Delineating the roles of vinculin in neural crest development and neurocristopathies using human induced pluripotent stem cell (hiPSC) models

Abstract

Vinculin (VCL) is a key adopter protein for the focal adhesion (FA) assembly and is associated with regulating various cellular processes. We identified a loss-of-function mutation in VCL from patients with Hirschsprung (HSCR) and cardiac outflow tract (OFT) defects. We further revealed this mutation alters VCL protein conformation and disrupts FA assembly in neural crest cells (NCCs) derived human-derived induced pluripotent stem (hiPSC), perturbing NCC development. Through the NCC-specific Vcl-knockout (Vcl-KO) mouse model, we demonstrated perturbed TGF-β and stress-activated MAPK pathways are associated with disrupted mesenchymal (MES) to vascular smooth muscle cell (VSMC) transition of cardiac NCCs (CNCCs) in Vcl-KO mice and deletion of the Vcl gene in NCCs affects the migration and differentiation of enteric NCCs (ENCCs), interfering the formation of the OFT and the enteric nervous system (ENS), respectively.

However, revealing the complete relationship between VCL and NCC-associated disease is still challenging. The limited number of NCCs accessible in mouse embryos and the lack of inducible NC-specific Cre mouse line hamper more detailed analyses of the molecular mechanisms underlying the disease pathogenesis and the roles of VCL at different stages of NC development.

It was hypothesised that the molecular and cellular processes underlying human CNCCs (hCNCCs) and ENCCs (hENCCs) development could be recapitulated in vitro using the hiPSC model, and that supported the studies of the molecular mechanisms underlying VCL in human OFT and ENS development.

The first part explored how VCL knockdown (KD) affects hCNCCs development. A hiPSC line with a doxycycline-inducible Cas9 expression platform was utilised, enabling a cell stage-specific gene deletion via clustered regularly interspaced short palindromic repeats/Cas9 system. When VCL was KD in hCNCCs, the MES-to-VSMC differentiation was compromised with the downregulation of TGF/SMAD2 and MAPK/p38 pathways. Adding a p38 antagonist and p38 agonists abolished and favoured the MES-to-VSMC transition of hCNCCs, respectively. Importantly, the VCL-associated MES-to-VSMC transition defect solely affected CNCC lineage but not the second heart field progenitors. Activating the p38 pathway in CNCCs derived from a patient-specific iPSC line expressing the defective VCL rescued their VSMC differentiation defect, representing a potential therapeutic target.

The second part focused on how VCL may affect the migration behaviour and neuronal differentiation capacities of hENCCs. Fluorescent-activated-cell-sorting-enriched hiPSC-derived ENCCs were subjected to wound healing assay, revealing downregulated VCL impairs hENCCs migration by perturbing cell projections and FA assembly. VCL-KD in hENCCs also completely abolished the formation of enteric neurons. However, it is likely because VCL is required for the cell-cell aggregation of hENCCs, which is essential for initiating neuronal differentiation. Deletion of VCL after the onset of neuronal differentiation, VCL did not alter the yield of enteric neurons. In summary, VCL is vital for cell-cell and/or cell-matrix interactions, in turn mediating the migration and neuronal differentiation of hENCCs for forming functional ENS.

In sum, explicating human NCC development and the associated congenital diseases using hiPSC provides new insights into the molecular mechanisms and defines a potential therapeutic target for these diseases.

(485 words)