Protocadherin 20 Is a POU Class 2 Homeobox 3 Target Gene Required for Proper Tuft Cell Microvillus Organization

Tuft cells play protective roles in infection, inflammation, and tumorigenesis through the secretion of cytokines and eicosanoids. Tuft cells are known for their tall, blunt microvilli, thought to be analogous to mechanosensory hair cell stereocilia; however, a functional role for the microvillar apparatus has not been identified. POU Class 2 Homeobox 3 (POU2F3) is the master regulator transcription factor for tuft cells, yet how POU2F3 drives formation of this unique structure is unknown. Here, we aimed to identify POU2F3 target genes and commonalities between tuft and hair cells to better understand this unique structure.

POU2F3 chromatin immunoprecipitation sequencing was performed on tuft cells and compared with the hair cell transcriptome. Tuft cell RNA sequencing datasets were interrogated for hair cell structural and mechanosensory genes; expression was validated. Intestinal and gallbladder tuft cells were examined using multiple light and electron microscopy modalities. Protocadherin 20 (PCDH20) was knocked down in mouse models, and ultrastructural analyses were performed. The tuft cell cytoskeleton was modeled using AlphaFold3 prediction.

Genes encoding structural and mechanosensory proteins common to both tuft and hair cells, including Pcdh20, were identified. Imaging localized PCDH20 to tuft cell microvilli and hair cell stereocilia. Genetic ablation of Pcdh20 in mice resulted in structural defects in tuft cell microvilli, including loss of rigidity and organization. Molecular modeling suggests PCDH20 homodimers link adjacent microvilli.

Pcdh20 is a POU2F3 target gene in tuft cells, critical to maintain the rigid microvillar apparatus. These findings, together with the shared expression of mechanosensory components like transmembrane channel-like protein 1, support the hypothesis that tuft cells could have mechanosensory capabilities analogous to cochlear hair cells.