Loss of Twist1 leads to disruption of ciliary length, endocytic vesicle dynamics, and cell–cell junctions during neural tube formation

BACKGROUND: Endocytosis constitutes a fundamental cellular process governing development through coordinated regulation of plasma membrane remodeling and ciliogenesis, processes essential for cell shape changes and tissue development. Although Twist1 null embryos display complete cranial neural tube (NT) closure defects and conditional knockout in neuroectoderm disrupts cranial neural crest cell fate determination and delamination, the function of TWIST1 in NT morphogenesis remains unknown. We investigated the basis underlying neuroectodermal morphological abnormalities in TWIST1 mutant embryos, specifically the formation of ectopic lateral bending points and cellular disorganization, by examining Twist1's role in cilia formation, adherens junction integrity, and endocytic vesicle dynamics. RESULTS: Immunofluorescence analysis revealed that cytosolic TWIST1 colocalizes with β-catenin and endocytic regulators LRP2 and RAB11B along the apical surface of cranial neuroectoderm. Twist1 knockout resulted in reduced ciliary length and number. Quantitative polymerase chain reaction (PCR) and Western blot analyses demonstrated upregulation of RAB11B and β-catenin at mRNA and protein levels in Twist1 mutants. This molecular dysregulation coincided with increased accumulation of apical endocytic vesicles and altered expression profiles of endocytic component genes, ultimately modifying the apical neuroectodermal cell-cell junctions. CONCLUSION: Our findings establish TWIST1 as a crucial factor for neuroectodermal morphology, demonstrating its importance in ciliogenesis, endocytic vesicle dynamics, and cell-cell integrity.