138 Expression profiling of the ventral colon, pelvic flexure, and dorsal colon epithelium by RNA-seq

Abstract

The equine large intestine is essential for nutrient absorption and as a location for microbial digestion. The pelvic flexure is an important anatomical landmark that divides distinct microbial communities in the equine hindgut. Previous work from our group showed that several taxonomic groups were restricted to the cecum and ventral colon. Since no physical barrier separates these compartments from the more distal areas, we became curious about alternative mechanisms that support the segregation of different microbial groups. Physiological differences in each compartment likely result in different requirements from the microbiota at each location. This study evaluated gene expression in the mucosal and submucosal layers of the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). Samples were collected from 3 4-year-old American Quarter Horses, and total RNA was isolated. Single-end libraries were prepared using the NEBNextⓇ Ultra II Directional RNA library kit and sequenced on an IlluminaⓇ NextSeq 550 platform. The sequencing generated an average of 22.5 million reads per sample (range 14.5 to 30.9). Gene expression analysis was performed using the Galaxy platform by aligning quality trimmed sequence reads to EquCab3 using the HISAT2, assembling transcripts with StringTie, and quantifying transcript abundance with Salmon. DESeq2 was used to assess differential expression between the intestinal compartments. Expression (present in 2 of 3 biological replicates) was detected in at least one intestinal compartment for 21,987 transcripts and ranging from 14,766 to 15,134 transcripts per sample. A total of 13,944 transcripts were detected in all 5 areas, and 739 transcripts demonstrated a tissue-restricted expression pattern (detected above threshold in only on anatomical location). Gene ontology was used to identify the functional roles of transcripts expressed in all or only one hindgut compartment. Transcripts expressed in all tissues were associated with internal cell metabolic processes, which are common despite cell type. In contrast, transcripts with tissue-restricted expression patterns were associated with cellular differentiation and secretion processes. Matrix metalloproteinases, transcription factors, and integrin proteins were identified as differentially expressed (P < 0.05) in the PF compared with the surrounding VC and DC compartments. Continued investigation of these transcripts in the equine hindgut will enhance our understanding of digestive physiology and support future studies of interactions between host and microbiota.