The role of EphA4/ephrin-A1 signaling in enteric neural crest interaction

Abstract

Enteric neural crest cells (ENCCs) form the enteric ganglia and neuronal network during enteric nervous system (ENS) development. Ret and p75 are well-known ENCC markers that are important in ENCC survival and cell fate commitment. Apart from premature differentiation and reduction of progenitor cell pool, failure of ENCCs to completely colonize the gut during ENS development can contribute to aganglionosis in Hirschsprung’s disease (HSCR). Eph/ephrin is a family of cell surface receptor and ligand molecules that mediate forward and reverse signaling. Cranial and trunk neural crests express Ephs and ephrins that regulate cell migration through repulsion or attraction. Protein expression of Ephs and ephrins in mouse developing gut has been recently discovered. However, the role and mechanism of Eph/ephrin signaling in ENCC interaction and migration were not well-understood. Mutation in EFNA1, EPHA5 and EPHB2 were also reported in human HSCR patients, suggesting that Ephs and ephrins are required in ENS development. In this project, I aimed to elucidate the role of EphA4/ephrin-A1 signaling in ENCC interaction by analyzing ENCC RNA-seq datasets, studying protein interaction of wildtype and mutant ephrin-A1 with Ret and p75 and investigating the functional role of EphA4/ephrin-A1 signaling in vitro.

Bioinformatic analysis of Wnt1Cre ENCC bulk RNA-seq dataset and Sox10Cre ENCC single cell RNA-seq dataset of E12.5 embryonic guts showed that Eph and ephrin genes were expressed in subpopulations of ENCCs. From cell lineage plot, EphA4 expression was widespread in gliogenic, neurogenic and bi-potential populations of ENCCs. Ephrin-A1 was expressed in gliogenic and bi-potential populations with high expression level, but it was completely absent in the neurogenic population. Ephrin-A1 co-expressed with ENCC markers Ret and p75. Ephrin-A1 expression was particularly correlated with the expression of p75 visualized in correlation matrix.

GPI-linked Ephrin-A ligands recruit co-receptors to exert intracellular signals upon binding to EphA receptors. Co-receptors identified includes Ret and p75. Protein-protein interaction experiments were done to study the link between ephrin-A1, Ret and p75. Results showed that ephrin-A1 could interact with Ret and p75 in HEK293T cells. These protein interactions suggested that Ret and p75 could be involved in ENCC interaction and migration by partnering with ephrin-A1. Interactions between ephrin-A1-H135Q mimicking human HSCR patient mutation and Ret and p75 were not altered.

Furthermore, cell segregation assay suggested that EphA4/ephrin-A1 interaction mediated forward and reverse repulsive signaling. Ephrin-A1 mutation seemingly exaggerated the repulsion between ephrin-A1- and EphA4-expressing cells. Preliminary results showed that ephrin-A1 mutation could also alter the phosphorylation level of Ret, p75 and FAK. As FAK affects cell-cell interaction through integrin signaling, ephrin-A1 mutation could be causing defect in ENCC migration by dysregulating ENCC cell-cell interaction through FAK.

Taken together, I highlighted the novelty of Eph and ephrin expression in ENCCs and protein interaction of ephrin-A1 with ENCC markers, Ret and p75. My study also suggested the possible mechanism of EphA4/ephrin-A1 repulsive signaling in affecting ENCC interaction and migration.