Genetic and Mechanical Regulation of Intestinal Smooth Muscle Development

Abstract

The gastrointestinal tract is enveloped by concentric and orthogonally aligned layers of smooth muscle; however, an understanding of the mechanisms by which these muscles become patterned and aligned in the embryo has been lacking. We find that Hedgehog acts through Bmp to delineate the position of the circumferentially oriented inner muscle layer, whereas localized Bmp inhibition is critical for allowing formation of the later-forming, longitudinally oriented outer layer. Because the layers form at different developmental stages, the muscle cells are exposed to unique mechanical stimuli that direct their alignments. Differential growth within the early gut tube generates residual strains that orient the first layer circumferentially, and when formed, the spontaneous contractions of this layer align the second layer longitudinally. Our data link morphogen-based patterning to mechanically controlled smooth muscle cell alignment and provide a mechanistic context for potentially understanding smooth muscle organization in a wide variety of tubular organs.

Keywords: Bmp; Hedgehog; cell orientation; differentiation; gut development; mechanical forces; morphogenesis; patterning; smooth muscle.

Figure 1.. Hedgehog signaling patterns circumferential smooth muscle in a concentration-dependent manner

(A) Transverse cross-sections and corresponding whole-mount images of smooth muscle differentiation and alignment in the chick midgut. At E6, the nascent inner layer first appears and aligns in the circumferential direction, perpendicular to the gut proximal-distal axis. At E11, the outer layer differentiates and aligns longitudinally, parallel to the proximal-distal axis. (B) Cross-sections of explanted midguts treated with cyclopamine or recombinant Shh. (C) Quantification of smooth muscle actin (αSMA) pattern across the radial thickness of the gut tube based on immunofluorescence from cross-sections in (B). Endoderm boundary is highlighted by white dashed line and mesothelium boundary is highlighted by orange dashed line in (B) control sample. (D) Fluorescent in situ hybridization (FISH) of PTCH1 expression in equivalent sections to (B). (E) Quantification of PTCH1 FISH signal across the radial thickness as in (C). Images are representative of at least 4 replicate guts per treatment. Scale bars=50μm. See also Figure S1.

Figure 2.. Hedgehog acts through Bmp signaling to inhibit subepithelial smooth muscle.

(A) FISH of BMP4 expression in midgut explants treated with cyclopamine or recombinant Shh from E5 for 72 hours. (B) Quantification of radial expression of BMP4 from images in (A). (C) Sections from explanted midguts cultured as in (A) and immunostained for pSmad1/5/9. Dotted lines denote endoderm-mesenchyme boundary. (D) Quantification of radial pattern of SMA and nuclear pSmad1/5/9 immunofluorescence from sections in (C). (E) Sections from the jejunum of E13.5 chick midguts electroporated with RCAS-Noggin at E2.5. (F) Gene expression levels determined by qPCR from E5 guts cultured for 72 hours with noted treatments. Fold change expression is relative to GAPDH. Error bars are mean +SEM. *p<0.05 by t-test. (G) qPCR data from E5 guts treated for 12 hours with 1μg/mL Bmp4. (H) Model for Hh/Bmp-mediated patterning of the circumferential layer. Hh (both Sonic and Indian ligands) are expressed by the endoderm and activate Bmp4 expression in the subjacent non-muscle mesenchyme where their concentrations are highest. Bmp4 subsequently acts at high concentrations to inhibit smooth muscle formation in this subepithelial compartment through repression of MYOCD and MRTFA/B, whereas Hh acts through promoting MYOCD expression to induce muscle formation at a distance further away where inhibitory Bmp levels are lower. Images are representative of at least 4 replicate guts per treatment. Scale bars=50μm. See also Figure S2.

Figure 3.. Outer longitudinal smooth muscle differentiation depends on local Bmp inhibition

(A) Cross section of control and Noggin-treated midgut explants, arrowhead denotes precocial muscle (representative of n=6 replicate guts). (B) Quantification of radial smooth muscle pattern based on SMA staining from (A). (C) FISH of BMP2/7 in the midgut showing expression in mesothelium (left arrowheads) and NOG expression in neurons and muscle (rigth arrowheads). (Right) FISH of Nog and Grem1 on mouse duodenal sections. (D) Section and whole mount immunofluorescence of RCAS-Bmp2 and control mock electroporated jejunal midguts segments stained with SMA or Tagln (representative of n≥5 guts per treatment). Note the electroporations performed in early lateral plate mesenchyme tend to more effectively target the outer mesenchyme. (E) Quantification of radial smooth muscle pattern from experiment in (D). Dashed grey line denotes the boundary occupied by enteric neurons between the inner (to the left) and outer (to the right) muscle layers. (F) Immunostained E16 mouse duodenum in whole mount and section (representative of n=6 double mutants). (G) Quantification of radial smooth muscle pattern and nuclear pSmad1/5/9 from sections in (F). Dashed grey line denotes the boundary between muscle layers. (H) Model of outer layer muscle development. After formation of the inner layer, there are two opposing Bmp gradients that inhibit muscle formation within the inner and outer mesenchyme. Just prior to and during the formation of the outer longitudinal layer, Bmp antagonists are expressed by myenteric neurons and the inner muscle layer, reducing Bmp activity locally within the outer layer of mesenchyme and allowing for muscle differentiation. Scale bars=50μm. See also Figures S1–S4.

Figure 4.. Continuous strain from differential growth aligns inner circumferential smooth muscle

(A) Jejunal section before and after radial cut at E5 (prior to smooth muscle differentiation) to reveal circumferential opening angle and residual strain. (B) Immunostained whole mount midgut explants following tubular or flat (zero-strain) culture and both conditions after application of longitudinal stretch. Accompanying quantification of actin alignment based on SMA staining. 90°=circumferential orientation and 0°=longitudinal. (C) Whole mount images of inner layer ileum segments following electroporation of viral constructs perturbing proliferation rates along with alignment and opening angle measurements. (D) Whole mount images of midgut segments from embryos treated with aphidicolin or DMSO in ovo. Cell alignment quantifications are relative to the proximal-distal (long) axis of the gut. Error bars are mean ± SD, ***P<0.001 by two tailed t-test. n≥5 measurements (alignment) and n≥3 (opening angle) per sample from at least 3 samples for each condition. Scale bars=50μm. See also Figure S5.

Figure 5.. Cyclic strain aligns outer longitudinal smooth muscle

(A) Whole mount image of immunostained inner and outer muscle layers when contractions are blocked with nifedipine (100μM) and ML-7 (45μM) or increased with BayK8644 (1μM) during outer layer differentiation. (B) Whole mount of midguts intubated with tungsten rods cultured with nifedipine and cyclically stretched compared to intubated unstretched controls. Quantifications of alignment determined as in Figure 4 were obtained from n≥3 measurements per sample from at least 5 samples per condition. Inner and outer layers were distinguished by the presence of the neural plexus separating them. Scale bars=50μm. See also Figure S5 and S6.

Figure 6.. Mechanical control of helical muscle in the murine esophagus

(A) Whole mount projection of mouse esophagi from control and mutant embryos. (B) Quantification of longitudinal tension in the esophagi of control and mutant embryos at E12. (C) Quantification of the longitudinal opening angle of esophagi at E12, as defined by the reflex angle relative to the straight (uncut) tube (θ=180°). Each point represents a different mouse. (D) Whole mount stain of muscle layers in mouse esophagi after culturing from E12.5 for 72 hours with contraction inhibitors (nifedipine=60μM and ML-7=45μM). Quantifications of cell alignment were performed on at least 3 different regions for each of at least 6 separate explants per treatment. (E) Whole mount of misaligned and inverted inner and outer layers of explanted chick midgut following application of exogenous stretch. Error bars are mean±SD, ***p<0.001 by two tailed t-test. Scale bars=50μm. See also Figure S7.

Figure 7.. Model for mechanically controlled smooth muscle alignment

(A) Residual strains in the mesenchyme lead to circumferential tensions that align differentiating muscle circumferentially. Once formed, the cyclic contractions of the inner layer align the outer layer in the perpendicular, longitudinal, direction. (B) In the esophagus of mice, longitudinal tension and longitudinal residual strain are present in the mesenchyme and result in an inner left-handed helical array of muscle. Contractions of the inner layer cause the outer layer to align perpendicularly in a right-handed helix. (C) When Nog is deleted from the mesoderm (along with one or two copies of Grem1) there is increased longitudinal strain in undifferentiated mesenchyme and the inner layer aligns longitudinally. The longitudinal contractions of this layer align the outer layer circumferentially. This inversion of muscle layers is phenocopied by applying longitudinal stretch to the chick small intestine.