The overall goals of these studies are to gain an understanding of the molecular and cellular biology of intestinal development and the roles of glucocorticoids (GC) in the ontogenic process. It appears that GC have two distinct actions on the developing intestine: early effects wherein they advance the timing of initiation of enzymic changes, and late effects, wherein they control only the rate of change. Each of these actions could potentially be exerted at the transcriptional level or at any post-transcriptional level (e.g., message stabilization, translation of post-translational processing). Moreover, these effect of GC could involve either the proliferating crypt epithelial cells, the differentiating villus cells, or the underlying mesenchymal cells.
In Specific Aims #1 and #2, sucrase/isomaltase (SI) will be used as a developmental marker to investigate the molecular and cellular site of both early and late effects of GC. These studies, together with Specific Aim #3, are also designed to distinguish between two general models that have been proposed for intestinal development.
In Specific Aims #4 and #5 the hope is to generate potentially powerful tools for subsequent studies of this ontogenic process. The details of the five Specific Aims are as follows: 1. To determine whether critical features of rat intestinal development previously deduced from measurements of sucrase enzyme activity represent control at the level of mRNA accumulation. specifically, to investigate; a) whether early effects of GC on SI mRNA are mediated via crypt cells; b) whether SI mRNA concentrations display increasing responsiveness to GC during the first two postnatal weeks; c) whether SI mRNA concentrations cease responding to GC on approximately day 18; d) the precise time course of enhanced expression of SI mRNA following GC administration during the first two postnatal weeks. 2. To use sucrase enzyme activity and quantitation of Si mRNA to determine whether: a) """"""""late"""""""" effects of GC are due to action at the level of the crypt or the villus; b) the timing of loss of GC responsiveness is delayed by adrenalectomy and accelerated by administration of GC. 3. To identify regulatory gene products (RGPs) of GC action on the small intestine by screening of an intestinal cDNA library with appropriately subtracted probes, and to use the clones thus obtained as probes to determine: a) whether RGPs elicited by GC on immature intestine also appear during normal and GC-independent development; b) the cellular site (crypt vs. villus; epithelium vs. mesenchyme) of appearance of RGPs. 4. To systematically screen a number of inbred and wild strains of mice to identify one in which intestinal development is either delayed or accelerated. If such a strain is identified, it would be used in a subsequent grant period to complement the data generated from above studies by investigating the molecular genetics of intestinal development. 5. To isolate and characterize the rat SI gene. In particular, to obtain potential regulatory sequences (e.g., the 5' flanking region) to enable studies of the mechanisms of control of SI expression during both precocious elicitation of GC and GC-independent development.
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