Intestinal fibrosis in aganglionic segment of Hirschsprung's disease revealed by single‐cell RNA sequencing

Abstract

Background Hirschsprung's disease (HSCR) is a relatively common congenital disability. Accumulating extracellular matrix (ECM) prompts intestinal fibrosis remodelling in the aganglionic segments of HSCR. The contributions of various cellular subsets in the fibrogenesis of HSCR segments are poorly understood. Methods Single‐cell transcriptomics from 8 aganglionic segments and 5 normal segments of 7 HSCR subjects and 26 healthy segments of seven healthy donors were analysed. Fibrotic phenotype and alterations were explored using differential expression analysis and single‐cell trajectory analysis. Fibrosis‐related transcription factors were inferred through single‐cell regulatory network inference. Bulk transcriptomic data, proteomic data, immunohistochemistry (IHC) and real‐time polymerase chain reaction were used to validate the alterations in the HSCR intestine. Results Various collagen, fibronectin and laminin protein‐coding genes expression were up‐regulated in the stromal and glial cells of the HSCR intestine. The number of fibroblasts and myofibroblasts in the aganglionic segments increased, and more myofibroblasts were activated at an earlier stage in HSCR segments, which infers that there is an intestinal fibrosis phenotype in HSCR segments. The fibrotic regulators POSTN , ANXA1 and HSP70 were highly expressed in the ECM‐related cellular subsets in the transitional segments and aganglionic segments. The transcription factor regulatory network revealed that fibrosis‐related and megacolon‐related NR2F1 in the fibroblasts and glial subsets was up‐regulated in the aganglionic segment. Conclusions This work identifies intestinal fibrosis and related regulators in aganglionic segments of HSCR; hence, anti‐fibrotic therapy may be considered to prevent HSCR‐associated enterocolitis (HAEC), relieve intestinal stricture and improve cell therapy.

Unravelling the conundrum of nucleolar NR2F1 localization: A comparative analysis of NR2F1 antibody-based approachesin vitroandin vivo

“…In humans, NR2F1 haploinsufficiency caused by deletion or loss-of-function mutations results in a monogenic neurodevelopmental disorder named Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) [4][5][6] . Characterized by intellectual disability, developmental delay, and visual impairment 3,19,22 , BBSOAS is often associated with additional features, such as epilepsy, autistic traits, hypotonia, oromotor dysfunction, craniofacial malformations, and hearing impairment [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] . NR2F1 is now listed among the 10 top genes causally linked to rare hereditary optic neuropathies 21 .…”

“…NR2F1 is now listed among the 10 top genes causally linked to rare hereditary optic neuropathies 21 . In addition, NR2F1 has recently been linked to Hirschsprung disease 22 and Waardenburg syndrome type IV 23 , as well as to the development of lymphoid and cardiac cells 24 , but its precise role in these disorders remains to be investigated.…”

“…Atrophy Syndrome (BBSOAS) 4–6 . Characterized by intellectual disability, developmental delay, and visual impairment 3, 19, 22 , BBSOAS is often associated with additional features, such as epilepsy, autistic traits, hypotonia, oromotor dysfunction, craniofacial malformations, and hearing impairment 4–20 . NR2F1 is now listed among the 10 top genes causally linked to rare hereditary optic neuropathies 21 .…”

More than missing neurons: Intestinal fibrosis in Hirschsprung disease

“…The recent study of Shiwei He and colleagues shed light on this subject by identifying cellular changes that occur in the aganglionic segment of HSCR patients. 5 By using single cell RNA sequencing (scRNA-seq), they provide for the first time, the transcriptomic profile of different cell populations in both aganglionic and ganglionic segments. Single cell suspensions were prepared from these regions and analysed using the 10X Chromium system.…”