Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure

Abstract

Planar cell polarity (PCP) controls the convergent extension cell movements that drive axis elongation in all vertebrates. Though asymmetric localization of core PCP proteins is central to their function, we currently understand little about PCP protein localization as it relates to the subcellular behaviors that drive convergent extension. Here, we have used high magnification time-lapse imaging to simultaneously monitor cell intercalation behaviors and the localization of the PCP proteins Prickle2 and Vangl2. We observed the expected asymmetric enrichment of PCP proteins, but more interestingly, we also observed tight temporal and spatial correlation of PCP protein enrichment with contractile behavior in cell-cell junctions. These patterns of localization were associated with similar pattern of protein turnover at junctions as assessed by FRAP. In fact, dynamic enrichment of PCP proteins was linked more strongly to junction behavior than to spatial orientation. Finally, recruitment of Prickle2 and Vangl2 to cell-cell junctions was temporally and spatially coordinated with planar polarized oscillations of actomyosin enrichment, and all of these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation of the closing vertebrate neural tube.

Frequency and synchrony of actomyosin oscillation during PCP-dependent convergent extension

“…Our data also provide important new insights into the function of PCP signaling in vertebrate CE, which is an important issue given the implication of PCP genes in human neural tube defects (Wallingford et al, 2013). These results here are consistent patterns we observed recently in neural epithelial cells (Butler and Wallingford, 2018), which again is significant for demonstrating that the dynamic spatial and temporal patterns of Pk2 localization are conserved across cell fate (ectoderm vs. mesoderm) and tissue type (epithelial and mesenchymal). These results are also consistent with findings that anteroposterior patterning is central to the organization of CE in both Drosophila and Xenopus (Paré et al, 2014, Ninomiya et al, 2004, Zallen and Wieschaus, 2004.…”

“…We knocked down Pk2 using an antisense morpholino-oligonucleotide that has been validated in both convergent extension and polarization of multiciliated cells Wallingford, 2018, Butler andWallingford, 2015). Pk2 knockdown strongly disrupted both the elongation and the planar orientation of cells in the dorsal mesoderm, consistent with disruption of other core PCP proteins in dorsal gastrula mesoderm (Jessen et al, 2002, Wallingford et al, 2000( Figures 4A-4D).…”

“…To explore the molecular control of these oscillations, we turned our attention to the PCP signaling network, which has been implicated in control of both deep and superficial actomyosin dynamics in the dorsal mesoderm Davidson, 2011, Shindo andWallingford, 2014). Recently, we showed that the core PCP protein Pk2 controls CE in neural epithelial cells, where it displays a tight temporal and spatial correlation with actomyosin at shrinking junctions (Butler and Wallingford, 2018). We therefore used mosaic expression to generate labeled cells that were directly apposed to unlabeled cells in the dorsal mesoderm ( Figure 3A).…”

“…GFP-Pk2 plasmids were constructed and designed as described previously in (Butler and Wallingford, 2015), and the Myl9-GFP plasmid in (Shindo and Wallingford, 2014). The Pk2 MO used disrupts splicing of the Pk2 gene (Butler and Wallingford, 2015) and severely disrupts CE of Keller explants and neural tube closure in intact Xenopus embryos; both phenotypes are rescued by co-expression of the wild-type Pk2 gene (Butler and Wallingford, 2018). Furthermore, these phenotypes are recapitulated by expression of a Pk2 dominant-negative (Butler and Wallingford, 2018) and are consistent with CE defects in a genetic mutant in the single prickle homolog in Ciona (Jiang et al, 2005).…”

PCP-dependent transcellular regulation of actomyosin oscillation facilitates convergent extension of vertebrate tissue

“…To explore the molecular control of these oscillations, we turned our attention to the PCP signaling network, which has been implicated in control of both deep and superficial actomyosin dynamics in the dorsal mesoderm (Kim and Davidson, 2011) (Shindo and Wallingford, 2014). In addition, we recently showed that the core PCP protein Pk2 controls CE in neural epithelial cells, where it displays a tight temporal and spatial correlation with actomyosin at shrinking junctions (Butler and Wallingford, 2018). We therefore used mosaic expression to generate labeled cells that were directly apposed to unlabeled cells in the dorsal mesoderm ( Figure 3A).…”