Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

Abstract

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5- 3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.

Parallel patterns of cell-specific gene expression during enterocyte differentiation and maturation in the small intestine of the rabbit

“…It is not always possible to state exactly the relationship between the pattern of mRNA accumulation as measured by in situ hybridisation and the transcription of a gene. In this instance however, the incorporation in RNA precursors into villus cells [44], and the rapid induction of calbindin expression by 1,25, (OH),D, in vitamin-D,-deficient chickens [49], suggest that the pattern of mRNA accumulation measured by this method here and in previous studies [16][17][18][19]491 may reflect a generalised pattern of transcriptional activity within these cells. The clear differences in the levels of each mRNA at a given point during a enterocyte differentiation and maturation are therefore likely to correspond to the level of transcription of that gene during its migration along the crypt-villus axis.…”

Renewal of the intestinal epithelium: New aspects as indicated by recent ultrastructural observations

“…In this case, a 3 day latent period was already known to precede epithelial denudation (Quastler, 1956). Further evidence in this direction was obtained in a study of RNA synthesis using radioautography (Uddin et al, 1984). This study showed that nucleolar RNA synthesis as well as nucleoplasmic RNA synthesis in the columnar cells become reduced to negligible levels in the upper half of the crypts that is long before reaching the absorptive stage.…”

Hypertranscription in Development, Stem Cells, and Regeneration

“…1b), but we would today consider these cells to be progenitors committed to the myeloid lineage (proerythroblasts and myeloblasts). Progenitor cells in the crypts of the small intestine have been shown to undergo high levels of nascent transcription (Jao and Salic, 2008; Uddin et al, 1984). Recent reports highlight the importance of increased ribosomal RNA and protein synthesis for the transition between germline stem cells and proliferative progenitors in the Drosophila ovary (Sanchez et al, 2016; Zhang et al, 2014), although hypertranscription was not specifically analyzed.…”