HABP4/VIM-Mediated Focal Adhesion Assembly Regulates Enteric Neural Crest Cell Migration via the FAK/SRC Pathway

The enteric nervous system (ENS) arises from enteric neural crest cells (ENCCs), and defects in this process can lead to Hirschsprung's disease (HSCR), a congenital disorder characterized by the absence of enteric neurons in the distal colon. Our previous work has identified HABP4 as a potential susceptibility gene for HSCR; however, the mechanisms by which it regulates ENS development remain unclear.

Single-cell RNA sequencing combined with immunofluorescence staining was used to characterize the spatiotemporal expression pattern of HABP4 in mouse intestinal tissues. HABP4-knockdown human embryonic stem cell-derived neural crest cells (hESC-derived NCCs) were generated to evaluate its role in ENS development, and cell migration was assessed using Transwell assays. Protein-protein interaction network analysis integrated with transcriptomic profiling was performed to identify HABP4-interacting partners and downstream signaling pathways, which were further validated by AlphaFold-based structural modeling and co-immunoprecipitation. In addition, pharmacological rescue experiments were conducted to examine the functional involvement of key downstream effectors, and the pathogenicity of HABP4 variants identified in HSCR patients was further evaluated.

HABP4 was highly and specifically expressed in ENCCs. Knockdown of HABP4 significantly impaired the migratory capacity of hESC-derived NCCs. Transcriptomic analysis revealed significant enrichment of focal adhesion-related gene sets following HABP4 knockdown. Mechanistically, HABP4 interacted with VIM to promote focal adhesion assembly and activate the FAK/SRC signaling cascade, thereby facilitating ENCCs migration. Notably, treatment with a focal adhesion kinase (FAK) agonist partially rescued the migration defects induced by HABP4 knockdown, and the HABP4 variant (R379W) identified in HSCR patients was found to impair ENCCs migration.

HABP4 is localized at the leading edge of migrating ENCCs, where it interacts with VIM to promote focal adhesion assembly and activate FAK/SRC signaling.