Regulation of embryonic lineages and morphogenesis by Wnt signaling

Our early studies assessed molecular processes underlying the formation of the dorsal signaling center, also known as the Spemann organizer.  This analysis revealed an essential role for secreted Wnt proteins in vertebrate axis determination.  Our group continued to study Wnt pathways for a number of years and obtained valuable insights related to several Wnt signaling components, from secreted Wnt antagonists and Frizzled receptors to transcription factors of the TCF family (Sokol et al., 1991; Sokol, 1996; Itoh et al., 2005; Hikasa et al, 2010).

Our group has uncovered an important function of noncanonical Wnt signaling in regulating convergent extension movements during vertebrate gastrulation and neurulation (Sokol, 1996, 2000).  This discovery served as a basis for subsequent molecular characterization of the Wnt/PCP pathway.  More recently, we linked Wnt/Planar cell polarity (PCP) signaling to apical constriction, another fundamental morphogenetic event that leads to embryonic tissue folding (Ossipova et al., 2014, 2015).

Noncanonical signaling during neural crest specification

The neural crest (NC) is a population of stem-cell-like cells that form in vertebrate embryos at the neural plate border andmigrate to diverse locations in the body to give rise to multiple cell types. NC-specific genes are activated by signaling interactions between the surrounding embryonic tissues, including the epidermis, neuroectoderm and underlying mesoderm.  During the same time period, premigratory NC progenitors restructure their cytoskeleton and cell junctions in a process known as epithelial-mesenchymal transition (EMT), which is a prerequisite for their migratory behavior.  We are studying how cell adhesion and cell shape changesduring EMT are connected to NC specification (Ossipova and Sokol, 2011).

The establishment of apicobasal and planar cell polarity (PCP) in the embryonic ectoderm

We have shown the involvement of apical-basal and planar cell polarity proteins in cell fate determination during epidermal differentiation, neurogenesis and neural crest development, using mouse, human and Xenopus progenitor models (Dollar et al., 2005; Ossipova et al., 2009; Lake and Sokol, 2009).  We have recently shown that Wnt proteins can direct PCP in the vertebrate neural plate (Chu and Sokol, 2016).  These studies provided insights into asymmetric neural progenitor divisions during neurogenesis, multiciliated cell differentiation in the epidermis and mechanistically connected PAR, Notch and Wnt signaling to PCP.

Planar cell polarity is essential for neural tube folding. We have established a new model for neural plate PCP along the anteroposterior axis and found that this polarity requires both Wnt and Myosin II/ROCK signaling.  We have also characterized a new mediolateral axis of PCP that is likely involved in the regulation of apical constriction in the neural plate.  Our results suggest the involvement of PCP proteins in radial cell intercalations during neural plate closure (Ossipova et al., 2014, 2015; Sokol, 2016).

 How do core PCP proteins segregate?

 We have shown that Wnt proteins can serve as such cues and are required for neuroectodermal PCP (Chu and Sokol, 2016). In a live imaging study, we observed that fluorescently labeled Pk3 initially polarized in the posterior region of the neural plate (NP) and later polarized in the anterior NP (Mancini et al., 2021).  A surgical incision in the middle of the NP disrupting neuroectoderm (but not underlying mesendoderm) prevented the establishment of PCP in the anterior NP. These findings indicate that PCP is induced by a signal spreading from the posterior embryonic region. Supporting this conclusion, transplanted blastopore lip caused PCP reversals in the anterior NP (Mancini et al., 2021). These observations demonstrate that a PCP cue from the dorsal lip is both necessary and sufficient for PCP induction.

We discovered a feedback regulation between core PCP proteins that contributes to their segregation to opposite sides of the cell.  Using a new in vivo proximity biotinylation approach, we found that the apical polarity protein Par3 is planar polarized in the NP and promotes the formation of the anterior Vangl2-Pk3 complex by recruiting Pk3 to the apical surface (Chuykin et al., 2018). With this technique, we also demonstrated a negative role of Fz3 in the formation of the Vangl2-Pk3 complex, which is mediated by a novel phosphorylation of Vangl2 (Chuykin et al., 2021). This regulatory feedback contributes to the anterior restriction of the PCP complex in neuroepithelial cells

How does cell polarization modulate Myosin II contractile activity ?

Based on published literature and our observations, we hypothesize that PCP signaling locally modulates actomyosin contractility to produce anisotropic forces needed for morphogenesis. Once a tissue becomes polarized, core PCP complexes may engage downstream effectors that are necessary to change cell shape leading to NP folding (Nishimura et al., 2012; Ossipova et al., 2014; Sokol, 2016). During the past funding period, we focused on relatively understudied Prickle proteins and identified molecules that link Pk3 to cell junctions. These include the apical determinant Par3 (Chuykin et al., 2018) and the LIM domain protein Wtip (Chu et al., 2016). Wtip belongs to the Zyxin/Ajuba family implicated in neural tube closure actomyosin dynamics, and mechanotransduction (Chu et al., 2018; Ibar et al., 2018; Rauskolb et al., 2014).  We next developed a new proximity biotinylation approach in Xenopus embryos based on anti-GFP single domain antibody (sdAb)-mediated targeting of BirA and this screen led to the isolation of SSX2IP/ADIP as a Wtip-binding protein (Reis et al., 2021).These findings point to SSX2IP as a potential mediator of Wtip effects on NP morphogenesis.

How do actomyosin contractions produce forces that instruct morphogenesis?

To modulate cell shape, contractile actomyosin complexes need to localize at cell junctions.  We recently discovered that the localization of the LIM domain protein Wip to intercellular junctions likely depends on a mechanical force (Chu et al. 2018, the figure shows Wtip puncta in neighboring cells). Wtip, a Prickle3-interacting protein containing LIM domains, can sense Myosin II activity and intercellular tension (Chu et al., 2018).  Moreover, Wtip is localized in ectodermal cells in a very peculiar manner, by forming cytoplasmic aggregates or puncta opposing each other across the cell membrane as shown in the image.  This localization may indicate that neighboring cells mechanically communicate with each other through Wtip, a possible molecular link to planar polarity proteins.

Our recent studies have focused on LMO7 protein that is also sensitive to mechanical force. A mutation in the fly homologue of LMO7, smallish, has morphogenesis defects in Drosophila embryonic epidermis (Beati et al., 2018). Importantly, LMO7 is capable of triggering apical constriction in Xenopus ectoderm (Matsuda et al., 2021).  Of note, LMO7 is distributed in ectoderm as a double line adjacent to the apical junction, mimicking the distribution of nonmuscle Myosin II heavy chain (NMIIA). Thus, LMO7 is a good candidate for regulating Myosin II-dependent morphogenesis. We hypothesize that LMO7 regulates the organization of Myosin II, and the distribution of Wtip. Importantly, we recently found that LMO7 recruits Myosin II heavy chain to the cell cortex, supporting our hypothesis (Matsuda et al., 2021). At the tissue level, our model is that planar polarity and force balance are responsible for neural plate folding in the mediolateral axis but not in the anteroposterior axis.