The Extracellular Matrix is Abnormal in the Presumptive Aganglionic Bowel of Isls Mice

A variety of defects involving components of the extracellular matrix have been found in ls/ls mice [200-202] and in human patients with Hirschsprung's disease [203, 204]. A common feature that unites these abnormalities is that they all involve an over-abundance and/or maldistribution of constituents of basal laminae. Molecules that have been noted to be over-abundant include laminin, collagen type IV, nidogen non-sulfated glycosaminogly-cans, and proteoglycans. In the developing colon of fetal ls/ls mice, the abnormal molecules are diffusely distributed throughout the mesenchyme of the colon and the surrounding pelvis and are not, for the most part, aggregated in formed basal laminae [200-202]. The mucosal basal lamina of the terminal and distal colon, however, is also thickened relative to that of a wild-type fetus of the same age. The location of the accumulated molecules of the extracellular matrix is in the paths both of vagal crest-derived cells migrating down the bowel [205] and of sacral crest-derived cells approaching the gut [206]. Double-label electron microscopic immunocytochemis-try, moreover, has revealed that crest-derived cells, identified by their expression of HNK-1 immunoreactivity, migrate through the enteric mesenchyme of the developing bowel in contact with what appears to be diffuse tufts of electron-opaque material that is laminin-immunore-active [207].

The over-abundance of laminin and type IV collagen can be detected in the colon of Is/ls mice at an earlier age [200] than that when crest-derived cells colonize the terminal colon in wild-type mice [199]. This timing and the fact that the extracellular matrix molecules accumulate in the path of incoming crest-derived cells are consistent with the possibility that the abnormal extracellular matrix in ls/ls mice (and by analogy in patients with Hirschsprung's disease) contributes to the pathogenesis of aganglionosis. This suggestion, however, presumes that the accumulation of laminin and other constituents of the extracellular matrix is a primary event rather than a secondary response to the absence of neurons and/or their precursors.

Recent studies with ls/ls mice have indicated that, at least in that model, the accumulation of molecules of the extracellular matrix in the fetal bowel is probably due to an increase in their biosynthesis [202]. mRNAs encoding the (1 and -y1 subunits of laminin, as well as the a1 and a2 chains of collagens type IV, were found by quantitative Northern analysis to be increased in the colons of ls/ls mice. Transcripts encoding laminin a1 were also found to be increased; however, the abundance of mRNA encoding the a1 chain was so much less than that of the (1 and y1 subunits that the a1 protein had to be evaluated quantitatively with reverse transcription and the competitive polymerase chain reaction (RT-cPCR). The abundance of mRNA encoding laminin a1 was develop-mentally regulated and declined as a function of age after E11; nevertheless, at all ages the abundance of mRNA encoding laminin a1 was higher in the ls/ls colon than in an age-matched wild-type colon or in the small intestine of the same Is/ls animals. The location of the cells responsible for the bulk of the biosynthesis of laminin a 1 and (31 and the a2 chain of collagen type IV was found by in situ hybridization (with 35S-labeled antisense riboprobes) to change as a function of developmental age. In the fetal colon, transcripts of mRNA encoding these molecules are first concentrated in the endodermal epithelium; however, by day E15, the transcripts are more abundant in mesenchymal cells of the outer gut wall than in the epithelium. More mRNA was found in the colonic mes-enchyme of the ls/ls colon than in the wild-type colon at an equivalent age.

To determine whether the increase in mRNA encoding subunits of laminin is a primary or secondary event, the expression of laminin-1 in E15 and newborn c-ret knockout mice were compared with that in age-matched ls/ls and wild-type animals. The assumption behind this comparison was that the aganglionosis that occurs in both Is/ls and c-ret knockout mice does so for different genetic reasons. In c-ret knockout mice, the entire bowel distal to the rostral foregut becomes aganglionic because early crest-derived precursors lack functional Ret receptors and thus cannot respond to GDNF [101-104, 109]. In the ls/ls mice the animals lack EDN3 and the agangli-onic region is restricted to the colon [185]. If the increase in transcripts of laminin and the associated accumulation of laminin and other molecules of the extracellular matrix in the colon of ls/ls mice were to be a secondary response to the absence of neural precursors, then one would expect to see the same increase in the aganglionic bowel of c-ret knockout mice. In contrast, the increase in mRNA encoding laminin subunits should not occur in the aganglionic bowel of c-ret knockout mice if the change is ls/ls-specific and related to an effect of the absence of EDN3 on the colonic mesenchyme. No difference from controls either at E15 or in newborn mice was detected by RT-cPCR in the abundance of mRNA encoding laminin a1 in the c-ret knockout colon [202]; furthermore, the over-abundance of immunocytochemi-cally visualizable laminin characteristic of the ls/ls colon was seen in c-ret knockout mice.

The results of these experiments suggest that the increase in abundance of mRNA encoding components of the extracellular matrix occurs in Is/ls mice as a primary effect of the genetic defect in EDN3, and is not a consequence of the aganglionosis. The observations also suggest that at least one isoform of laminin that is present in excess in the Is/ls mouse is laminin-1 (a1-^1-y1). It should be noted that the accumulation of laminin-1 and other molecules of the extracellular matrix is not limited to the colon, although it occurs there. The excess of these molecules is also found in the pelvic mesenchyme that surrounds the terminal bowel. As a result, the abnormal extracellular matrix is located in the paths both of the vagal crest-derived cells that descend within the gut and of the sacral crest-derived cells that approach the bowel within the pelvis. The location, as well as the Is/ls specificity of the abnormal matrix, therefore, are compatible with the possibility that it contributes to the pathogenesis of aganglionosis. Whether the extracellular matrix defects are actually contributory to the condition, however, remains the be confirmed.

Although molecules of the extracellular matrix have been demonstrated to inhibit the migration of crest cells in a number of locations, including the dorsolateral path between the ectoderm and the somites [208, 209], the posterior sclerotome [210-212], and the perinotochordal mesenchyme [213], in none of these regions have the inhibitory effects been linked to accumulations of components of basal laminae [208, 211, 212, 214]. In fact, the extracellular matrix in these regions behaves rather differently from that of either the aganglionic ls/ls [142] or Hirschsprung's bowel [9, 215]. The aganglionic bowel in each of these conditions is heavily innervated both by axons of neurons from the more rostral hypoganglionic gut and from extrinsic ganglia [142]. The defect in the colon of ls/ls mice and patients with Hirschsprung's disease thus impedes its colonization by crest-derived cells, but it does not antagonize the ingrowth of axons.

In contrast, the other regions that normally exclude crest-derived cells also inhibit the outgrowth of axons [214]. It also seems paradoxical that laminin-1 should be one of the molecules that is overly abundant in a zone where crest-derived cells fail to migrate. Laminin is a favorable substrate for the adherence of crest-derived cells [81, 216]; moreover, laminin-1 also stimulates the migration of cells away from the neural crest itself [82, 217]. Antibodies to integrins that block attachment of crest-derived cells to laminin [218, 219], as well as antibodies that bind to a laminin-proteoglycan complex [220] inhibit cranial crest cell migration in vivo. The abundance of laminin in the aganglionic ls/ls colon, therefore might be expected to promote rather than inhibit the colonization of this region of the bowel by cells from the neural crest. On the other hand, the abundance of laminin-1 in the aganglionic colon of Is/ls mice and human patients with Hirschsprung's disease could explain why this region of the gut is so well innervated by extrinsic axons; laminin promotes neurite extension and axonal growth [221-226].

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