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Research Highlight: Understanding the Key Role of a EMT Master Regulator, Twist1, during Mouse NCC Delamination and EMT.

Posted by , on 11 July 2022

Epithelial to mesenchymal transition (EMT) is an essential process in multiple steps of embryogenic morphogenesis and various pathological conditions. As an example, EMT is involved in gastrulation and neural crest cell (NCC) development during embryogenesis. EMT is also crucial for wound healing, tissue fibrosis and cancer progression. In addition to the cellular and molecular changes facilitating the transformation from epithelial cells into mesenchymal cells, EMT has also been associated with stemness, therapeutic resistance and tumor heterogeneity specifically in the context of malignancy. Due to the inherent differences between different species, cell types and biological contexts, there are variations within phenotypic changes and the underlying molecular mechanisms of different EMT programs.

NCC are an embryonic progenitor cell population that gives rise to numerous cell types and tissues, such as craniofacial bone and cartilage, in vertebrates. Pre-migratory NCCs delaminate from the neuroepithelium via EMT, following which NCCs migrate throughout the embryo and undergo differentiation. Currently, we have limited understanding of the EMT process that gives rise to migratory NCCs in mammals because many NCC EMT related findings in non-mammalian species have not been successfully replicated in mammalian species.

Transcriptional factor Twist1 is one of the major master regulators shown to be involved and to play an important role in EMT throughout both development and carcinoma progression. Previous studies on the role of Twist1 during mammalian NCC development using various mouse models have been thoroughly summarized and reviewed (Zhao and Trainor, 2022). In short, Twist1 null mice exhibit embryonic lethality around E11.5 associated with craniofacial defects such as malformed branchial arches and facial primordia (Chen et al.,2007; Chen and Behringer, 1995). Upon careful experimental testing, these phenotypes were believed to be caused by abnormality of NCC migration and differentiation. Consistent with these in vivo findings, mutations in Twist1 in humans lead to Saethre-Chotzen syndrome, which is characterized by craniosynostosis and cleft palate. In a recent publication “Twist1 Interacts with Beta/Delta-Catenins During Neural Tube Development and Regulates Fate Transition in Cranial Neural Crest Cells”, however, Bertol and her colleagues further depict the neuroectodermal expression profile of Twist1 during early mouse embryogenesis and illustrate potential functions of Twist1 in mouse cranial NCC delamination and EMT.

Key findings

  1. During mouse embryogenesis between E8.5 and E9.5, Twist1 is detected in vesicle-like structures on the apical side of the neuroepithelium/neural plate. Interestingly, such apical expression of Twist1 coincides with the expression pattern of B-catenin and Claudin-1 suggesting an association of Twist1 with adherens and tight junctions in the neuroepithelium. Furthermore, a physical interaction between cytosolic Twist1 and B-catenin is demonstrated by co-immunoprecipitation. When Twist1 is deleted in whole embryos, apical B-catenin in vesicle like structures become diffused and mostly cytosolic in the apical neuroepithelial cells of the neural plate.
  2. Consistent with other studies, Twist1 is also found to be expressed in migratory cranial NCCs at E8.5, E9.5 and E10.5. When Twist1 is conditionally deleted in cranial premigratory NCCs at early E8.5, cranial migratory NCCs are observed throughout the embryos, but there is a fewer number of migratory NCCs in the frontonasal and pharyngeal processes between E9.5 and E11.5. This observation is later confirmed by severe frontonasal prominence defects and neural tube closure abnormalities.
  3. Examinations of remaining post-delamination migratory NCCs in neural tube explants from Twist1 conditional knockout mice reveal that the majority of migratory NCCs exhibits epithelial morphologies, significant cell-cell adhesions and continuous junctional signals of ZO1. Moreover, migratory cranial NCCs in vivo show increased E-cadherin expression, and Specc1 (an actomyosin cytoskeleton regulator) expression is reduced in the hindbrain and first pharyngeal arch. These data indicate disrupted EMT during the delamination of cranial NCCs in an absence of Twist1 expression.
  4. To study the importance of Twist1 phosphorylation in craniofacial tissue development, the researchers have also generated four Twist1 phospho-incompetent mouse lines. Phenotypic characterizations of these mutants demonstrate that S18/20 and S68 phosphorylation sites are critical for craniofacial development.

In summary, the paper contributes a valuable collection of data to fill our knowledge gap of how NCC delamination and EMT are regulated in mammalian species. To my knowledge, this is the first publication that directly studies the role of Twist1 specifically in early NCC development via using Wnt1-Cre and Wnt1-Cre2 driven conditional knockout mouse models. Although I find some parts of the paper slightly confusing regarding the interpretation of certain data and the relevance of IRF6 data to the rest of the paper, the data itself is still very intriguing and thought-provoking. Interestingly, Zeb2 null mutant mouse embryos exhibit similar phenotypes of persistent E-cadherin expression in migratory cranial NCCs (Putte et al., 2003). Similar to Twist1, neither Snail1 or Zeb2 conditional knockout in pre-migratory NCCs completely inhibits NCC delamination and EMT (Murray and Gridley, 2006; Rogers et al., 2013). These previous findings in combination with the proposed function of Twist1 in the completion of mouse cranial NCC EMT could suggest that perhaps EMT master regulators act synergistically in waves to promote the complete transition from neuroepithelial cells to mesenchymal migratory NCCs.

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