Study of the effect of Hirschsprung disease associated ephrinA1 and EphB2 mutations on cell-cell interaction

Abstract

During enteric nervous system development, enteric neural crest cells (ENCCs) migrate and sequentially differentiate rostral-caudally along the gut, this process is termed ENCC colonization. Incomplete ENCC colonization would contribute to Hirschsprung disease, a congenital disorder characterized by the aganglionic gut. Eph/ephrin bidirectional signaling promotes cell migration, cells contact repulsion and attraction. Although Eph/ephrin signaling is known for its participation in nervous system development events such as pathfinding during branchial arch neural crest cells migration, little is known about the functional role of Eph/ephrin signaling in ENCC development. Mutations in EPHA5, EPHB2 and EFNA1 had been identified in Hirschsprung disease patients, suggesting a possible role of Eph/ephrin signaling in ENCC colonization. Previous investigation on Ephs and ephrins mRNA expression showed that ephrinA1 expression level is the highest among all Ephs and ephrins in the developing gut, while EphB2 is the most abundant Ephs and ephrins in ENCCs. Given the Hirschsprung disease-associated mutations in ephrinA1 and EphB2, it is hypothesized that the mutations may pose defects on ENCCs’ interaction with each other as well as surrounding cells in the gut. This study focused on the ephrinA1-H134Q and EphB2-S421L mutations found in Hirschsprung disease patients, aimed at investigating the functional role of Eph/ephrin signaling on ENCC colonization, the impact of the mutations on cell-cell interaction and Eph/ephrin signaling mechanism. To examine the response of migrating ENCCs upon interaction with Ephs and ephrins, stripe assay was performed with ex vivo gut explant culture. Transfected cell lines expressing ephrinA1, mutated ephrinA1, EphB2, mutated EphB2, and their corresponding partners, EphA4 and ephrinB2 were co-cultured in in vitro segregation assay to investigate the influence of the mutations on cell-cell interaction. Stripe assay showed fewer ENCCs observed on EphA4 stripes, more ENCCs observed on ephrinA1 stripes, indicating that migrating ENCCs would repulse from EphA4 and are attracted towards ephrinA1. The results show that ENCCs respond to Ephs and ephrins in the extracellular environment, suggesting that ENCCs may utilize Eph/ephrin signaling during interaction and migration. Segregation assay demonstrated that EphA4/ephrinA1 and EphB2/ephrinB2 co-cultures would form well-segregated, compact cell clusters, while EphA4/ephrinA1-H135Q and EphB2-S421L/ephrinB2 co-cultures form loosely packed, interconnecting cell clusters. The results suggest that ephrinA1 and EphB2 mutations failed to properly segregate in co-cultures, indicating that the mutations caused defect in cell-cell interaction and subsequent cell behaviors. Western blot analysis on cell lysate of mixed EphA4/ephrinA1 and EphA4/ephrinA1-H135Q cells showed enhanced phosphorylated EphA4 level in EphA4/ephrinA1-H135Q lysate compared with EphA4/ephrinA1 lysate, suggesting that mutation in ephrinA1 lead to an increase in activated EphA4 level, possibly due to abnormality in cell-cell repulsion to terminate the interacting EphA4/ephrinA1, which explain the cell segregation defect observed. The study shows that migrating ENCCs respond to extracellular Ephs and ephrins and that the defect in cell-cell interaction caused by mutations associated with Hirschsprung disease may alter ENCC migration during colonization. The study provides clues for the functional role of Ephs and ephrins in ENCC colonization, as well as the possible influence of the mutations on ENCCs' interaction with surrounding cells and each other.