Villification: How the Gut Gets Its Villi

Authors: Amy E. Shyer,1* Tuomas Tallinen,2,3* Nandan L. Nerurkar,1 Zhiyan Wei,2 Eun Seok Gil,4 David L. Kaplan,4 Clifford J. Tabin,1† L. Mahadevan2,5,6,7,8†

The villi of the human and chick gut are formed in similar stepwise progressions, wherein the mesenchyme and attached epithelium first fold into longitudinal ridges, then a zigzag pattern, and lastly individual villi. We find that these steps of villification depend on the sequential differentiation of the distinct smooth muscle layers of the gut, which restrict the expansion of the growing endoderm and mesenchyme, generating compressive stresses that lead to their buckling and folding. A quantitative computational model, incorporating measured properties of the developing gut, recapitulates the morphological patterns seen during villification in a variety of species. These results provide a mechanistic understanding of the formation of these elaborations of the lining of the gut, essential for providing sufficient surface area for nutrient absorption.

In amniotes, the primitive midgut is established as a cylinder with an outer mesenchymal layer and an inner, luminal endoderm. As development proceeds, distinct radial layers of smooth muscle differentiate. In parallel, the luminal surface of the gut transforms from a smooth surface to a convoluted morphology. In humans, as well as in mice and birds, this leads to an organized array of fingerlike projections termed intestinal villi (1, 2) although a variety of morphologies such as ridges, zigzags, and honeycombs occur in other species (3–5). Early work suggested a mechanical basis for villus formation (6); however, systematic biological or physical studies of this hypothesis are lacking.

Morphogenesis and Differentiation of the Chick Midgut

Until embryonic day 7 (E7), the gut tube, with its inner endodermally derived epithelium and outer mesenchymal layer, maintains a smooth luminal surface (Fig. 1A). At E8, as the first layer of circumferentially oriented smooth muscle begins to form, inward buckling of the tube leads to longitudinal ridges that increase in number until E13, when the differentiation of this layer is complete (Fig. 1B). At this point, a second longitudinally oriented layer of muscle differentiates just exterior to the circular layer, while the previously formed ridges fold into parallel zigzags over 3 days (Fig. 1C). Last, at E16, as a third longitudinally oriented muscle layer differentiates just interior to the circular layer, bulges arise from the zigzag pattern that presage the formation of villi (Fig. 1D). The coincident emergence of luminal ridges, zigzags, and villi with the sequential forma- tion of smooth muscle layers suggests that smooth muscle differentiation and epithelial morphogenesis might be linked.

Ridges Form Because of Muscle-Constrained Azimuthal Growth of the Endoderm-Mesenchyme Composite

The notion that differential growth of layered tissues can lead to epithelial buckling is classical (7, 8) and has been evoked, for example, to ex- plain longitudinal ridge formation in healthy and diseased adult trachea and esophagus (4, 9). To investigate the tissue interactions that lead to the ridge patterns in the embryonic gut, we sur- gically separated the layers and observed the ef- fects on their respective morphologies. When the muscle was separated from the combined mesen- chymal and epithelial layers at different stages from E8, when the circular muscle layer first forms, to E12 just before the first longitudinal muscle layer forms, we found that the mesen- chyme and attached epithelium unfold (Fig. 2A). This indicates that relative growth of these layers leads to reversible elastic compression when con- strained within the muscle layer; indeed the ratio of the inner circumference of the once-attached muscle layer to the outer circumference of the separated mesenchyme and endoderm, the cir- cumferential stretch ratio, consistently averages to 0.55 across the developmental stages from E8 to E12 (Fig. 2B). However, the separation of the endoderm from the composite of mesenchyme and muscle does not abolish ridge pattern in the mesenchyme (Fig. 2C).

Taken together, these results support a model that the circular muscle layer, once differentiated, forms a stiff constraint mechanically preventing the free azimuthal expansion of the mesenchyme and endoderm; further growth of these tissues relative to the muscle layer leads to azimuthal compression and buckling. This suggests that absent muscle differentiation, the gut tube would expand freely radially without ridge formation. To test this, we developed an in vitro culture sys- tem for gut growth. When segments of E6 guts with smooth lumens and no muscle layers were cultured for 48 hours in vitro, they differentiated to form a ring of circumferential smooth muscle and parallel luminal folds, indistinguishable from in ovo E8 guts (Fig. 2D). When E6 guts were cultured in the presence of 10 mM AG1295 or FK506, drugs known to block the differentiation of smooth muscle but that act through distinct signaling pathways (10, 11), they did not form a smooth muscle layer and concomitantly did not form luminal folds (Fig. 2D). Importantly, these compounds did not influence proliferation or lead to an increase in cell death when compared with guts grown with the vehicle (dimethyl sulf- oxide, DMSO) alone (fig. S1); indeed there was a significant increase in the outer circumference of guts lacking circular smooth muscle when compared with control gut samples, confirming that blocking smooth muscle differentiation eliminates circumferential restriction of the outward expan- sion of the gut tube. As a control, gut segments grown in vehicle alone developed a layer of cir- cular smooth muscle and formed luminal folds. Quantifying the constraint provided by the mus- cle, we find that the ratio of inner circumference of the muscle layer in the control samples to the outer circumference of the gut segments cultured with either compound to be 0.53 on average (Fig. 2D), a ratio that agrees closely with the stretch ratio obtained from surgical separation of the layers, in- dicating that tissue differentiation into smooth muscle provides most of the circumferential constraint.

with either compound to be 0.53 on average (Fig. 2D), a ratio that agrees closely with the stretch ratio obtained from surgical separation of the layers, in- dicating that tissue differentiation into smooth mus- cle provides most of the circumferential constraint.

Because smooth muscle begins active peristalsis once it forms, the contractility of muscle could drive epithelial buckling in addition to, or instead of, functioning as a passive barrier to expansion. To test this, we cultured E6 gut segments with either sodium nitroprusside (SNP), a com- pound shown to inhibit smooth muscle contraction during peristalsis, or motilin, known to enhance peristaltic smooth muscle contraction (12, 13). After 48 hours in culture, neither compound affected the formation of luminal ridges, suggesting that the spontaneous contractility of smooth muscle is not required for epithelial buckling (Fig. 2E).

Fig. 2. Differentiation of circularly oriented smooth muscle is necessary for maintenance and development of ridges. (A) Transverse slices from E8, E10, and E12 whole guts (left) are surgically separated along the junction of the mesenchyme and the circular smooth muscle (dotted line). When separated from the muscle, the luminal ridges in the mesenchyme and attached endoderm unfold (middle) and expand, whereas the detached muscle remains invariant (right). The outer circumference of the unfolded mesenchyme and endoderm (blue arrowhead) is larger than the inner circumference of the separated muscle layer (green arrowhead). (B) Inner circumference of muscle layer (green line) compared with outer circumference of mesenchyme and endoderm (blue line) over time, along with the compression ratio (bar graph). (C) Surgical separation of endoderm from mesenchyme and muscle at E10 does not abolish ridge pattern. (D) (Top left) Experiment schematic of E6 gut cultured for 48 hours. (Bottom) Transverse sections of a fresh E8 gut or E6 guts cultured in DMSO alone or with either 10 mm AG1295 or 10 mm FK506 for 48 hours and labeled with DAPI (blue) and SMA (green). (Top right) Quantification of compression from E8 muscle shows the ratio of the inner circumference of the circular muscle at E8 (green arrowhead) to the resulting mesenchyme outer circumference (blue arrowhead). (E) Transverse sections of guts labeled as in (D); culturing E6 guts in the presence of either SNP or motilin does not affect ridge formation. (F) Transverse sections of guts labeled as in (D), cultured in silk tubes of 380-mm inner diameter (top) or 300-mm inner diameter (middle) or cultured in 300 mm and extracted before fixation (bottom). n > 3 for all culture experiments; error bars represent one SD. Scale bars = 100 mm.

Last, to assess whether the role of the circular smooth muscle layer as a stiff barrier is sufficient to drive luminal folds, we sought to mimic its role in samples where smooth muscle development was blocked. To do so, we constrained radial gut growth by using porous silk tubes, synthesized by spinning silk fibroin around a reciprocating ro- tating mandrel with the inner diameter of the circular smooth muscle (14). E6 gut segments cul- tured inside of silk tubes in the presence of either AG1295 or FK506 for 48 hours did not form a muscle layer and, when given sufficient room to expand in silk tubes of 380-mm inner diameter, still did not form luminal ridges (Fig. 2F). However, when the segments were grown in AG1295 and FK506 and restricted by a silk tube of inner diam- eter of 300 mm, they formed ridges similar to those seen in control guts in spite of the lack of smooth muscle (Fig. 2F). This demonstrates that the me- chanical barrier function of the circumferential smooth muscle is sufficient for luminal ridge for- mation. Moreover, upon removal from the con- fining silk tube, these ridges were quickly lost, just as they vanished from gut tubes upon surgical re- moval of the circumferential muscle layer (Fig. 2F), corroborating our previous finding that continued mechanical constraint is required for the main- tenance of luminal ridges.

Zigzag Intermediates Form in Response to Additional Muscle-Constrained Longitudinal Growth in Endoderm-Mesenchyme Composite As described earlier, at E13 a second longitudinally oriented muscle layer forms; simultaneously the ridges buckle into zigzags. Previous work has shown that a thin layer atop an elastic substrate may take on a zigzag topography when it is com- pressed biaxially (15–17), suggesting that the longitudinal muscles in conjunction with the pre- viously established circumferential muscle com- press the gut biaxially. To investigate whether the longitudinal muscle layer generates longi- tudinal compression, we surgically separated the muscle layers from the endoderm-mesenchyme composite at different developmental stages. At E12, before longitudinal muscle or zigzags have formed, the separated mesenchyme and attached endoderm have about the same axial length as the muscle to which they were attached (Fig. 3A). However, after longitudinal muscle layer dif- ferentiation, at E13, E14, and E15, the ratios of the length of separated muscle to the mesen- chyme and endoderm were about 0.75, 0.69, and 0.55, respectively (Fig. 3A), showing that the endoderm-mesenchyme is increasingly com- pressed longitudinally as this muscle layer forms. Conversely, separation of the endoderm from the mesenchyme and muscle at E14 did not abolish the zigzag pattern, suggesting that this interaction is not required for maintenance of the zigzags (Fig. 3B).

To directly test whether the development of the outer longitudinal layer is required for the formation of zigzags, we resorted again to our in vitro culture system. When E12 gut segments, with a single, circumferential smooth muscle layer and parallel ridges, were cultured for 48 hours, they differentiated a longitudinal smooth muscle layer and underwent morphogenesis to form zig- zags, similar to those seen in guts harvested at E14 (Fig. 3C). In the presence of either AG1295 or FK506, the longitudinal muscle layer failed to differentiate, and concomitantly the zigzag pat- tern did not form, suggesting that the longitudinal layer is required to induce zigzags. These com- pounds only block further smooth muscle for- mation and leave established layers intact, so the ridge patterns remain (Fig. 3C). Additionally, when differentiation of this longitudinal muscle was blocked, the length of the gut increased signifi- cantly compared with control gut segments; the ratio of the length of control gut segments to those cultured in the presence of either compound was on average 0.66; that is, this longitudinal muscle layer compressed the mesenchyme and attached