Elucidating the Role of Mechanics in Neural Plate Convergent Extension

Neural tube formation is crucial for the proper development of the brain and spinal cord and its failure results in congenital disorders known as neural tube defects (NTDs). Several known genetic mutations are associated with NTDs but the physical mechanisms by which they affect neural tube morphogenesis remain unclear. The neural tube begins as an epithelial sheet on the embryo surface called the neural plate that undergoes a series of shape changes to form an elongated tubular structure, internalized within the embryo. Integrated behaviors of embryonic cells orchestrate these tissue-level deformations. Our study aimed to identify the cell behaviors accompanying early neural plate shaping in Xenopus laevis embryos when the tissue elongates in the anterior-posterior axis while narrowing in a perpendicular mediolateral axis. Through observation and quantification of local cell and tissue mechanical strains, we identified the emergence of distinctive spatiotemporal patterns of cell behavior. Cells undergo oriented rearrangements within the medial neural plate whereas at its lateral edges, cells assume an elongated morphology. Among the mutations associated with human NTDs, planar cell polarity (PCP) pathway mutations are known to inhibit plate narrowing and elongation and prevent cell rearrangements in vertebrate models of human development. As a cell’s local tissue mechanical environment can influence its behaviors, we sought to determine whether the lack of rearrangement in PCP-compromised embryos might be due to the lack of tissue deformation. We tested how wild type and PCP-compromised plate cells behave in altered tissue strain environments. We find that medial plate cell rearrangement is an intrinsic program independent of tissue extension; however, lateral cell elongation is likely strain dependent. PCP compromised cells in a narrowing and extending tissue assume an elongated morphology compared to wild type cells, becoming stretched in the direction of tissue elongation. These distinctive behaviors under similar mechanical conditions suggest that the PCP pathway mediates a cell's response to its mechanical microenvironment, guiding morphology during plate shaping. This dissertation exposes a role for tissue mechanics in the PCP-mutant phenotype and provides a framework to test the interplay between tissue mechanics and planar patterning in guiding cell behaviors during neural tube morphogenesis

Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure

Neural tube closure is a critical developmental event that relies on actomyosin contractility to facilitate specific processes such as apical constriction, tissue bending, and directional cell rearrangements. These complicated processes require the coordinated activities of Rho-Kinase (Rock), to regulate cytoskeletal dynamics and actomyosin contractility, and the Planar Cell Polarity (PCP) pathway, to direct the polarized cellular behaviors that drive convergent extension (CE) movements. Here we investigate the role of Shroom3 as a direct linker between PCP and actomyosin contractility during mouse neural tube morphogenesis. In embryos, simultaneous depletion of Shroom3 and the PCP components Vangl2 or Wnt5a results in an increased liability to NTDs and CE failure. We further show that these pathways intersect at Dishevelled, as Shroom3 and Dishevelled 2 co-distribute and form a physical complex in cells. We observed that multiple components of the Shroom3 pathway are planar polarized along mediolateral cell junctions in the neural plate of E8.5 embryos in a Shroom3 and PCP-dependent manner. Finally, we demonstrate that Shroom3 mutant embryos exhibit defects in planar cell arrangement during neural tube closure, suggesting a role for Shroom3 activity in CE. These findings support a model in which the Shroom3 and PCP pathways interact to control CE and polarized bending of the neural plate and provide a clear illustration of the complex genetic basis of NTDs

In situ mechanotransduction via vinculin regulates stem cell differentiation.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate

The non-canonical Wnt-PCP pathway shapes the caudal neural plate

The last stage of neural tube (NT) formation involves closure of the caudal neural plate (NP), an embryonic structure formed by neuromesodermal progenitors and newly differentiated cells that becomes incorporated into the NT. Here, we show in mouse that, as cell specification progresses, neuromesodermal progenitors and their progeny undergo significant changes in shape prior to their incorporation into the NT. The caudo-rostral progression towards differentiation is coupled to a gradual reliance on a unique combination of complex mechanisms that drive tissue folding, involving pulses of apical actomyosin contraction and planar polarised cell rearrangements, all of which are regulated by the Wnt-PCP pathway. Indeed, when this pathway is disrupted, either chemically or genetically, the polarisation and morphology of cells within the entire caudal NP is disturbed, producing delays in NT closure. The most severe disruptions of this pathway prevent caudal NT closure and result in spina bifida. In addition, a decrease in Vangl2 gene dosage also appears to promote more rapid progression towards a neural fate, but not the specification of more neural cells.This work was supported by grants from the Instituto de Salud Carlos III (PS09/00050, CP08/00111, CPII14/00033, PI14/01075 and PI17/00693 to P.Y.-G.) co-financed by the European Regional Development Fund “A way to achieve Europe”; the Andalusian Health Service, Junta de Andalucia (Servicio Andaluz de Salud, Junta de Andalucia; PI-0438-2010 to P.Y.-G.); the Andalusian Regional Ministry of Economy, Science and Innovation (Consejerıa de Econom ́ ıa, Innovacio ́ ́ń, Ciencia y Empleo, Junta de Andalucıá ́; P11-cts-7634 to P.Y.-G.); the National Institutes of Health (R01 HD044750 to L.A.D.); and the National Science Foundation (CMMI-1100515 to L.A.D.). In addition, D.S.V. was supported by the “Biomechanics in Regeneration” Training Program from the National Institute of Biomedical Imaging and Bioengineering (BiRM T32 EB003392)

The non-canonical Wnt-PCP pathway shapes the mouse caudal neural plate.

The last stage of neural tube (NT) formation involves closure of the caudal neural plate (NP), an embryonic structure formed by neuromesodermal progenitors and newly differentiated cells that becomes incorporated into the NT. Here, we show in mouse that, as cell specification progresses, neuromesodermal progenitors and their progeny undergo significant changes in shape prior to their incorporation into the NT. The caudo-rostral progression towards differentiation is coupled to a gradual reliance on a unique combination of complex mechanisms that drive tissue folding, involving pulses of apical actomyosin contraction and planar polarised cell rearrangements, all of which are regulated by the Wnt-PCP pathway. Indeed, when this pathway is disrupted, either chemically or genetically, the polarisation and morphology of cells within the entire caudal NP is disturbed, producing delays in NT closure. The most severe disruptions of this pathway prevent caudal NT closure and result in spina bifida. In addition, a decrease in Vangl2 gene dosage also appears to promote more rapid progression towards a neural fate, but not the specification of more neural cells