EXTRACELLULAR-MATRIX AND ADHESIVE MOLECULES IN THE EARLY DEVELOPMENT OF THE GUT AND ITS INNERVATION IN NORMAL AND SPOTTING LETHAL RAT EMBRYOS

The distribution of fibronectin (FN), laminin (LM), J1/tenascin, chond roitin sulphate proteoglycan (CSPG), neural cell adhesion molecule (NC AM), neurofilament (NF) and the HNK-1 epitope were studied immunohisto chemically in the developing mid- and hindgut of E12.5-E16.5 rat embry os. Over this period the gut wall changed from a uniform mesenchyme to an annular organisation. FN and LM remained widely distributed, but J 1/tenascin became concentrated in mesenchyme outside the nascent circu lar muscle layer, and CSPG declined in, and NCAM increased in, the cir cular layer. Protease and fixation treatments suggested that CSPG coul d mask other molecules such as LM. This re-organisation proceeded bidi rectionally, as a rostrocaudal wave which was met in the colon by a ca udorostral wave. The caecum, however, was conspiciously delayed in all maturation events and also showed mesenchymal and serosal epithelial labelling for the cell-adhesion-related HNK-1 epitope, which was absen t elsewhere, This period also covered the appearance of enteric neuron s, recognised by HNK-1 and NF antibodies. Cells labelled by these anti bodies appeared in a unidirectional rostrocaudal wave, from the duoden um at E12.5 to the rectum at E16.5. This wave was not in exact synchro ny with the wave of intestinal maturation, but lagged behind so that n euronal cells first appeared in increasingly mature micro-environment at progressively more caudal levels. These cells initially were positi oned imprecisely about mid-way across the gut mesenchyme layer and wer e not clearly related spatiotemporally to any of the above molecules. Slightly later, however, this neural region was broadly defined by rel atively low levels of both CSPG and J1/tenascin. The final position of the myenteric neurons was very precise, and was related to a thin J1/ tenascin layer and to a step in NCAM labelling intensity. Litters of p ups, of which 25% would be expected to be spotting lethal homozygous e mbryos which develop total colonic and caecal aganglionosis, showed no difference in any of the molecules studied, but in 4 embryos out of 1 2, the progress of the rostrocaudal wave of neuron appearance was dist inctly slowed even in the duodenum and proximal small intestine, regio ns well outside the final aganglionic zone. The observations suggest t hat this Hirschsprung's-disease-like regionalised defect is a result o f a generalised abnormality which does not ee involve gross changes in extracellular matrix and NCAM expression.