Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus

Abstract

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here, we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via sequential elongation and expansion processes. This starts with a synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial-cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.

Fig. 1

Tracheal tubulogenesis process. (a) Azan staining of trachea at E18.5. Length was defined as the distance from the larynx to the main branch. (b) Gross morphology of developing trachea. Tube length (c). External diameter (d). Data represent means ± SEM (n ≥ 3). (e) Schematic model of trachea tubulogenesis. Red and blue arrows indicate length and diameter expansion. f 3D-luminal structures of whole tracheas reconstructed from micro-CT images using IMARIS. See also Supplementary Movie 1. Quantification of the length (g), circumference (h), and luminal-surface area (i) (n ≥ 4). Change in luminal surface area (i, upper panel) and the fold change (i, lower panel) over time are shown. Data represent means ± SEM (n ≥ 3). j Sections of BrdU-incorporated developing trachea were stained for BrdU (green) and TOPRO-3 (blue). Dotted lines indicate epithelium–mesenchyme boundary. k Rate of proliferating cells determined by cumulative BrdU-incorporation assay. Data represent means ± SEM (n ≥ 4). l The numbers of epithelial cells (upper panel) and fold changes (lower panel) in whole trachea over time. Data represent means ± SEM (n ≥ 3). A anterior; D dorsal; L jeft; P posterior; R right; V ventral; cart cartilage; SM smooth muscle; Epi epithelium; Mes mesenchyme; Tr trachea; Es esophagus. Scale bar = 500 μm (b, f), 200 μm (a; sagittal view), 50 μm (a; transversal view, j)

Fig. 2

Low-proliferative luminal-surface enlargement with epithelial rearrangement after E14.5. a Azan staining of developing trachea epithelium. b Representative 3D reconstructed images of an epithelial-cell cluster (25 cells). See also Supplementary Movie 2. c Apical-surface area. Numbers represent means. (n = 217, 253, and 189 in three embryos for E14.5, E16.5, and E18.5, respectively). center line, box limits, and whiskers represent mean, 25 and 75% confidence limits, and min and max values, respectively. d Proportion of luminal cells in total epithelial cells. Data represent means ± SEM (n ≥ 3) (n = 528, 732, and 1045 in three embryos for E14.5, E16.5, and E18.5, respectively). e Integration of quantitative values revealed that modest increases in cell numbers, apical enlargement, and apical emergence contributed to luminal-surface enlargement. f Optical image of tracheal epithelium in Nkx2.1^CreERt2; LSLKras^G12D trachea and littermate control at E18.5. Whole tracheas were stained for CDH1 (green) and TOPRO-3 (red). g Gross morphology of Nkx2.1^CreERt2; LSL-Kras^G12D trachea and littermate control. Yellow arrows indicate trachea length. Tracheal tube length (h). External diameter (i). Data represent means ± SEM (n ≥ 3). P values (^**P < 0.01, ^*P < 0.05) show the significance with the Tukey-Kramer method (c, d) or Student’s t test (h, i). NS not significant. Scale bar = 500 μm (g), 20 μm (f), 5 μm (b)

Fig. 3

Wnt5a-Ror2 signaling is involved in tube elongation. a Gross morphology of a developing Wnt5a^−/− trachea and littermate control. b Tracheal tube length (control; n = 4–6, Wnt5a^−/−; n = 3). c In situ hybridization for Wnt5a mRNA in developing tracheas. d Gross morphology of Foxg1^Cre; Wnt5a^flox/flox trachea (n = 4), SHH^Cre; Wnt5a^flox/flox (n = 3) trachea, and littermate control (n = 7) at E12.5. Gross morphology of Ror1 and/or 2^−/− trachea and littermate control at E18.5 (e) and 12.5 (f). g Tracheal tube length (control; n = 4, Ror1^−/− = 5, Ror2^−/− = 5, Ror1^−/−; Ror2^−/− = 3). h Ror2 and SMA expression in developing tracheas. Sections were stained for Ror2 (green), SMA (red), and TOPRO-3 (blue). Lower panels show higher magnification images of dotted squares. Dotted lines indicate epithelium–mesenchyme boundary. i Gross morphology of Foxg1^Cre; Ror2^flox/flox trachea and littermate control at E12.5. j Tracheal tube length (control; n = 5, Foxg1^Cre, Ror2^flox/flox; n = 9). Yellow arrows indicate trachea length. D dorsal; V ventral; Epi epithelium; Mes mesenchyme; Tr trachea; Es esophagus. P values (^*P < 0.05, ^**P < 0.01) show the significance with Student’s t test (b) or the Tukey-Kramer method (g). Scale bar = 500 μm (a, d, e, i), 200 μm (c, e, h; upper panel), 10 μm (h; lower panel)

Fig. 4

Wnt5a-Ror2 signaling regulates SM morphogenesis by synchronizing the radial SM-cell polarity. a SM morphology in Wnt5a^−/− trachea and control at E12.5. Whole tracheas were stained for SMA (red), TOPRO-3 (blue). Coronal (upper panel) and transversal (lower panel) sections. b Array of SM cells. Apical view (upper panel) and lateral view (lower panel). See also Supplementary Movie 3. c, d Polarity of Ror2-positive SM cells, determined by the Golgi-apparatus position relative to the nucleus. Sections were stained for GM130 (white), Ror2 (green), and DAPI (blue). White-dotted lines show the contours of cell membranes. Quantifications in three embryos are shown below. c Developmental time-course analysis in wild type (E10.5; n = 115, E11.5, n = 214, E12.5; n = 236). d In Wnt5a^−/− trachea at E11.5 (control; n = 99, Wnt5a^−/−; n = 73). e Polarity of Ror2-negative or positive (arrowheads) SM cells in Foxg1^Cre; Ror2^flox/flox trachea at E11.5: the lower panel shows magnified images of the upper panel. f, g Mosaic targeting of SM cells using Tagln1^Cre line. GFP (green), SMA (red), DAPI (blue) in control (f) and Ror2^flox/flox (g). Arrowheads indicate Ror2-negative SM cells. h Time-lapse sequence of epithelial sphere (red) and SM cells (magenta, cyan, gray, yellow, blue, and green) in Matrigel. Cells were labeled by Image J. ^*Cells keeping at a distance from spheres. See also Supplementary Movie 4. i Approaching distance of SM cells to sphere in h. Numbers represent means (control; n = 23, Wnt5a^−/−; n = 29). j Distribution of angles reflecting directionality to sphere in h. Scale bar = 100 μm (e, left), 10 μm (a–c; upper, d; upper, e; right upper, g; upper, h), 3 μm (c; lower, d; lower, e; lower, g; lower panels)

Fig. 5

Wnt5a-Ror2 signaling controls contractility of SM and restricts radial expansion during tube elongation of developing trachea and esophagus. a Representative time-lapse sequence of contractile movement of tracheas. The black-dotted squares in upper are magnified in lower. White-dotted lines indicate epithelium–mesenchyme boundaries. See also Supplementary Movie 5. Maximal contractility (b) and contraction time (c) in a. Columns represent means ± SEM (n ≥ 3). Numbers represent means. For box and whisker plot, center line, box limits and whiskers represent mean, 25 and 75% confidence limits, and min and max values, respectively. (d) Thickness of tracheal mesenchyme in Tbx4^Cre, R26R^H2B-EGFP/+. Transversal views were shown. Red-arrows indicate thickness of mesenchyme. (e, f). External diameter of tracheal tube. (Control; n = 4–6, Wnt5a^−/−; n = 3, Ror1^−/− = 5, Ror2^−/− = 5, Ror1^−/−; Ror2^-/- = 3). Each column represents the means ± SEM. P values (^*P < 0.05, ^**P < 0.01) show the significance with Student’s t test (b, e) or the Tukey-Kramer method f. NS not significant. (g) Gross morphology of the esophagus at E12.5. Yellow arrows indicate the length of the esophagus. h SM tissue architecture of digestive organs at E12.5. Whole organs were stained for SMA (white). Coronal sections were shown. A anterior, P posterior. See also Supplementary Movie 6. i, j The polarity of subepithelial cells of digestive organs from wild type (i) and Wnt5a^−/− (j). Ror2 (green), GM130 (white), and DAPI (blue). Dotty squares were magnified in right panels. Lines indicate boundaries between epithelium and mesenchyme. Red arrow indicates the position of Golgi-apparatus relative to nuclei. Scale bar; 50 μm (d), 20 μm (h), 10 μm (e, i, j; left), 2 μm (i, j; right panels)

Fig. 6

Cartilage development expands the tracheal but not esophageal diameter and promotes epithelial rearrangement for luminal enlargement. a Gross morphology of Foxg1^Cre; Sox9^flox/flox trachea and littermate control. Yellow arrows indicate length and diameter of the tracheas. Measurement of tracheal tube length (b) and external diameter (c). (Control; n = 5–8, Foxg1^Cre; Sox9^flox/flox; n = 3 or 4). d Azan staining of transversal sections. e Azan staining of Foxg1^Cre; Sox9^flox/flox trachea epithelium. f Representative 3D reconstructed images of epithelial-cell cluster (20 cells) of Foxg1^Cre; Sox9^flox/flox or littermate control at E18.5. (left, apical view; middle, oblique view; right, lateral view). See also Supplementary Movie 7. g Apical-surface area at E18.5. center line, box limits, and whiskers represent mean, 25 and 75% confidence limits, and min and max values, respectively. Numbers represent means. (Control, n = 119, Foxg1^Cre; Sox9 ^flox/flox, n = 107). h Pseudostratification index at E18.5. center line, box limits, and whiskers represent mean, 25 and 75% confidence limits, and min and max values, respectively. Numbers represent means (Control, n = 130; Foxg1^Cre; Sox9^flox/flox, n = 84). (i) Azan staining of developing esophageal epithelium. P values (^**P < 0.01, ^*P < 0.05) show the significance with Student’s t test (b, c, g, h). NS not significant. Scale bar = 500 μm (a), 50 μm (d), 10 μm (f), 5 μm (i)