Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves

Abstract

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Adult Bovine-Derived Small and Large Intestinal Organoids: In Vitro Development and Maintenance

“…Tis characteristic has also been reported for other types of organoids such as the human and murine liver [45,46]. Te highest level of FABP2 expression was in ileal organoids followed by jejunal and large intestinal organoids in this study, which is in accordance with a previous report that evaluated gene expression in various locations of intestinal tissues collected from Jersey calves [47]. Indistinguishable levels of Muc2 gene expression across jejunal, ileal, colonic, and rectal organoids was also in agreement with a previous study using tissues collected from 4-year-old cows [48].…”

Transcriptional changes detected in fecal RNA from neonatal dairy calves of different breeds following gastrointestinal disease of varying severity

“…This is relevant to the current study in that Rosa et al [ 12 , 13 ] demonstrated that even under nondiarrheic conditions, RNA isolated from neonatal dairy calf feces is derived from a considerable number of IECs as evidenced by mRNA expression of KRT8 . This held true in our study in that KRT8 mRNA abundance exceeded that from other genes of interest, suggesting that a considerable amount of fecal RNA originated from IECs.…”

“…In addition to disrupting tight junction proteins, nitric oxide is implicated in the induction of enterocyte apoptosis and an accelerated rate of intestinal epithelial cell (IEC) shedding potentially exceeding the rate of IEC production and disturbing intestinal homeostasis [42,43]. This is relevant to the current study in that Rosa et al [12,13] demonstrated that even under nondiarrheic conditions, RNA isolated from neonatal dairy calf feces is derived from a considerable number of IECs as evidenced by mRNA expression of KRT8. This held true in our study in that KRT8 mRNA abundance exceeded that from other genes of interest, suggesting that a considerable amount of fecal RNA originated from IECs.…”

“…However, the inflammatory response observed by Rosa et al in fecal RNA was delayed compared to the pattern of fecal scores and proinflammatory blood biomarkers. Accordingly, further research of transcriptional alterations is warranted to study the intestinal response to inflammation and its variation over time in neonatal dairy calves of different breeds experiencing diverse manifestations of GI disease [ 12 , 13 ].…”