Dysregulation of Gli Signaling Underlies Degective Enteric Nervous System Development

Abstract

During enteric nervous system (ENS) development, coordination of neurogenesis and gliogenesis require an appropriate balance between the proliferation and differentiation of enteric neural crest cells (NCCs). Defects in these processes may lead to intestinal aganglionosis, Hirschsprung (HSCR) disease in humans. We have previously shown that aberrant Hedgehog signaling interferes both proliferation and differentiation of NCCs and results in disease predisposition, but the underlying molecular and cellular events remain unclear. In this study, we further delineated the roles of the three key Hedgehog effector genes namely Gli1, Gli2 and Gli3 in ENS development. Western blot analysis with FACS-sorted NCCs revealed that Gli signaling is transiently activated during NCC differentiation. Using an in vivo reporter of Gli activity, we demonstrated that Gli activity is elevated in the early committed ENCCs. Transient high Gli signaling is required for the proper ENS development, as constitutively high Gli activity by conditional ablation of Suppressor of Fused (Sufu) in NCCs resulted in impaired neuronal differentiation, axonal fasciculation, gangliogenesis and early onset of glial lineage differentiation of NCCs. The ratio of Gli2 activator (Gli2A) and Gli3 repressor (Gli3R) was particularly critical for ENS development, as they restrained the differentiation and cell cycle progression of NCCs. Aberrantly high Gli2A to Gli3R ratio in the NCCs resulted in dysregulation of cell cycle gene expression and an increase in S and G2/M phase, accompanied by early onset of NCC differentiation. Similarly, Gli3Δ699/ Δ699 mice, where a truncated form of Gli3 that mimics Gli3R was constitutively produced, exhibited reduced neuronal and glial differentiation, suggesting that forced expression of Gli3R was deleterious to NCC differentiation. Taken together, our findings suggest that transient activation of Gli activity promotes cell cycle progression and initiates NCC differentiation by regulating multiple proliferative signaling pathways. Aberrant Gli activity interferes NCC development and may contribute to HSCR pathogenesis.