).

Artificial peristaltic waves caused cloacal contractions
In the same specimens shown in Figure 2 (D156C), in which blue light was irradiated at the middle of hindgut, the reaction of the cloaca was also recorded to examine whether the hindgut-derived waves caused acute contractions of this tissue. As previously reported and explained above, the acute and intense contraction of cloaca pulls the hindgut simultaneously, which is displayed as a horizontal line in kymograph (Shikaya et al., 2022). Importantly, the kymograph of the D156C-hindgut exhibited reiterated horizontal lines (shown in dotted black lines) coupled with the arrival of the optogenetically produced waves from the hindgut (Figures 2B,D, median; 77.8%, average; 51.6%, Supplementary Movie S3), indicating that the hindgut-derived waves caused the acute contractions in cloaca. Together, we concluded that in the developing hindgut, the cloacal acute contractions are mediated by the arrival of hindgut-derived peristaltic waves at least at E12.
In the normal hindgut, wave-triggered cloacal contractions do not give impact reciprocally to the hindgut except for the aforementioned pulling effect (recognized as a horizontal line in kymograph). Is this attributed to a refractory period of the hindgut juxtaposed to the cloaca? The optogenetic approach developed in this study allowed us to address this question. Focal irradiation was given to the cloacae in the D156Cexpressing guts ( Figure 3A), and motility in the hindgut was analyzed by kymography ( Figure 3A). We found that blue light successfully induced artificial contractions in the cloacae, which was recognized by horizonal lines (dotted blue lines) in the kymograph (median and average; 65%, Figure 3B, Supplementary Movie S4). However, these cloacal contractions induced very few, if any, additional waves in the hindgut, which would have been detected as leftward slanting lines ( Figure 3C, median; 0%, average; 6.3%). Given that the hindgut possesses a potential to accommodate peristalsis in both directions (as revealed in Figure 2), these observations imply that in the normal embryonic gut, the wave-mediated signal is transmitted unidirectionally from the hindgut to the cloaca, for which the refractory period of the hindgut is irrelevant. The uni-directional signaling from hindgut to cloaca revealed in the current study using embryos is reasonable, considering that in adults inter-luminal contents conveyed through the rectum need to be excreted out from the cloaca.

Contraction-ceased isolated cloacae resumed rhythmic contractions by blue light irradiation
We previously reported that the cloaca ceases its contractions when it is separated from the hindgut (Shikaya et al., 2022). To clarify whether the isolated cloaca had lost or retained their contractile potential, we stimulated the isolated cloacae using the optogenetic method developed in this study. From D156C-expressing guts, the cloacae were isolated from the hindguts and subjected to blue light irradiation ( Figure 4A). After transferring into a Petri dish, the cloacae were allowed to rest for another 10 min followed by video recordings for 10 min each before (control) and during irradiation ( Figure 4A). Relative intensity of contractions was calculated using the Stack Difference of ImageJ as previously reported ( Figure 4B) (Shikaya et al., 2022). For quantification, a peak that appeared above the relative intensity 2 within 6.5 s after photo-stimulation was defined as the lightinduced peak of contractions. While an isolated D156C-cloaca almost ceased its contraction (median; 1 peak/10 min, average; 1.4 peaks/10 min, n = 5), it was re-activated by pulsed blue light (10 times/10 min) with high responsivity to the irradiation rhythm (median; 10 peaks/10 min, average; 9.8 peaks/10 min) (Figures 4B-D, Supplementary Movie S5). Responsivity was calculated by the number of light-induced contractions to photo-stimulation (10 times). Accordingly, intervals for the repeated peaks in the irradiated cloaca were predominantly around 60 s ( Figure 4E; 5 specimens were shown in different colors). The isolated cloaca therefore retains its contractile potential when removed from the rest of the gut.
Isolated D156C-cloaca with drug-provoked aberrant contractions was able to respond to pulsed irradiation To determine whether the ceased-or reactivated contractions of isolated cloaca were mediated by gap junction signals, the isolated cloacae were treated with carbenoxolone (CBX), a drug Frontiers in Physiology frontiersin.org 06 widely used to inhibit gap junction function, followed by videorecording for 10 min ( Figure 5A). The specimen was allowed to rest for 10 min after administration with CBX to avoid possible effects by culture medium turbulence. We found that the CBXtreated cloaca exhibited active but irregular contractions with intense amplitudes compared with control (water-treated) cloacae (Figures 5B,C; median; 12.5 peaks/10 min, average; 12.8 peaks/10 min, Supplementary Movie S6). Intervals of these contraction cycles in the CBX-treated cloacae were random ( Figures 5B,D). These observations raised a possibility that in the normal gut, the cloaca possesses a latent ability to undergo spontaneous contractions, which are suppressed by gap junction-mediated signals, and this suppression is temporarily released at the time of the arrival of hindgutderived waves.
Given that the isolated D156C-cloacae retained a potential to respond to external stimuli (blue light) as shown in Figure 4, we expected that the CBX-provoked aberrant contractions with irregular intervals could be entrained by regular pulse of irradiation. D156C-cloacae were isolated from the hindguts and soaked in the medium containing CBX, and blue light pulses were delivered every 1 min for 10 min as described above ( Figure 6A). Contractions were recorded before and during blue light delivery, and compared between mCherryand D156C-cloacae. Markedly, the D156C-cloacae responded significantly to the rhythm of optogenetic stimuli ( Figure 6B, Supplementary Movie S7) with responsivity of 68% in average (median 80%, n = 4) being much higher than control mCherrycloacae (average; 12.5%, median; 10%, n = 4) ( Figure 6C). The number of intervals around 60 s was prominent in the photoactivated D156C-cloacae compared with that of pre-photo D156C-cloacae and photo-activated mCherry (control)-cloacae ( Figures 6D,E). The total number of peaks did not change significantly between before and during irradiation in control/ mCherry (12.5 and 11 peaks in average, respectively) and D156Ccloacae (9.8 and 11.3 peaks in average, respectively) ( Figures  6F,G). With irradiation, 7.5 peaks (average) were evoked in the D156C-cloacae suggesting that CBX-induced random contractions were reduced in number ( Figure 6G). Together, the isolated D156C-cloacae with CBX-provoked aberrant contractions could respond to the blue light to exhibit regular contractions. Frontiers in Physiology frontiersin.org 07

Discussion
We have succeeded for the first time to evoke functional peristaltic waves by optogenetically activating the gut muscle layer. The results obtained with this method have indicated that the coordination between the hindgut and cloaca is mediated by the hindgut-derived peristaltic waves. The D156C variant, but not the wild type, of ChR2 efficiently evokes a local contraction and following peristalsis in the hindgut. Our method also allows the isolated cloaca, which would normally cease its acute contractions, to resume contractions with the rhythm of light irradiation. Furthermore, the cloacae undergoing CBXprovoked aberrant contractions can respond to the external stimuli and implement rhythmic contractions with light pulse.

ChR2(D156C) efficiently evokes gut contraction and peristalsis in the hindgut
ChR2 and its variants have been used mostly in the field of neurosciences. Recently, ChR2-optogenetics has also been applied to study gut peristalsis. While wild type and/or H134R were able to evoke peristalsis when expressed in ENS (Hibberd et al., 2018;Perez-Medina and Galligan, 2019), no report has been provided in which peristalsis was produced by targeting the gut muscle layer. We have successfully produced local contractions and subsequent peristalsis by optogenetically activating muscle layer cells including smooth muscles in the hindgut using the D156C variant known to show extremely large photocurrents (Dawydow et al., 2014). Since D156C-
Frontiers in Physiology frontiersin.org 08 electroporated splanchnopleural cells might also have contributed to interstitial cells of Cajal (ICCs) thought to be a pace maker, the possibility that D156C-optogenetically evoked peristalsis was initiated by ICCs cannot be excluded. Combined with optogenetic activation of ENS, our method should lay the groundwork for understanding the intricate regulatory network between the ENS and smooth muscle layer during gut peristalsis. In chickens, one study was previously reported in which developing motor neurons were optogenetically manipulated using wild type ChR2 (Kastanenka and Landmesser, 2010).
It is unknown why C128S/D156A (CSDA), which is characterized by very long open-state lifetime, is unable to evoke artificial waves in our study (Figure 2) (Berndt et al., 2009;Bamann et al., 2010). This variant is known to be activated by a low light power of 8 μW/mm 2 (Yizhar et al., 2011b), which is similar to the light density in our optogenetic apparatus (approximately 10 μW/ mm 2 ). One possible explanation is that the extremely long openstate lifetime of CSDA (τ = 29 min) compared with D156C (τ = 76 s) might negatively affect the excitation of muscle layer cells/smooth muscles (Yizhar et al., 2011a;Dawydow et al., 2014).

FIGURE 5
Gap junction inhibitor CBX caused abnormal contractions in contraction-ceased isolated cloaca. (A) Cloaca was dissected from the gut and subjected to video recording for 10 min in normal medium, followed by a 10 min rest and second recording in water (control)-or CBX-containing medium. (B) Relative intensity of cloacal contractions (n = 6). Orange lines and red arrowhead are as shown in Figure 4. (C) The number of contractions during 10 min-recording in water-or CBX-treated cloaca. Water (before, after): medians, 0, 3/10 min; averages, 0, 3/10 min. CBX (before, after): medians, 0, 12.5/10 min, averages, 0.2, 12.8/10 min. p values were calculated by Wilcoxon rank-sum test. (D) Intervals of the contractions in the water-or CBX-treated cloaca. Dots in different colors correspond to different individuals (n = 6). No contractions were detected in three specimens treated by water. *, p < 0.05; **, p < 0.01.
Frontiers in Physiology frontiersin.org The coordination between the hindgut and cloaca is mediated by the peristaltic waves When an optogenetically evoked peristaltic wave arrives at the endpoint of the hindgut, it induces an acute contraction in the cloaca. Together with our previous report showing that the cloaca ceases its contractions when isolated from the hindgut (Shikaya et al., 2022), the findings obtained in the current study demonstrate that the cloacal contractions are triggered by the hindgut-derived peristalsis.
When the hindgut-connected (normal) cloaca is optogenetically activated locally, its contractions do not influence the hindgut peristalsis, whereas the hindgut has a potential to accommodate bidirectionally propagating waves revealed in this study ( Figure 2B). These observations suggest that in the normal gut, signal transmission upon the wave arrival at the cloaca is unidirectional, which is reasonable considering the directional transportation of the urine-feces complex out from the cloaca during the excretion in adults.

Gut optogenetics for a possible therapeutic tool
Optogenetic analyses with the isolated cloaca, which normally ceases its contractions, have further provided three novel findings. One is that the isolated cloaca is able to respond to external stimuli. Second, the ceased contractions of the separated cloaca are mediated, at least partly, by gap junction, since CBX causes aberrant contractions reflecting its latent contraction potential. One explanation for the cessation in the isolated CBX-free cloacae is that suppressors of the smooth muscle contractions, such as the nitric oxide known to be synthesized in smooth muscles or ENS (Llewellyn-Smith et al., 1992;Teng et al., 1998), spread through gap junctions, and CBX inhibits this spreading, resulting in spontaneous contractions. Third, even with the CBXprovoked aberrant contractions, the isolated cloacae can artificially be controlled to implement rhythmic contractions in accordance with blue light irradiation. It is conceivable that in the normal gut, the cloaca is restrained from spontaneous contractions by gap junction-mediated signals, and it is likely that this restraint is temporarily relieved when the hindgut-derived wave arrives. How the wave arrival relieves the gap-junction mediated signals and how such signaling temporarily operates have yet to be clarified.
In summary, the optogenetic method optimized for the gut muscle layer in chicken embryos has provided a powerful tool to decipher the mechanisms by which the gut contractions and peristalsis are regulated. The method also offers a means for noninvasive therapeutic control of gut peristalsis in gut motility-impaired patients.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by The Institutional Animal Care and Use Committee Approve # 202205.

Author contributions
YS, MI, and RT conducted experiments with guts. SU analyzed quantification. YS, MI, and YT wrote the paper. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. All authors contributed to the article and approved the submitted version.

Funding
This work was supported by JSPS KAKENHI Grant Numbers; 20K21425, 20H03259, 19H04775, 21K06198, and also by Research Foundation for Opto-Science and Technology, and SENSHIN Medical Research Foundation, and SPIRITS 2022 of Kyoto University. YS is an ex-fellow of JSPS.