J. Moore1, M. Fu1, H. Wood1, K. Kuruvilla1, A. Gosain1 1University Of Colorado Denver, Surgery, Aurora, CO, USA
Introduction: The enteric nervous system (ENS) governs all gastrointestinal function, including gut motility, nutrient absorption, and stool excretion. ENS development relies on the proliferation, differentiation and migration of neural crest cells (NCCs) through the extracellular matrix (ECM). Failure of NCC migration leads to Hirschsprung disease (HSCR), a congenital disorder characterized by the absence of enteric ganglia in the distal colon, resulting in functional bowel obstruction. Alterations in ECM composition may contribute to failure of NCC migration in the pathogenesis of HSCR.
ECM proteomics data from our laboratory has identified 48 proteins with significant differential expression in HSCR patient aganglionic bowel vs. age-matched controls. Among these, Focal Adhesion Kinase (FAK) was downregulated, whereas Collagen IV ?1, integrin ?1, and actin-related protein 2/3 complex were upregulated. Based on these findings, we hypothesized that elevated Collagen IV levels interfere with NCC migration by disrupting the FAK signaling pathway, which is essential for actin cytoskeleton remodeling and cell migration.
Methods: Intestinal ENS smooth muscle and myenteric plexus sheets were collected from postnatal day 2-15 wildtype mice and used for wholemount immunofluorescence (IF) or dissociated to generate neurospheres in culture. Neurospheres were then loaded onto dishes coated with fibronectin (control) or increasing concentrations of Collagen IV (1-50 μ g/cm2). Following attachment, glial cell line-derived neurotrophic factor (GDNF) was added to induce cell migration. Migration distance from the center of the neurosphere was measured and lamellipodia formation on migrating NCCs (RET+) was examined with IF staining for phalloidin.
Results: Wholemount IF confirmed Collagen IV ?1 is expressed around and Integrin ?1 is expressed in enteric neurons?. We observed a significant reduction in NCC migration on Collagen IV-coated dishes compared to fibronectin (FIGURE, p<0.0001). NCC migration was further decreased with increasing concentrations of Collagen IV, indicating that high expression of Collagen IV may inhibit the NCC migration (p<0.0001). At higher concentrations of Collagen IV, there was a marked reduction in lamellipodia formation in NCC.
Conclusion: High concentrations of Collagen IV inhibit NCC migration in vitro. The inhibitory role of Collagen IV on NCC migration in the context of HSCR may be through the FAK signaling pathway which is responsible for lamellipodia formation via actin-related protein 2/3 complex. This signaling pathway is the focus of ongoing work. Our findings highlight the critical role of the ECM in HSCR pathogenesis and ENS regenerative medicine strategies.