Cilk1 Is Essential for Mesenchymal Cilia Maintenance and Epithelial-mesenchymal Crosstalk in Intestinal Villus...

Intestinal development is intricately governed by epithelial-mesenchymal crosstalk, with Hedgehog (Hh) signaling playing a pivotal role. The formation of mesenchymal clusters, driven by epithelial Hh signals, is critical for villus morphogenesis. However, the specific role of primary cilia within the mesenchyme during this process remains insufficiently understood. Ciliogenesis-associated kinase 1 (Cilk1) is crucial for maintaining primary cilia required for Hh signal transduction. This study examines the role of Cilk1 in mesenchymal ciliogenesis and cluster formation during intestinal development.

We used both Cilk1 knockout (Cilk1^KO) and tissue-specific Cilk1 deletion models, along with conditional Ift88 knockout mice, to examine the role of primary cilia in intestinal development. Immunohistochemistry, in situ hybridization, quantitative real-time PCR (qRT-PCR), and bulk RNA sequencing (RNA-seq) were employed to evaluate the presence of primary cilia, mesenchymal cluster formation, and the expression of signaling pathway activity. The tissue-specific deletion of primary cilia was achieved by crossing Ift88 floxed mice with Cre driver lines targeting the intestinal epithelium (Villin^Cre) and mesenchyme (Gli1^CreERT2). Postnatal deletion experiments validated Hh responsiveness in postnatal intestinal mesenchyme.

Our findings demonstrate that, although Cilk1 is not required for the initial formation of primary cilia in both the epithelium and mesenchyme at embryonic day 12.5 (E12.5), it is crucial for maintaining mesenchymal cilia beyond E14.5-a critical stage for villus morphogenesis. Deletion of Cilk1 led to the loss of mesenchymal cilia, resulting in impaired Hh signaling, defective mesenchymal clustering, and abnormal villus formation. Transcriptomic analysis revealed disruption of multiple signaling pathways including marked downregulation of BMP ligands, significant enrichment of Wnt pathway alterations (NES = 1.52; false discovery rate [FDR] q = 0.0038), and changes in retinoic acid signaling, whereas fibroblast growth factor (FGF) signaling remained unaffected. Tissue-specific deletion of Ift88 further confirmed the necessity of mesenchymal cilia for proper cluster formation and Hh signal transduction. Activation of Hh signaling through administration of the agonist SAG partially restored mesenchymal cluster formation in Cilk1^KO intestines, consistent with the multi-pathway disruption identified by RNA-seq. Postnatal deletion experiments confirmed that Cilk1 is required for Hh responsiveness in postnatal intestinal mesenchyme.

Cilk1 is essential for maintaining mesenchymal primary cilia and ensuring effective signaling required for intestinal villus morphogenesis. Disruption of Cilk1 impairs Hh-mediated mesenchymal clustering, with additional effects on BMP and Wnt pathways contributing to the developmental defects. These findings establish the critical role of Cilk1-dependent mesenchymal ciliogenesis in epithelial-mesenchymal communication during intestinal development.