Gut peristalsis and cell proliferation are required for caecum elongation.

a, Representative images of whole chicken embryos (upper panels) and digestive tracts (lower panels) at embryonic day 10 (E10) and E12. Scale bars, 10 mm. b, Growth of the gut, forelimb, body, and eye between E10 and E12. Each point represents the mean value from three embryos. c, Schematic illustration of the chicken digestive tract. d, Developmental changes in a pair of caeca from E8 to E12 (left). Graphs (right) show changes in caecal length and width (mean ± 95% CI) at E8 (n = 16), E10 (n = 24), and E12 (n = 20). e, Kymographs of peristaltic waves in E9 (left) and E11 (right) caeca. The horizontal axis and vertical axis represent position along the caecal axis and time, respectively. Bar graphs (right) show the frequencies of peristaltic waves passing through the proximal region of the caecum indicated by the dotted area in the schematic. f, Ex-vivo culture of the caecum. Left column: representative images of caeca at E10 (upper) and after 3 days in culture (lower); a schematic of the culture system is shown at the top. Right column: kymographs corresponding to each condition. Proximal (P)-distal (D) axes are indicated in both images and kymographs. Scale bar, 1 mm. g, Changes in caecal length during ex vivo culture (mean ± 95% CI; n = 6). h, Representative images of the caecum showing the whole view (upper) and a fragmented view (lower). Regions along the caecum are annotated and numbered; region 2, used in subsequent analyses, is indicated by a white rectangle. Scale bar, 1 mm. i, Changes in peristaltic contractions under the indicated pharmacological conditions: DMSO (1:1000), nifedipine (30 μM), or aphidicolin (10 µM). j, Frequencies of peristaltic waves in caecal fragments under the conditions shown in i. Data were analyzed using Welch’s t-test. k, Morphological changes in caecal fragments from E10 (upper) to E10 + 3 days in culture (lower) under the same conditions as in j. Scale bar, 500 µm. l, Growth rates (%) in length (left) and width (right) of caecal fragments under the same conditions as in j (mean ± SE).

Optogenetically-triggered peristaltic contractions are sufficient for caecum elongation.

a, Genetic introduction of ChR2 into caecum by in-ovo electroporation. b, Expression of ChR2(D156C)-mCherry in the right caecum. Scale bar, 1 mm. c, Blue-light stimulations of ex-vivo cultured caecal (P2) fragment with/without ChR2 (+/-). d, Optogenetic induction of peristaltic contractions in the control (DMSO, 1:200). The red trace indicates changes in fragment contraction. Black arrowheads mark spontaneous contractions, and blue bars indicate the timing of blue-light illumination. e, Optogenetic induction of peristaltic contractions in the presence of Ani9 (50 µM). Note that spontaneous contractions are suppressed, whereas responsiveness to blue light is retained. f-i, Long-term (22h) responses of ChR2(-) or ChR2(+) fragments to blue-light pulses under the indicated conditions: f, ChR2(-), DMSO (1:200), 120-s interval; g, ChR2(-), Ani9 (50 μM), 120-s interval; h, ChR2(+), Ani9 (50 μM), 120-s interval; i, ChR2(+), Ani9 (50 μM), 30-s interval. j, Frequencies of the contractions under each condition: DMSO, n = 6; Ani9, n = 6; Ani9 ChR2 (120s-BL), n =7; Ani9 ChR2 (30s-BL), n =6. k, P2 fragments after 24 h of incubation under the indicated conditions. Scale bar, 500 μm. l-o, Growth ratio (length and width) after 24h of incubations under the indicated conditions: l, n=11; m, n =12; n, n = 6; o, n = 12. Boxplots show the median (center line), IQR (25th–75th percentiles), and whiskers indicate 1.5xIQR. Statistical tests: j, Kruskal-Wallis test followed by Dunn’s multiple-comparison test with Holm correction; l-m, two-side Wilcoxon signed-rank test.

Neither cell hypertrophy nor cell density changes account for the anisotropic growth of caecum.

a, Transverse cross-section and lateral views of whole-mount immunostaining of smooth muscle (aSMA, red) and nuclei (DAPI, blue) in E10 caecum. Dotted outlines display circumferential smooth muscle (CSM) layer. Scale bar, 50µm (Left panel); 10µm (right panels). b, Transverse cross-section of caecal (P2) fragments before (E10) and after (E10+3) cultured with DMSO, Nifedipine, or Aphidicolin. Red, α-SMA; blue, DAPI. Mean areas of CSM layer are shown below each image (n=4). Volume increase ratios are defined as (the cross-sectional area increase ratio) × (the length increase ratio). Scale bars, 100μm. c, Immunofluorescence staining of E10 + 3-day cultured P2 fragments for cell proliferation (pHH3, green), smooth muscle (αSMA, red), and nuclei (DAPI, blue). Inset, magnified images. White arrows show proliferating cells. Scale bars, 100 μm (left columns), and 10µm (right columns). d, Box plots of proliferating cell density [/10,000 µm²] under each condition (n = 6 for each). Statistical significance was assessed by Mann–Whitney U test (p < 0.05). e, Genetic labeling of CSM cells by in-ovo electroporation in P2 fragment. Fluorescent signals (red, Gap-Orange; green H2B-EGFP) are detected from lateral view of P2 fragment. E10 and E10+3 cultured fragment with DMSO, Nifedipine, or Aphidicolin are shown. Scale bar, 100 μm. f, The size of CSM cells before and after culture with drugs. Representative image of a smooth muscle cell labelled fluorescently (left). Schematic showing the definition of minor/major axis of a cell (middle), and box plots of cell minor/major axis length in the condition of E10 and E10+3 with DMSO, Nifedipine, and Aphidicolin (right two). In minor axis, n = 70 cells in total from 3 individuals (E10), n=49 cells in total from 3 individuals (DMSO), n=91 cells in total from 3 individuals (Nifedipine), and n=38 cells from 3 individuals (Aphidicolin). In the major axis, n=68 cells in total from 3 individuals (E10), n = 40 cells from 3 individuals (DMSO), n = 96 cells from 3 individuals (Nifedipine), and n = 38 cells from 3 individuals (Aphidicolin). g, The cell density of cells in circumferential smooth muscle layers. Representative images of E10, DMSO, Nifedipine, or Aphidicolin conditions, transverse and lateral view of caecum, stained against α-SMA (red) and DAPI (blue). The white squares (20 µm per side) indicate ROIs. Schematic illustrating transverse and lateral views are shown upper. h, Box plots of cell density [/400 µm²] in transverse (left) and lateral (right) views in the four conditions. n=12 cells from 3 individuals for all conditions. Statistical significance was assessed by Mann–Whitney U test.

Peristalsis-independent circumferential migration and division of CSM cells.

a, Representative image of P2 fragment labelled fluorescently (red, Gap-Orange; green H2B-EGFP) from lateral view (top panel), with a white box indicating a single cell selected for kymograph analysis. Scale bar, 100 µm. Kymograph profiles the movement of a smooth muscle cell along the circumferential axis with white dots on the cell poles (middle). Scale bar, 50 µm. The line graph shows the relative positions of nuclei within cells over time (lower). A schematic of the live-imaging system is shown left. (B) The same analyses as b, were conducted in Nifedipine condition. c, Histograms show the circumferential velocities of smooth muscle cells in DMSO (left) and Nifedipine (right) conditions. Red vertical lines represent medians, which were assessed statistically by Mann–Whitney U test. d, Kymographs showing a dividing smooth muscle cell in DMSO (left) and Nifedipine (right) conditions along the circumferential axis. The asterisks indicate the nuclei of newly generated cells. e, Quarter rose plots showing cell division orientation in DMSO (left) and Nifedipine (right) conditions. Schematic defining cell division orientation is shown left, and graphical abstract of smooth muscle cell mitosis is shown below.

Peristalsis-dependent longitudinal rearrangement of CSM cell population

a, The daughter cell tracking. The longitudinal trajectories of daughter cells after mitosis, with the image of a cell at the verge of mitosis shown above (left). Time points 30 minutes and 6 hours after mitosis are indicated with red lines. Paired scattered plots overlaid on box plots show the profiles of distances between daughter cells 30 min. and 6 hr. after mitosis in DMSO (left) and Nifedipine (right) conditions (right). b, The longitudinal trajectories of a cell population. c, Divergence analysis. Definition of Divergence Index (DI) is shown left, while the meaning of DI is summarized in the right column. d, The divergence analysis in DMSO condition. Representative image of the fluorescently labeled P2 fragment from the lateral view, with white rectangle indicating the region for cell sampling, and with the trajectories from time 0 hr. to time 12 hr. and DI shown below (left column). Trajectories and DI in other samples (right). e, The divergence analysis in Nifedipine condition. f, Box plots of DI. g, Summary figure illustrating how the peristalses contribute to the circumferential elongation of the gut, and what changes arise upon inhibition of peristalsis. Statistical tests: a, Mann–Whitney U test comparing distance changes between two conditions; f, one-sample Wilcoxon signed-rank test against 1.00.