Comprehensive timeline of mesodermal development in the quail small intestine

Abstract

Background: To generate the mature intestine, splanchnic mesoderm diversifies into six different tissue layers each with multiple cell types through concurrent and complex morphogenetic events. Hindering the progress of research in the field is the lack of a detailed description of the fundamental morphological changes that constitute development of the intestinal mesoderm.

Results: We used immunofluorescence and morphometric analyses of wild-type and Tg(tie1:H2B-eYFP) quail embryos to establish a comprehensive timeline of mesodermal development in the avian intestine. The following landmark features were analyzed from appearance of the intestinal primordium through generation of the definitive structure: radial compartment formation, basement membrane dynamics, mesothelial differentiation, mesenchymal expansion and growth patterns, smooth muscle differentiation, and maturation of the vasculature. In this way, structural relationships between mesodermal components were identified over time.

Conclusions: This integrated analysis presents a roadmap for investigators and clinicians to evaluate diverse experimental data obtained at individual stages of intestinal development within the longitudinal context of intestinal morphogenesis.

Figure 1. Schematic depicting the intestinal primordium, primitive intestinal tube and adult intestine

A: Transverse section through an embryonic day (E) 2.1 quail embryo equivalent to Hamburger and Hamilton (HH) stage 14. At this stage, the intestinal primordium is open and comprised of the splanchnic mesoderm (red; SpM), endoderm (yellow; En) and an intervening endothelial plexus (green; EP). B: At E6, the intestine is completely closed and composed of a mesothelium (orange; Mes), a two layered endothelial plexus (green; EP), a visceral smooth muscle layer (red; viSM), and endoderm (yellow; En). C: In the adult intestine, villi are lined with a mucosal epithelium (yellow; Mu) and contain a lamina propria (LP) composed of capillaries, a lymphatic lacteal, and connective tissue. A four-layered muscularis externa (ME) surrounds the lamina propria. A serosal membrane (Se) lines the coelomic surface.

Figure 2. Early basement membrane dynamics in generation of the mesenchymal compartment

Schematics in left column depict quail embryos at the stage isolated and the red line denotes the plane of section. A–B: At E1.9, continuous basement membranes (arrowheads) lined the splanchnic mesoderm and endoderm with multiple apparent points of contact (arrows). Asterisk denotes a rare mesenchymal cell. C–D: The outer basement membrane (white arrowheads) began to break down at E2.1 and mesenchymal cells were more common (asterisks). The endodermal basement membrane (yellow arrowhead) remained solid. E–F: At E2.2, there were multiple mesenchymal cell layers (asterisks) and the outer basement membrane was dispersed (white arrowheads). Yellow arrowhead denotes the endodermal basement membrane. G–J: At E3.5, both the outer epithelial (white arrowhead) and endodermal (yellow arrowhead) basement membranes were continuous in the open and closed intestinal regions. Scale bars: 50µm (A, C, E, G, I) and 10µm (B, D, F, H, J). Ec, ectoderm; En, endoderm; FL, forelimb; H, head; Hrt, heart; HL, hindlimb; L, lumen; LC, lateral cavity; LPM, lateral plate mesoderm; M, mesenchyme; nc, notochord; NT, neural tube; OE, outer epithelium; S, somites; SoM, somatic mesoderm; SpM, splanchnic mesoderm.

Figure 3. Basement membrane dynamics throughout gut tube closure and mesenchymal differentiation

Schematics in left column depict quail embryos at the stage isolated and the red line denotes the plane of section. A–B: At E5, the outer epithelial basement membrane appeared dispersed (white arrowhead). Yellow arrowhead denotes the endodermal basement membrane. C–D: At E6, both the outer (white arrowhead) and endodermal (yellow arrowhead) basement membranes were unbroken. E–F: At E10, villi (V) were present and both basement membranes were continuous (arrowheads). G–H: At E16, the mesenchyme was condensed (compare F and H). The outer basement membrane was robust and unbroken (white arrowhead) while the mucosal basement membrane weakly stained with laminin (yellow arrowhead). Scale bars: 50µm (A, C, E, G,) and 10µm (B, D, F, H). En, endoderm; FL, forelimb; H, head; HL, hindlimb; L, lumen; Le, leg; M, mesenchyme; Mes, mesothelium; Mu, mucosa; OE, outer epithelium; V, villi; W, wing.

Figure 4. Mesothelial differentiation

Schematic in upper-left corner depicts the region of the gut tube that was imaged. A–C: At E3.5, the outer epithelium (arrowheads) was stratified (asterisks) and the basement membrane was continuous (arrows). No cytokeratin staining was evident at this time. D–F: At E4, the outer epithelium was a single cell layer thick (arrowheads) with a continuous basement membrane (arrows). Cytokeratin staining was weakly positive. G–I: At E5, laminin staining in the outer basement membrane was dispersed (arrows). Cytokeratin staining was present at low levels. J–L: At E6, laminin staining (arrows) was unbroken and cytokeratin staining was robust within the mesothelium (arrowheads). Scale bars: 10µm (A–L). DM, dorsal mesentery; En, endoderm; L, lumen; M, mesenchyme; OE, outer epithelium.

Figure 5. Expansion of the mesenchymal compartment over time

A: Graph of the distance between the outer epithelial and endodermal basement membranes measured at key stages between E1.9 and E8. The dashed line represents the time period over which the outer basement membrane was dispersed. Solid lines indicate a continuous outer basement membrane was present. B: Four regions along the anterior-posterior axis of the small intestine (SI 1–4) were analyzed individually for mesenchymal cross-sectional area between E8 and E16. The cross-sectional area of each region was graphed independently. C: Small intestinal length measured between E6 and E16 (left y-axis, black circles). Fold change in intestinal length over the same time period (right y-axis, grey triangles). D: Photomontage of isolated small intestines with mesentery and blood vessels removed and pinned out to demonstrate their length.

Figure 6. Differentiation of visceral smooth muscle

A–D: At E6, faint staining for α-SMA and γ-SMA defined the outer circular muscle layer. Asterisks represent SMA-negative mesenchymal cells bordering the outer circular muscle layer. E–H: Robust staining for α-SMA marked the outer circular and outer longitudinal muscle layers. γ-SMA was observed in the outer circular but not the outer longitudinal layer. SMA-negative submucosal mesenchyme was still present (asterisk). I–L: By E14, the inner circular layer (α-SMA-positive, weak γ-SMA) was evident. Asterisk denotes SMA-negative submucosal mesenchyme. M–P: At E16, four muscle layers were present including the inner longitudinal layer. All layers stained for both α-SMA and γ-SMA. Double asterisks denote submucosal neuronal plexus. Limited SMA-negative submucosal mesenchyme was present (asterisk). Arrowhead in M indicates SMA-positive staining within the villi. Q–T: In the adult intestine, the four visceral smooth muscle layers were directly subjacent to the lamina propria (arrow) with no intervening submucosal mesenchyme. The outer circular layer was α-SMA-negative. Scale Bars: 50µm (A, E, I, M, Q) and 10µm (B–D, F–H, J–L, N–P, R–T). En, endoderm; IC, inner circular; IL, inner longitudinal; LP, lamina propria; L, lumen; M, mesenchyme; Mes, mesothelium; Mu, mucosa; OC, outer circular; OL, outer longitudinal; V, villi.

Figure 7. Generation of a two-tiered endothelial plexus

Laminin (basement membrane marker; red), QH1 and eYFP (endothelial markers; green) immunofluorescence. A–B: At E2.1, an endothelial plexus marked by QH1 (arrowheads) was present between the endoderm and splanchnic mesoderm. C–F: Sections through Tg(tie1:H2B-eYFP) quail intestinal primordia. C–D: At E3, the endothelial plexus (arrowheads) was detected in the middle of the multilayered mesenchyme. E–F: At E6, the endothelial plexus was organized into two concentric layers below the endoderm and mesothelium, respectively (arrowheads). Scale bars: 50µm (A, C, E) and 10µm (B, D, F). DA, dorsal aorta; En, endoderm; L, lumen; M, mesenchyme; Mes, mesothelium; nc, notochord; NT, neural tube; OE, outer epithelium; SpM, splanchnic mesoderm.

Figure 8. Endothelial plexus remodeling during villi formation

Schematic in upper-left corner depicts the regions of the intestine that were sectioned. E10 intestines were isolated from Tg(tie1:H2B-eYFP) embryos. A–F: Villi were present in the anterior region of the intestine. The endothelial plexus (eYFP-positive) was organized in two concentric rings (arrowheads) but did not extend into the villi. G–L: In the posterior small intestine, ridges but no villi were identified. The endothelial plexus remained organized in two concentric rings (arrowheads). Scale bars: 50µm (A–L). DM, dorsal mesentery; L, lumen; M, mesenchyme; Mes, mesothelium; Mu, mucosa, V, villi.

Figure 9. Extension of endothelial cells into the villi

Images are of sections through Tg(tie1:H2B-eYFP) quail intestines. A–B: At E14, eYFP-positive endothelial cells (arrowheads) were localized within the base of the villi in low numbers. C: The outer endothelial plexus was substantial at E14 (arrows). D–E: By E16, endothelial cells had reached the tip of the villi (arrowheads) and were present in high numbers. F: Thinning of the outer endothelial plexus was observed (arrows). Scale bars: 50µm (A, D), 10µm (B–C, E–F). L, lumen; M, mesenchyme; Mes, mesothelium; Mu, mucosa; V, villi.

Figure 10. Development of large blood vessels of the small intestine

All panels are whole mount images of eYFP fluorescence in isolated gut tubes from Tg(tie1:H2B-eYFP) quail. A–B: At E6, eYFP-positive endothelial cells were evident in the wall of the small intestine in a honeycomb pattern. C–D: At E10, mesenteric vessels were visible (arrows) but large vessels on the small intestine proper were not observed. E–F: At E11, major vessels near the surface of the small intestine were present (arrowheads) extending from the mesentery (arrows). G–J: Major small intestinal vessels (arrows) displayed further branching at E12 and E13 (arrowheads). Scale bars: 1mm (A, C, E, G, I); 200µm (B, D, F, H, J). C, caeca; SI, small intestine; Ven; ventriculus.

Figure 11. Muscularization of small intestinal blood vessels

Schematic in upper-left corner represents the small intestine (SI), blood vessels (BV) and the orientation of sections (black slice). A–F: Sections from Tg(tie1:H2B-eYFP) intestines. A–B: At E12, eYFP-positive endothelial cells subjacent to the coelomic surface were in close proximity to the visceral smooth muscle layers (OC, OL) but were not invested by vascular smooth muscle cells. C–D: At E14, vascular smooth muscle cells (α-SMA-positive, arrowheads) arranged in a single layer were identified surrounding eYFP-positive endothelial cells localized near the coelomic surface of the small intestine. E–F: At E16, the vascular smooth muscle cells appeared more mature and were in multiple layers surrounding endothelial cells (arrowheads). G–H: QH1 staining of a wild type adult quail small intestine revealed mature vessels with multiple layers of vascular smooth muscle cells in large arteries (arrowheads) but only a single layer in veins (arrows). The second tier of blood vessels at the base of the villi was also muscularized in the adult (yellow arrowhead). Scale bars: 50µm (A, C, E, G) and 10µm (B, D, F, H). A, artery; L, lumen; Mes, mesothelium; Mu, mucosa; OC, outer circular muscle layer; Ve, vein; V, villi.