Immunolocalization of α-integrin subunits and extracellular matrix components during human colonic organogenesis

GASTROENTEROLOGY 1996;110:58–71

Immunolocalization of a-Integrin Subunits and Extracellular Matrix Components During Human Colonic Organogenesis BRIAN K. DIECKGRAEFE,* SAMUEL A. SANTORO,‡ and DAVID H. ALPERS* *Division of Gastroenterology, Department of Internal Medicine, and ‡Department of Pathology and Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, Missouri

Background & Aims: The role of cell adhesion molecules in colonic organogenesis remains poorly understood. This study examined the expression of a-integrin subunits and extracellular matrix ligands during human colonic development. Methods: Standard immunohistochemistry was used to characterize extracellular matrix and a-integrin subunit expression during development. Results: At 9 weeks, type-IV collagen and laminin were present underlying epithelium, around vascular structures, and surrounding inner circular muscle layer fibers. Fibronectin was uniformly expressed in the mesenchyme. Tenascin distribution was restricted to the presumptive muscle layer and, later, to the villus core and muscularis mucosae. The 9-week epithelium expressed a2, a3, a5, and a8, and, by 11 weeks, a9 . a3, a6, and a8 expression was accentuated at the basal membrane. During transition from pseudostratified to simple columnar epithelium, a vertical a2 gradient formed. Mesenchymal cells expressed a5 and a8 by 9 weeks. The developing muscularis (propria and mucosae) showed accentuated a5 expression. By 16 weeks, a8 expression localized to the muscularis mucosae and villus core. Mesenchymal vascular elements stained strongly with anti-a2 and a6 by 9 weeks. Conclusions: These observations show the complexity and overlap of adhesive receptor expression and ligands during development and reveal early cell commitment to the formation of specific structures.

I

n development, the human colon progresses from a simple cylinder of mesenchyme, through a stage of primitive villus formation, to the adult crypt-containing organ. These events have been carefully detailed by light1 and electron microscopy.2 Morphogenesis is a complex process involving cell proliferation, adhesion, migration, recognition, differentiation, and cell death. Coordination of these events leads to the ordered arrangement of cell groups and differential gene expression. Understanding of the developmental anatomy surpasses knowledge concerning the cell-cell and cell–extracellular matrix (ECM) interactions that control cell behavior during colonic development. / m4433$0035

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The interactions of cells with ECM components are mediated by adhesive receptors. The integrins are a group of structurally related transmembrane proteins that mediate a variety of cell-cell and cell-ECM interactions.3 – 5 Integrins are heterodimers made up of distinct a and b subunits. Multiple different a and b subunits have been identified that can pair in restricted combinations, leading to more than 20 distinct receptors. The b1-containing integrins represent the largest subfamily identified and can be found paired with a1 – 9 and with aV . Ligand binding redundancy is considerable, with at least four fibronectin binding receptors in the b1 subfamily. It has been speculated that this structural and functional redundancy has evolved to mediate the complex adhesive requirements of morphogenesis.6 Fibronectin, laminin, collagens, and other ECM components are under tight temporal and spatial developmental control. The b1 integrins are widely distributed in human,7 chicken,8 and Xenopus tissues.6 Multiple examples show the importance of b1 integrins in cell migration and morphogenesis. Chick neuritogenesis on collagen substrates is effectively blocked by either monoclonal anti-b1 –integrin antibody or rabbit polyclonal antiserum.9 Similar approaches implicate b1 integrins in somite organization,10 neural crest11 and myoblast migration,12 gastrulation,13,14 and eye development.15 Drosophila integrins, originally called position-specific antigens,16,17 play key roles in development. The common b subunit (PS3) is encoded by the lethal myospheroid gene.18 Nonlethal mosaic constructs show aberrant wing and eye development.19 Attempts to systematically examine coexpression of ECM molecules and specific a-integrin subunits during development in nonhuman models have been limited because of the paucity of reagents. On the other hand, studies in human organs have been hampered by the unavailability of tissues. In this study, attention has been Abbreviations used in this paper: ECM, extracellular matrix; MAb, monoclonal antibody. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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Figure 1. Developmental H&E staining. H&E staining of (A) 9-week, (B) 11-week distal, (C) 12-week, (D) 13-week, (E ) 16-week, and (F ) 19week colon specimens. The 9-week colon consisted of a simple tube of pseudostratified epithelium (A, arrowhead) with a slit-like lumen. Ridges (defined as thickening of the epithelial layer projecting into the lumen) were present. Mesenchyme (bar) surrounded the epithelium and was covered by a single cell layer mesothelium (arrow). Indentation of epithelial ridges by mesenchyme resulted in folds (B, arrow) by 11 weeks. Condensation of cells was seen in the region of the presumptive muscle layer, forming the inner circular muscle layer (*). The 12-week colon (C) showed knob-like projections at the base of the primary villi (arrow) and represented developing crypts. The developing Auerbach’s plexus was present (arrowhead ) just outside the innercircular muscle layer. The 13-week fetal colon (D) had simple columnar epithelium. Cyst-like spaces developed within the epithelium (arrow). Goblet cells developed around 12 weeks and increased numbers were evident (arrowheads). Splitting of primary villi resulted in amplification of villus numbers. The 16-week fetal colon (E ) showed extensive splitting of the crypts (arrowheads). The interval development of a muscularis mucosae was evident (bar). The 19-week fetal colon (F ) had less prominent crypt division. Vascular structures were evident in the submucosa (arrows, E and F ).

focused on some of the a subunits that pair with the b1 subunit because these receptors interact predominately with ECM components believed to be of developmental significance. To better understand the role of integrins in cell substrate interactions during colonic development, the expression of a-integrin subunits and potential ECM ligands was examined by immunohistochemistry. / m4433$0035

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Materials and Methods Tissues Fetal colon specimens were obtained after legal termination of pregnancy between fetal ages 9 and 19 weeks. Fetal tissues were used in accordance with institutional and state guidelines. Specimens were obtained immediately after curet-

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tage, and the midcolon was removed after identification of the ileocecal thickening. Fetal age was determined by maternal dating and verified by standard ultrasound dating. Tissues were embedded in OCT compound (Miles Diagnostics, Elkhart, IN), frozen in liquid nitrogen, and stored at 070⬚C until sectioning.

Tissue Processing Frozen sections were prepared from the fetal colon of 9–19 weeks of gestation. Sections were cut at 6–8 mm, fixed in acetone at 020⬚C for 5–10 minutes, and rinsed three times in phosphate-buffered saline (PBS). Sections were then blocked with PBS containing 5 mg/mL of bovine serum albumin and 1% normal goat serum (Gibco BRL, Gaithersburg, MD). Primary antibody was added at the appropriate dilutions for 60 minutes, followed by two washes in PBS. Slides were then treated with the appropriate biotin-conjugated second antibody (Vector Laboratories, Burlingame, CA). After three washes, the sections were processed using the standard Vectastain ABC Elite protocol (Vector Laboratories) and developed with 3,3ⴕ-diaminobenzidine. After dehydration, the noncounterstained slides were mounted with Accu-Mount 60 (Baxter Healthcare Products, McGaw Park, IL). A Nikon MicrophotFX microscope was used to examine and photograph the specimens. Appropriate control sections were processed as above with the omission of the primary antibody or substitution of nonimmune control serum. Control sections did not show significant background staining under the conditions used. In some cases, nickel chloride enhancement (0.08% NiCl in the 3,3ⴕ-diaminobenzidine development solution) was used for detection of low-level expression.

Antibodies The following antibodies were purchased from Telios Pharmaceuticals (San Diego, CA): mouse monoclonal antibody (MAb) P1E6, recognizing human a2-integrin subunit (used at 1:1000 dilution); mouse MAb P1B5, recognizing human a3 integrin subunit (1:1000 dilution); mouse MAb P4G4, recognizing human a4-integrin subunit (1:1000 dilution); and mouse MAb P1D6, recognizing human a5-integrin subunit (1:2000 dilution).20 – 22 Rat MAb GoH3,23 recognizing human a6-integrin subunit (1:100 dilution), was purchased from AMAC Inc. (Westbrook, ME). Affinity-purified polyclonal antibodies raised to peptides derved from the cytoplasmic domains of human a8 and a9 were kindly provided by Drs. Lynn Schnapp, Elise Palmer, Dean Sheppard, and Robert Pytela (University of California, San Francisco) and were used at 1:100

dilution. ECM antibodies used were MAb antihuman collagen IV,24 clone CIV 22, used at 1:80 (Dako A/S, Glostrup, Denmark); rabbit polyclonal immunoglobulin G (IgG) antihuman fibronectin, used at 1:100 (Collaborative Biomedical Products, Bedford, MA); MAb A030 antihuman tenascin, used at 1:2000 (Gibco BRL); and rabbit polyclonal IgG antihuman laminin, used at 1:3000 (kindly provided by Dr. Joshua Sanes, Washington University School of Medicine, St. Louis, MO).

Results Sections representing the major morphogenic events previously described by other investigators1,2 were selected for examination. Key events in colonic development between 9 and 19 weeks can be found by H&E staining in Figure 1A–F. Figure 1A shows a transverse section from a 9-week fetal colon. It consisted of a simple tube of pseudostratified columnar epithelium with a slitlike lumen as indicated by the arrowhead. The epithelium averaged 3–5 cell layers thick. Ridges (thickening of the epithelial layer projecting into the lumen) can be seen forming. The epithelium was surrounded by mesenchyme, indicated by the bar, and the single cell layer outer mesothelium as shown by the arrow. Figure 1B was from a distal 11-week fetal colon. The epithelial ridges were indented by mesenchyme to form folds as indicated by the arrow. The epithelium remained pseudostratified. In the region of the presumptive muscle layer differentiation of cells forming the circular muscle layer of the muscularis externa was evident as indicated by the asterisk. Figure 1C shows the 12-week fetal colon. Developing crypts formed knob-like projections at the base of the primary villi as shown by the arrows. The epithelium was in transition with areas of both pseudostratified and columnar epithelium. Evidence of the developing Auerbach’s plexus was present, shown by the arrowhead, just outside the inner circular muscle layer. The 13-week fetal colon is shown in Figure 1D. The epithelium was now uniformly columnar. Cyst-like spaces developed within the epithelium, shown by the arrow, as previously described.2 Goblet cells developed around 12 weeks and increased numbers were evident here as indicated by the arrowheads. Splitting of primary villi resulted in amplification of villus numbers. The 16week fetal colon (Figure 1E) showed extensive splitting

䉴 Figure 2. Distribution of collagen-type IV and laminin. Localization of collagen-type IV in (A) 9-week, (B) 11-week distal, (C) 11-week proximal, (D) 12-week, (E ) 13-week, (F ) 16-week, and (G) 19-week fetal colon is shown. Anticollagen type IV stained the epithelial-mesenchymal interface at all the developmental times examined. The location of the epithelium, not stained, is indicated by the asterisk (A and D). Contrast this figure with the parallel H&E-stained sections in Figure 1. Discontinuous staining of inner circular muscle layer was seen as early as 9 weeks (A–C, arrowhead ) Auerbach’s plexus, not stained, was clearly visible at 12 and 16 weeks (D and F, large arrow) after the outer longitudinal muscle became collagen-type IV positive. Light staining of the muscularis mucosae is indicated by the arrowheads at 16 (F ) and 19 weeks (G). Laminin localization is seen in sections from (H ) 9-week, (I ) 11-week, and (J ) 19-week specimens. Laminin distribution mirrors collagen-type IV during development.

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of the crypts as indicated by the arrowheads. The interval development of a muscularis mucosae was evident as shown by the bar. By this time, the colon had become a highly organized structure with well-formed crypts and villi. The 19-week fetal colon (Figure 1F) was similar in appearance to the colon at 16 weeks except crypt division was less prominent. Vascular structures were evident in the submucosa as indicated by the arrows in Figure 1E and 1F and in some sections were seen extending into the villus core. Collagen IV and Laminin Figure 2 shows the immunolocalization of typeIV collagen and laminin. From 9 weeks onward, a strong reaction for type-IV collagen was seen at the epithelialmesenchymal interface below the pseudostratified epithelium (Figure 2A–G) The epithelium itself was negative. Type-IV collagen staining extended up the villus core. As early as 9 weeks, faint staining could be seen in the region of the presumptive muscle layers as shown by the arrowhead in Figure 2A. By week 11, the inner circular muscle layer was clearly highlighted by staining around the muscle bundles as shown by the arrowhead in Figure 2B and 2C. The outer longitudinal muscle bundles were not outlined by type-IV collagen until approximately week 12 (Figure 2D). By week 16, as indicated by the arrowhead in Figure 2F, band-like weak staining for type-IV collagen was present at the crypt base. Vascular structures were clearly defined by collagen staining at 9 weeks. Between 12 and 13 weeks, the pseudostratified epithelium converted to a simple columnar epithelium that covered the villi. Coincident with the development of villi, vascular structures were seen extending up the villus core to the villus tip. Laminin showed a pattern of staining essentially identical to that of type-IV collagen (Figure 2H–J). Localized deposition at the epithelialmesenchymal interface identified both molecules as components of the epithelial basement membrane. Fibronectin and Tenascin The distributions of fibronectin and tenascin were also analyzed in relation to colon morphogenesis (Figure 3F–J and 3A–E, respectively). Fibronectin was strongly expressed at the earliest stages of morphogenesis exam-

ined (9 weeks) (Figure 3F). At this time, the serosa, presumptive muscle layers, and mesenchyme were positive. The epithelium remained negative through 19 weeks (Figure 3J). After the development of villi, intense staining for fibronectin was present in the villus core. Tenascin was much more restricted in distribution than fibronectin. At 9 weeks, faint staining was concentrated in the region of the presumptive muscle layer, while the remainder of the colon was negative as indicated by the arrowhead in Figure 3A. By 11 weeks, tenascin staining remained restricted to the developing inner circular muscle layer as shown by the arrowhead in Figure 3B. Both muscle layers and several adjacent mesenchyme cell layers were identified in the tenascin-stained specimens at week 13 as indicated by the bar in Figure 3C. With the development of the muscularis mucosae at 16–19 weeks, tenascin was detected in a discrete band consisting of a few cell layers of the mesenchyme beneath the base of the intervillous area as shown by the arrows in Figure 3D and 3E. Some immunoreactivity was present in the villus core, which became strongly positive by 19 weeks, as shown in Figure 3E by the circles. Integrins The results in fetal colon are summarized in Table 1. a2 . In the fetal colon, anti-a2 reacted strongly with the epithelium as early as 9 weeks as indicated by the asterisk in Figure 4A. The multiple cell layers of the pseudostratified epithelium showed uniform staining. Cells in the region of the presumptive muscle layer showed light uniform staining, but it was not possible to identify individual muscle layers. Scattered positive cells were found throughout the mesenchyme that later condensed into easily discernible vascular structures below the epithelium. At 11 and 12 weeks, the epithelium was more complex but remained pseudostratified and positive for a2 expression (Figure 4B and 4C). As early as 11 and 12 weeks, small islands of a2-positive cells coalesced and formed a vascular band in the mesenchyme approximately midway between the epithelial base and the developing muscle layers (as shown by the arrow in Figure 4B). a2-Positive vessels extended into the core of the primitive villi. Between 11 and 12 weeks, fibers

䉴 Figure 3. Distribution of tenascin and fibronectin. Localization of colonic tenascin in fetal specimens at (A) 9 weeks, (B) 11 weeks, (C) 13 weeks, (D) 16 weeks, and (E ) 19 weeks of gestation is displayed. Fibronectin staining is shown at (F ) 9 weeks, (G) 11 weeks, (H ) 12 weeks, (I ) 13 weeks, and (J ) 19 weeks. Tenascin expression was limited at 9 weeks of gestation (A). Light reactivity was present in the region of the presumptive muscle layer (A, arrowhead ) and localized to the region of the inner circular muscle layer (B, arrowhead ) at 11 weeks. Tenascin reactivity was also evident in the mesenchyme inside the inner circular muscle at 13 weeks (C, bar) and in the muscularis mucosae (D and E, arrows) at 16 and 19 weeks and was strongly present in the villi near the tips (E, small circles) by 19 weeks of gestation. Extensive fibronectin deposition in the mesenchyme was present as early as 9 weeks of gestation (F ). This pattern remained constant during further morphogenesis, sparing the epithelium and Auerbach’s plexus through 19 weeks (J ).

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Table 1. Summary of Integrin Staining Epithelium a2: from 9 wk onward, expression gradient established by 13 wk a3, a6: from 9 wk onward, accentuation of staining at basal membrane a5: light staining at 9 and 11 wk a8: basal epithelial reactivity from 9 wk onward a9: intensely reactive by 11 wk; decreasing to 19 wk; accentuated at epithelial base Mesenchymal cells a5: diffusely reactive from 9 wk onward a8: diffusely reactive at 9 wk, coalesces into a band-like distribution, and migrates to the location of the muscularis mucosae by 19 wk Vascular elements a2 , a6 : reactive from 9 wk onward a3: minimally reactive small vessels, intensely reactive large vessels a5: slightly positive from 9 to 19 wk Muscularis mucosae and villus core a5, a9: reactive by 16 and 19 wk a8: strongly positive by 16 and 19 wk, accentuated in the villus core and tip Auerbach’s myenteric plexus a2, a3, a5, a6, a9: identifiable from 11 to 12 wk onward Muscularis propria a2, a3: lightly positive at 9 wk onward, a9- positive by 11 wk a5: positive by 9 wk a6: light staining of the presumptive muscle layer, decreasing with time a8: lightly positive by 12 to 13 wk, decreasing reactivity and essentially negative by 19 wk

of the inner circular muscle layer could be identified. Individual nerve bundles were seen clearly in Auerbach’s myenteric plexus by 12 weeks, just outside the inner circular muscle layer as shown by the arrow in Figure 4C. These bundles stained lightly for the a2 subunit. The epithelium of the colon at 13 weeks of gestation is in transition from a pseudostratified epithelium to a simple columnar surface (Figure 4D). At this time, there was a discernible gradient of a2 expression, with decreasing expression in the colonocytes moving from the crypt to the surface. Both the outer longitudinal and inner circular muscle layers and the myenteric nerve plexus were positive. The subepithelial and intervillous core vascular elements remained strongly stained for a2 . Changes oc-

curring between 13, 16, and 19 weeks of gestation are mainly the proliferation of the villi. The gross development of a muscularis mucosae occurs at about 16 weeks, but no significant a2 staining was evident (data not shown). The a2 epithelial gradient was preserved through at least 19 weeks of gestation (Figure 4E). Examination under high power revealed a2 expression by all epithelial cell populations. Goblet cells expressed a2 , but staining was not present in the region of mucin granules. a3 . Anti-a3 staining also was evident as early as 9 weeks (Figure 4F). The serosal cell layer stain was strongly positive at all the gestational ages examined. The epithelium was lightly positive at 9 weeks of gestation and strongly stained by 11 weeks (Figure 4G). Staining for a3 was particularly accentuated in the basal membrane of the epithelial cells adjacent to the mesenchyme, in contrast to the more uniform staining of a2 . The region of the presumptive muscle layer was faintly positive from 9 weeks and showed increased staining by 11 weeks. As the epithelium developed, no significant staining gradient was found in epithelium along the cryptvillus axis. In contrast to a2 , only extremely faint staining was observed in the developing vascular elements in the mesenchyme and developing muscle. Larger vessels in the adjacent tissue (figure not shown) showed intense positivity in the vessel wall. Myenteric plexus cells were lightly stained in the distal 11-week specimens (Figure 4G) and remained positive throughout the older specimens examined. Crypt branching can be seen at 19 weeks (Figure 4I). a4 . Under standard conditions, anti-a4 did not detectably react with any of the specimens examined. On overdeveloped specimens, very faint reactivity was seen in the villus core, in individual cells in the mesenchyme, and in the presumptive muscle layer (data not shown). The lymphoid aggregates found in control sections of adult colonic mucosa stained strongly with anti-a4 , showing that this antibody works well with the fixation and sectioning techniques used. a5 . In the 9-week fetal colon, anti-a5 reacted strongly with the cells of the innermost region of the presumptive muscle layer in the location of the devel-

䉴 Figure 4. Localization of a2, a3, and a5. The distribution of (A–E ) a2, (F–I ) a3, and (J–N ) a5 in 9-week (A, F, and J ), 11-week (B, G, and K ), 12-week (C and L), 13-week (D), 16-week (H and M), and 19-week (E, I, and N ) specimens is shown. Anti-a2 immunoreactivity of the epithelium was present from 9 weeks (A, *) onward, and a gradient of expression was established by 12–13 weeks (C and D) with more prominent staining at the crypt base. Vascular elements in the mesenchyme stained strongly for a2. Weaker immunostaining in the region of the developing muscularis propria was present (C, bar) and the region of Auerbach’s plexus (C, arrow). Strong a3 expression was detected in the epithelium. Compared with a2, accentuation of staining at the epithelial cell base was present (G, arrow). No gradient of epithelial expression became evident with epithelial maturation, and strong expression was maintained to week 19 (I ). Light expression of a3 was present in the developing muscularis propria and Auerbach’s plexus (G and H ). Anti-a5 immunostaining strongly identified the inner circular muscle layer at 9 weeks (J, arrow). Weaker staining throughout the mesenchyme was present. Light staining of Auerbach’s plexus (L, arrow) is highlighted by dark staining of the surrounding outer longitudinal and inner circular muscle layers. Development of the muscularis mucosae between 16 and 19 weeks can be identified by condensation of a5 -expressing cells at the crypt base (N, arrow).

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oping inner circular muscle layer (Figure 4J). The mesenchyme was diffusely reactive. The epithelium had very light staining at 9 and 11 weeks, which persisted with additional dilution of the antiserum (data not shown), consistent with low-level epithelial a5 expression. By 11 weeks of gestation, the most intense a5 staining was present in the region of the developing circular muscle layer (Figure 4K). Moderate mesenchymal staining was seen throughout development. By 12 weeks, a5 reactivity was seen in both the inner circular and outer longitudinal muscle layers (Figure 4L). The developing Auerbach’s plexus was weakly reactive, shown by the arrow in Figure 4L, and accentuated by strongly positive surrounding muscle layers. Vascular elements in the subepithelial mesenchyme and villus core stained slightly positively compared with the surrounding mesenchyme. This pattern persisted until 16 weeks, when subepithelial reactivity reflected the development of the muscularis mucosae (Figure 4M). Positive staining was seen at the crypt base and extended up the villus core as shown by the arrow in Figure 4M and N. a6 . At 9 weeks (Figure 5A), the epithelium itself was lightly positive with an especially strong reaction at the epithelial-mesenchymal interface. The presumptive muscle layer stained lightly, but a6 reactivity progressively decreased through 19 weeks. Vascular structures were strongly stained in the mesenchyme as shown by the arrowhead in Figure 5A. These elements remained strongly positive throughout the developmental period examined. Individual nerve bundles stained lightly from 12 weeks onward as shown by the arrow in Figure 5C. a8 . The fetal colon strongly expressed a8 during development. At 9 weeks, the mesenchyme was uniformly positive (Figure 5F). By 11 weeks, the a8-expressing mesenchyme had condensed into a thick band, which spared the immediate subepithelial region (Figure 5G). Antibody staining was seen at the epithelial-mesenchymal junction, indicating basal epithelial expression as shown by the arrowhead in Figure 5G. As villi develop during week 13, shown by the arrow in Figure 5I, subepithelial cells at the villus tip became strongly positive. By 16 weeks (data not shown) and 19 weeks (Figure 5J),

the positive cells localized to the region immediately below the crypts and extended into the villus core, corresponding in location and timing to development of the muscularis mucosae. The external muscle layers were lightly positive at 12 weeks (Figure 5H) and 13 weeks (Figure 5I), but minimal or no staining was evident by 19 weeks (Figure 5J). a9 . At 9 weeks (Figure 5K) light staining was observed in the serosal cell layer, but no staining was seen in the epithelial or mesenchymal compartments. By 11 weeks (Figure 5L), both the pseudostratified epithelium, shown by the arrow, and presumptive muscle layers expressed a9. Epithelial expression seemed accentuated at the basal membrane. The 13-week fetal colon (Figure 5M) expressed a9 strongly in the epithelium and in the external muscle layer and myenteric plexus. Expression decreased in the interval between 13 and 19 weeks. In a manner similar to a8 expression, light a9 staining of the muscularis mucosae was evident at week 16 (data not shown) and week 19 (Figure 5N) and extended into the villus core. Light a9 staining in the region of the muscularis mucosae and villus core occurred by 16 and 19 weeks, reducing the contrast between the epithelium and the background and making the positive epithelium appear less reactive.

Discussion Immunohistochemical analysis of tissue samples was used to study the expression of ECM ligands and adhesion molecules of the integrin family from weeks 9 to 19 during fetal development, a period when the colon evolves from a simple tube with a slit-like lumen to a complex structure with villi, covered by a simple columnar epithelium. The present results confirmed the morphogenic steps previously outlined by others1,2 and simultaneously localized expression of the integrin a subunits and potential ECM ligands. Uncomplexed a subunits are degraded,25 – 27 thus localization of a subunits detects the heterodimeric complex. Because a single a subunit can form a complex with more than one potential ECM ligand and some a subunits can potentially pair with more than one b chain, one can only infer

䉴 Figure 5. Localization of a6, a8, and a9. The distribution of (A–E ) a6, (F–J ) a8, and (K–N ) a9 in 9-week (A, F, and K ), 11-week (B, G, and L), 12-week (C and H ), 13-week (D, I, and M ), and 19-week (E, J, and N ) specimens is shown. Anti-a6 antibody recognized the epithelium as early as 9 weeks, with accentuation of basal staining. Mesenchymal vascular elements could be seen in the 9-week specimen (A, arrowhead ). Light staining in the region of the developing muscularis propria was seen at 9–13 weeks (A–D) but was absent at 19 weeks (E ). Auerbach’s plexus was lightly outlined (C, arrow). a8 expression was recognized throughout the mesenchyme at 9 weeks (F ). a8 expressing cells condensed into a band-like distribution by 11 and 12 weeks (G and H ), which localized to the region of the subepithelial and intervillus lamina propria (I and J ). Accentuation of staining at the villus tip could be seen at 13 weeks (I, arrow). Basal expression of a8 by the epithelium was seen at multiple developmental times (G, arrowhead ). a9 expression was minimal at 9 weeks (K ) but was evident in the epithelium and developing muscle layers by 11 weeks (L, arrow). This pattern remained unchanged until 16 and 19 weeks when light staining was seen in the region of the muscularis mucosae (N, arrow).

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potential physiological ligands and function from morphological data. Nevertheless, this study represents an initial step in detailing the availability and location of receptors during development. Type-IV collagen and laminin were distributed during colonic development, similar to patterns observed previously in fetal human small intestine28 and adult and developing rodent intestine.29 – 31 Both collagen IV and laminin were localized at the basement membrane underlying the epithelium. No differences were seen in staining patterns between specimens showing the immature pseudostratified epithelium or the mature simple columnar surface. Both antibodies stained numerous vascular structures in the mesenchyme and both antibodies stained a band in the presumptive muscle layer as early as 9 weeks, corresponding to the location of the inner circular smooth muscle. Evidence for the development of the outer longitudinal smooth muscle did not become obvious until 12 weeks by staining for type-IV collagen, laminin, tenascin, a5 , or a9. Developmental differences between the inner circular smooth muscle layer and the outer longitudinal layer are consistent with the separate development of the two layers observed in the rat/mouse chimeric intestine.32 Tenascin and fibronectin distribution during colonic development differed considerably from the localization of the basement membrane components laminin and type-IV collagen. Fibronectin was detected at 9 weeks throughout the mesenchyme. This pattern continued throughout development, sparing the epithelium and the region of the developing Auerbach’s plexus. It is known that fibronectin is synthesized early30 during intestinal morphogenesis and may provide a substrate for migration. Without a clear gradient, however, fibronectin is not likely to provide the actual migratory cue. We did not observe the disappearance of fibronectin from the colonic villi tips as reported by others30 in the rat small intestine. This disparity may indicate differences in either the species or in the organs studied. The potential complexity of the fibronectin role in morphogenesis is increased by the existence of isoforms. Multiple sequence variations are produced by alternative splicing of the premessenger RNA transcribed from a single gene.33 Splice selection is regulated in a tissue or cell type and developmentally specific fashion. These splicing events can potentially influence specific integrin interactions.34 The tight regulation of various forms of fibronectin in a tissue-specific fashion during development argues for potential unique roles during tissue morphogenesis. Our use of polyclonal antifibronectin antibodies was designed to be an initial step in developmental characterization and did not discriminate / m4433$0035

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between isoforms. In addition, because we cannot perfuse our specimens, we cannot exclude the possibility that some of the immunoreactivity for fibronectin represented reactivity with plasma fibronectin. Future studies using ribonuclease protection assays and splice-specific antibodies can address these specific questions. The role of fibronectin in colonic development is inferred by its extensive and early distribution. Fibronectins promote the migration of many cells in vitro. Recent a5-integrin subunit35 and fibronectin homozygous null mutations36 in mice have been achieved. In both cases, the homozygotes died as embryos, and minimal data are available on early gastrointestinal tract development. Deletion of the b1 gene by homologous recombination in F9 embryonal cells has also been accomplished.37 These studies suggested that the loss of b1 integrins severely affected morphological differentiation (epithelial formation and cell migration) but did not prevent tissue-specific gene expression in F9 cells induced by differentiation factors. Aside from the role integrins play in mediating cell adhesion and migration, evidence suggests they play a role in matrix assembly. Fibronectin matrix assembly is dependent on a5b1 integrins in a5-deficient CHO cells.38 However, embryonic cells from a5-null mice were capable of assembling extensive fibronectin matrices that were indistinguishable from matrices produced by wild-type cells.35 Tenascin expression was much more restricted. In the mouse, tenascin expression in the small intestine is regulated during development.39,40 Significant expression occurs shortly after villus formation and a vertical gradient develops with maximal expression occurring under the epithelial cells at the villus tip, probably modulated by the epithelial cell population.40 Similar expression patterns were found in the human colon. At 16 weeks, several weeks after primary villus formation, tenascin was weakly detected in the villus core, and a gradient was established by 19 weeks. Deposition at the base of the epithelial crypts corresponds anatomically and chronologically with the development of a muscularis mucosae. The presumptive muscle layer expressed tenascin early and it was detected at the earliest time we examined (9 weeks). By 13 weeks, strong expression was seen throughout the circular and longitudinal muscle layers, sparing the region of the nerve fibers and ganglion cells of Auerbach’s plexus. In contrast to fibronectin knockouts homozygous null mutation of the tenascin-C gene in mice resulted in no discernible anatomic or histological abnormalities.41 These mice developed normally without tenascin. Interpretation of negative knockout studies such as this are complicated by the need to consider WBS-Gastro

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other molecules having a duplicated function or potential compensatory changes in the expression or function of other molecules. The ECM molecules examined here are potential ligands for various integrin heterodimers. For a subunits 2–6 paired with b1 subunits, potential ligands defined include laminin (a2, a3, and a6), fibronectin (a3, a4, and a5), and collagen I/IV (a2 and a3). Integrins capable of binding to tenascin have not been clearly defined,42 but one recent report suggests that a9 may bind tenascin.43 The b1 subunit appears to pair with chicken a8,44 but the b pairing for human a8 remains to be determined. The distribution of integrin receptors in the adult colon and small intestine have been previously examined.45 – 48 Adult epithelial cells were found to express a2, a3, and a6. One study reported low level a5 expression by the epithelium,47 a finding confirmed in the fetal colon. Adult intestine muscularis mucosae and muscularis propria express a3 and a5.47 The classical fibronectin receptor, a5b1, has been suggested to play a major role in tissue morphogenesis.49 In the colon, early and extensive mesenchymal fibronectin deposition occurred from 9 weeks of gestational age. The pattern of expression of a5 throughout cells in the mesenchyme colocalized with fibronectin, whereas other potential fibronectin receptors (a3 and a4) had different distributions. Thus, mesenchymal fibronectin may play a role in early colon development, similar to that reported in other tissues,49 by providing a migratory substrate. Our results in the fetal colon are summarized in Table 1. a6 showed very strong localization at the basal epithelial membrane before and during conversion from an undifferentiated pseudostratified epithelium to a more mature simple columnar epithelium, suggesting that it may play a role as a basement membrane receptor. A similar function has been proposed for a6 during human kidney development.50,51 Both the a6b1 and a6b4 integrin complexes mediate cell adhesion to laminin. In the early stages of mouse submandibular gland epithelial development, a6 is present over the entire cell surface where it may contribute to homotypic cell–cell adhesion during the development of the immature epithelium.52 With further development, a6 expression becomes restricted to the basolateral aspect of the acinar cells and is turned off in cells not adjacent to the basement membrane. High-level expression on membranes adjacent to the basement membrane is consistent with its function as a laminin receptor. A similar high-level expression of a6 is found in developing kidney epithelial cells attached to the laminin A chain.51 The pattern of a3 expression differed considerably from that of a6 during colonic de/ m4433$0035

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velopment. The most notable difference was the persistent strong expression of a3 in the mature columnar epithelium. Some investigators47 have reported a gradient of a3 expression in the adult small intestine with increasing expression from the crypt to the villus tip. The present data showed little or no expression gradient during fetal colonic development. a2 expression in our fetal specimens showed development of a gradient during the transition to a simple columnar epithelium, with decreasing expression as cells migrate to the villus tip. It has been suggested53 that decreased adhesion secondary to reduced a2 expression might aid in surface cell exfoliation. The decrease or loss of a2 expression found in 37 of 96 colon adenocarcinomas,48 which correlated with advanced Dukes’ staging, might be ascribed to reversion to the phenotype of a cell that supports less adhesion. The developing muscle in fetal colon weakly expressed a2 throughout the time periods examined, in contrast to the lack of staining reported in adult the gut.45 a8 subunit distribution during early development showed extensive mesenchyme reactivity, similar to fibronectin distribution. Over time, this reactivity narrowed and localized to the subepithelial region and villus core in a pattern and time course similar to the development of the muscularis mucosae. The ECM ligands for a8 and a9 are not known. The sequence homology is greatest between human a8 and certain other members of the Arg-Gly-Asp integrin binding class (a5, aV, and aiib) in the human (B. Dieckgraefe, unpublished observations, December 1993) and chick,44 suggesting that an Arg-Gly-Asp–containing ECM molecule, like fibronectin, might serve as the physiological ligand. Because all the ECM molecules examined in the colon are positive in the region of the muscularis mucosae, few clues are provided to narrow the potential ligands. In the developing human colon, a8 subunit expression was found at the muscularis mucosae and the epithelial cell base. This basal epithelial expression was reproducible and was identical to the epithelial staining pattern described by others44 during the examination of chicken a8 expression in the developing gut, mesonephros, and Wolffian duct. a9 reactivity was found in both the colonic epithelium and smooth muscle layers, paralleling adult colonic expression, but like a8 the ECM ligand binding specificity is unknown. Original characterization of the human a954 revealed a widespread distribution in epithelial cells, including hepatocytes, squamous epithelium, Caco-2 and Tera-2 cells, and in smooth and skeletal muscle. In summary, by 9 weeks of gestation (the earliest time examined), the so-called undifferentiated mesenchyme showed significant commitment to specific structures WBS-Gastro

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when examined using functional markers like adhesive receptors or ECM ligands. Vascular elements, inner circular smooth muscle, and neuronal elements were clearly seen. Second, vertical villus gradients existed in the distribution of some ECM molecules, like tenascin, suggesting a possible role in regulating crypt-villus migration and differentiation. ECM molecules also may play a permissive role, allowing migration to occur in specific regions while other factors provide the migratory stimulus. Third, despite significant integrin redundancy with respect to ECM ligand binding, the human fetal colon revealed unique patterns of staining for all the a subunits examined, suggesting diverse physiological functions for these molecules. Finally, comparison of fetal integrin expression with adult expression generally revealed very similar staining patterns. Thus, the present data regarding integrin expression during colon organogenesis do not allow one to assign unique roles in development. Colocalization of integrin subunits and potential ECM ligands does not prove physiological association. These data do, however, begin to identify integrins with potential roles in colonic morphogenesis. Additional studies are needed to ascertain whether the same parallel patterns of expression and distribution occur during colonic wound repair and restitution.

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Received January 13, 1995. Accepted August 11, 1995. Address requests for reprints to: Brian K. Dieckgraefe, M.D., Ph.D., Gastroenterology Division, Box 8124, 660 South Euclid Avenue, St. Louis, Missouri 63110. Fax: (314) 362-8959. Supported in part by grants DK09100 and DK14038 from the National Institutes of Health and by an American Gastroenterological Association Foundation Merck Senior Fellow award.

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