Tennyson V M, Payette R F, Rothman T P, Gershon M D
Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
J Comp Neurol. 1990 Jan 15;291(3):345-62. doi: 10.1002/cne.902910303.
The terminal colon of the ls/ls mouse is aganglionic because an intrinsic defect prevents its colonization by cells migrating from the neural crest. Previous studies showed that laminin, type IV collagen, and glycosaminoglycans accumulate in the region of the presumptive aganglionic ls/ls bowel through which crest-derived cells would be expected to migrate. It was suggested that crest-derived cells might fail to enter the abnormal bowel because they receive inappropriate signals from a defective extracellular matrix. This hypothesis was evaluated by analyzing the ultrastructure of the extracellular matrix in mutant and control gut. Tissue was fixed in the presence of ruthenium red before or after selective enzymatic digestion. Heparan sulfate proteoglycan (diameter approximately equal to 15 nm) and chondroitin sulfate proteoglycan (diameter approximately equal to 20-50 nm) granules were found in both control and presumptive aganglionic gut. The heparan sulfate proteoglycan granules were primarily located within formed basal laminae, while chondroitin sulfate proteoglycan granules decorated plasma membranes and 5 nm hyaluronic acid microfibrils that formed a network in the extracellular matrix. At day E11.5, the mutant gut differed from the control in the following: 1) Hyaluronic acid microfibrils were longer and more numerous. 2) There were larger numbers of chondroitin sulfate proteoglycan granules associated with cell membranes and with hyaluronic acid microfibrils. By day E13 the spaces between mesenchymal cells of the outer wall of the control bowel contained a regular lattice of hyaluronic acid microfibrils studded with chondroitin sulfate proteoglycan granules. Instead of this lattice, tangles of excessively long hyaluronic acid microfibrils, coated more heavily than in the control with chondroitin sulfate proteoglycan granules, were found in the presumptive aganglionic gut. These results confirm that the extracellular matrix is abnormal in the presumptive aganglionic bowel of the ls/ls mouse; moreover, they also indicate that the defect involves not one, but several components of the extracellular matrix, as well as their distribution. The defective extracellular matrix is apparent at a time when crest-derived cells would be expected to be migrating in the terminal bowel and is located in their path. The observations thus support the idea that a localized abnormality of the extracellular matrix interferes with the colonization of the terminal bowel by crest-derived cells in the ls/ls mouse.
ls/ls小鼠的终末结肠无神经节，因为内在缺陷阻止了神经嵴来源的细胞对其进行定植。先前的研究表明，层粘连蛋白、IV型胶原蛋白和糖胺聚糖在预期的无神经节ls/ls肠段区域积聚，神经嵴来源的细胞本应通过该区域迁移。有人提出，神经嵴来源的细胞可能无法进入异常肠道，因为它们从有缺陷的细胞外基质接收到了不适当的信号。通过分析突变体和对照肠道中细胞外基质的超微结构来评估这一假设。在选择性酶消化之前或之后，将组织在钌红存在的情况下固定。在对照和预期的无神经节肠道中均发现了硫酸乙酰肝素蛋白聚糖（直径约等于15nm）和硫酸软骨素蛋白聚糖（直径约等于20 - 50nm）颗粒。硫酸乙酰肝素蛋白聚糖颗粒主要位于已形成的基膜内，而硫酸软骨素蛋白聚糖颗粒则修饰细胞膜和在细胞外基质中形成网络的5nm透明质酸微纤维。在胚胎第11.5天，突变体肠道与对照的不同之处在于：1）透明质酸微纤维更长且数量更多。2）与细胞膜和透明质酸微纤维相关的硫酸软骨素蛋白聚糖颗粒数量更多。到胚胎第13天，对照肠外壁间充质细胞之间的空间包含由硫酸软骨素蛋白聚糖颗粒点缀的规则透明质酸微纤维晶格。在预期的无神经节肠道中，未发现这种晶格，而是发现了过长的透明质酸微纤维缠结，其被硫酸软骨素蛋白聚糖颗粒覆盖的程度比对照更重。这些结果证实，ls/ls小鼠预期的无神经节肠段中的细胞外基质是异常的；此外，它们还表明缺陷涉及细胞外基质的几个成分及其分布。有缺陷的细胞外基质在神经嵴来源的细胞预期在终末肠段迁移时就很明显，并且位于它们的迁移路径上。因此，这些观察结果支持这样一种观点，即细胞外基质的局部异常干扰了ls/ls小鼠中神经嵴来源的细胞对终末肠段的定植。
