CHD9NB Is a Novel Brush Border Marker in the Intestine and Beyond
Authors: Burman Andreanna , Brown Monica , Watanabe Masahiko , Goldenring James , Kaji Izumi 
Published: May 2024

The public availability of single cell RNA sequencing datasets has accelerated discovery of novel gene markers of specific cell types. A recently published dataset of human small intestine and colon (GSE185224) revealed 14 distinct epithelial clusters, including tuft cell, a rare sensory cell population. We probed this dataset for novel tuft cell-specific makers and investigated the localization of candidate marker proteins. Using dataset GSE185224, Leiden clustering and UMAP visualization were generated, and tuft cell clusters were identified by previously known markers, e.g. POU2F3. Genes were systematically parsed through and CHD9NB (Chromodomain Helicase DNA Binding Protein 9 neighbor) was identified as a candidate novel tuft cell marker gene. CHD9NB encodes a 52 amino acid protein with no published information for function. Antibodies were raised against recombinant human CHD9NB. Paraffn sections of tissues and enteroids from healthy individuals and mice were immunostained against CHD9NB and brush border or cell structural markers. CHD9NB was expressed along apical actin bundles of intestinal tuft cells. Contrary to transcriptional level, which was lower in enterocytes than tuft cells, intense CHD9NB immunostaining was observed at the tips of microvilli along mature enterocytes in human and mouse small intestine and colon. This microvillus tip localization was suggested by the presence of CHD9NB above the ACTG1 staining in brush border, similar to EPS8. Less intense CHD9NB was detected at both the tips and base of microvilli of the developing brush border in intestinal crypts, suggesting a differentiation stage-dependent localization of CHD9NB in the intestinal epithelial cells. Differentiated human enteroids exhibited either villus-like (microvillus tip-specific) or crypt-like (present at both the base and tips) CHD9NB staining, likely suggesting a range of enteroid differentiation states. To determine whether CHD9NB is specific to the intestinal microvilli or more generally expressed in brush borders of other organs, sections of kidney, inner ear, and lung were stained. CHD9NB staining was specific to brush border-containing, OAT1+ proximal tubules of the kidney, and absent in brush border-lacking distal tubules and glomeruli. In contrast to tips of microvilli in enterocytes, CHD9NB was localized to the base of the brush border of proximal tubules. CHD9NB staining in the murine cochlea was specific to the DCLK1+ cells of the stria vascularis, rather than hair cells. In the lung, CHD9NB was detected along the ciliated cells of the human bronchiole. We propose CHD9NB as a novel brush border and intestinal tuft cell marker, with organ-specific localization patterns beyond the intestine. Further studies will explore the function of CHD9NB in enteroids. NSF GRFP; NIH R01DK128190; RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.