Supporting Data for "Delineating the roles of Vinculin in Neural Crest Development and Neurocristopathies using Human Induced Pluripotent Stem Cell (hiPSC)"

Vinculin (VCL) is a key adopter protein for focal adhesion (FA) assembly and is associated with regulating various cellular processes. We have previously identified a loss-of-function mutation in VCL from patients with Hirschsprung (HSCR) and cardiac cardiac outflow tract (OFT) defect. Subsequent analyses further revealed that this mutation alters protein conformation of VCL and disrupts FA assembly in human-derived induced pluripotent stems (hiPSCs) -derived neural crest cells (NCCs) and perturbs NCC development. With an NCC-specific Vcl knockout mouse model, we demonstrated that deletion of Vcl gene in NCCs affects the differentiation and migration of NCCs, interfering with the formation of OFT and enteric nervous system (ENS). The transcriptome analysis of cardiac NCCs (CNCCs) also revealed that perturbed TGF-β and stress-activated MAPK pathways are the potential causes underlying the disrupted mesenchymal (MES) to vascular smooth muscle cell (VSMC) transition, leading to the defect in VSMC differentiation of CNCCs in Vcl knockout mice. Besides, the loss of Vcl gene in enteric NCCs (ENCCs) perturbed the differentiation and migration of ENCCs, leading to the malformation of ENS.

However, there are still challenges to revealing the complete relation between VCL and NCC-associated disease. In particular, a limited number of NCCs are accessible in mouse embryos and lack an inducible NC-specific Cre mouse line; they hamper a more detailed analysis 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. Thus, the molecular mechanisms of how the loss of VCL may cause OFT and ENS defects in humans can be addressed by hiPSC model.

The first part of this project sought to explore how VCL knockdown (KD) affects the development of hCNCCs. A hiPSC line with a doxycycline-inducible Cas9 expression platform was utilized, enabling a cell stage-specific gene deletion via clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system. In the VCL-KD hCNCCs, the MES-to-VSMC lineage differentiation was compromised with downregulated p38 signalling. In concordant with this, p38 antagonist inhibited the MES-to-VSMC transition of hCNCCs, and p38 agonists could rescue the MES-to-VSMC differentiation defect of VCL-KD hCNCCs. In addition, VCL was essential for activating TGFb/SMAD2 and p38 pathways in hCNCCs; loss of VCL abolished these pathways, compromising the MES-to-VSMC transition of hCNCCs. With a patient-specific iPSC derived from a syndromic patient with ENS and OFT defects and carrying a loss-of-function mutation in VCL, I further illustrated that p38 agonists would be a potential therapeutic target for patients with OFT defects. Intriguingly, the VCL-associated MES-to-VSMC differentiation defect was lineage-specific, solely affecting the CNCC lineage, but not their neighbouring cells (second heart field progenitors).

The second part of this study 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 and found that down-regulation of VCL impairs the migration of hENCCs by perturbing cell projections and FA assembly. Additionally, VCL is required for the cell-cell aggregation of hENCCs to initiate neuronal differentiation, but not for supporting the subsequent neuronal maturation. Therefore, knocking down of VCL in hENCCs showed a greater impact on the formation of enteric neurons than those with VCL deletion at the later stage of neuronal differentiation. In summary, VCL is implicated in the migration and the neuronal lineage differentiation of hENCCs, for the formation of functional ENS.

In this project, human NCC development and the associated congenital diseases were modelled in vitro using hiPSC, providing new insights into the molecular mechanisms and defining a potential therapeutic target for these diseases.