Gli-Sox10 regulatory network in neural crest development and Hirschsprung disease pathogenesis

Abstract

In vertebrates, enteric neural crest derived cells (ENCC) undergo coordinated migration, proliferation and differentiation to form enteric nervous system (ENS). Hirschsprung (HSCR, aganglionic megacolon) disease is a complex congenital disease attributed to defective ENS development in human. Previously studies have shown that aberrant Hedgehog (HH) signaling interferes ENCC development and results in disease predisposition. However, the underlying molecular and cellular events remain unclear.

In mammals, three effectors, GLI-1, GLI-2 and GLI-3, determine HH signaling output. In this study, deep-targeted sequencing in 20 HSCR patients identified several novel mutations in GLI-1, GLI-2 and GLI-3, where various functional domains of GLI proteins are affected, leading to enhanced GLI activity and perturbed transactivation of Sox10.

Endogenous Gli activities in ENS were established utilizing Tg(GBS-GFP) reporter line. In this mouse model, transient activation of Gli signaling was observed only in subpopulations of enteric progenitors and glial progenitors during E11.5 to E12.5 while Gli activity remains low in the differentiated neurons and glial cells, suggesting a casual link between ENCCs differentiation and Gli activation.

Suppressor of Fused (Sufu) is a negative regulator of Hh signaling. In concordance with other systems, conditional ablation of Sufu in ENCCs induced high Gli activity which was defined by upregulation of the Hh target genes and increase of the ratio of Gli2 activator (〖Gli2〗^A) and Gli3 repressor (〖Gli3〗^R). Functional analysis of ENCCs revealed that aberrant Gli activity induces the early onset of neurogenesis and gliogenesis, which finally lead to a significant reduction of neurons and accompanied by an increase of glial cells at distal guts. Intriguingly, The ratio of 〖Gli2〗^Aand 〖Gli3〗^Rwas correlated inversely with neuronal versus glial lineage differentiation of ENCCs. In addition to differentiation defects, disrupted chain migration, impaired ENS organizations and defective axonal fasciculation were found in the Sufu mutants.

Subsequent mechanistic studies revealed that Gli binds directly to the Sox10 ENS specific enhancers to control its expression level, while Sox10 could also inhibit Sufu expression in ENCCs. Thus, a bi-directional regulatory loop between Sufu-Gli and Sox10 was involved to confer a precise regulation of ENCC differentiation.

Collectively, it is the first time identifying GLI mutations in HSCR patients and demonstrating that perturbed SOX10-SUFU-GLI regulatory nexus may contribute to HSCR pathogenesis by interfering neuronal versus glial lineage differentiation and cell migration. Overall, this study described a clinically important signaling cascade implicated in HSCR pathogenesis.