Olga Ossipova

LKB1 (XEEK1) regulates Wnt signalling in vertebrate development

Germline LKB1/STK11 mutations are associated with the cancer-prone Peutz–Jeghers syndrome (PJS) in humans, and nullizygosity provokes a poorly understood constellation of developmental perturbations in the mid-gestational mouse. To gain a better understanding of the processes regulated by LKB1, we have exploited the experimental merits of the developing Xenopus embryo. Here, specific inhibition of XEEK1, the Xenopus orthologue of LKB1, engendered developmental anomalies — shortened body axis and defective dorsoanterior patterning — associated previously with aberrant Wnt signalling. In line with this, LKB1/XEEK1 cooperates with the Wnt–β-catenin signalling in axis induction and modulates the expression of Wnt-responsive genes in both Xenopus embryos and mammalian cells. We establish that LKB1/XEEK1 acts upstream of β-catenin in the Wnt–β-catenin pathway in vivo. LKB1/XEEK1 regulates glycogen synthase kinase (GSK)3β phosphorylation and it is physically associated in vivo with GSK3β and protein kinase C (PKC)-ζ, a known GSK3 kinase. These studies show that LKB1/XEEK1 is required for Wnt–β-catenin signalling in frogs and mammals and provides novel insights into its role in vertebrate developmental patterning and carcinogenesis.

PAR-1 Phosphorylates Mind Bomb to Promote Vertebrate Neurogenesis

Generation of neurons in the vertebrate central nervous system requires a complex transcriptional regulatory network and signaling processes in polarized neuroepithelial progenitor cells. Here we demonstrate that neurogenesis in the Xenopus neural plate in vivo and mammalian neural progenitors in vitro involves intrinsic antagonistic activities of the polarity proteins PAR-1 and aPKC. Furthermore, we show that Mind bomb (Mib), a ubiquitin ligase that promotes Notch ligand trafficking and activity, is a crucial molecular substrate for PAR-1. The phosphorylation of Mib by PAR-1 results in Mib degradation, repression of Notch signaling, and stimulation of neuronal differentiation. These observations suggest a conserved mechanism for neuronal fate determination that might operate during asymmetric divisions of polarized neural progenitor cells.

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.

Cell polarity, Notch signaling and neurogenesis

Comment on: Ossipova O, et al. Dev Cell 2009; 17:222-33.

Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling

The vertebrate neural tube forms as a result of complex morphogenetic movements, which require the functions of several core planar cell polarity (PCP) proteins, including Vangl2 and Prickle. Despite the importance of these proteins for neurulation, their subcellular localization and the mode of action have remained largely unknown. Here we describe the anteroposterior planar cell polarity (AP-PCP) of the cells in the Xenopus neural plate. At the neural midline, the Vangl2 protein is enriched at anterior cell edges and that this localization is directed by Prickle, a Vangl2-interacting protein. Our further analysis is consistent with the model, in which Vangl2 AP-PCP is established in the neural plate as a consequence of Wnt-dependent phosphorylation. Additionally, we uncover feedback regulation of Vangl2 polarity by Myosin II, reiterating a role for mechanical forces in PCP. These observations indicate that both Wnt signaling and Myosin II activity regulate cell polarity and cell behaviors during vertebrate neurulation.

Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signaling, was necessary for neural plate folding, and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking plays key roles in diverse morphogenetic behaviors of individual cells and propagates in a tissue by a common mechanism that involves PCP.

Neural crest specification by noncanonical Wnt signaling and PAR-1

Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Our gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate.

Metastasis-associated kinase modulates Wnt signaling to regulate brain patterning and morphogenesis

Wnt signaling is a major pathway regulating cell fate determination, cell proliferation and cell movements in vertebrate embryos. Distinct branches of this pathway activate β-catenin/TCF target genes and modulate morphogenetic movements in embryonic tissues by reorganizing the cytoskeleton. The selection of different molecular targets in the pathway is driven by multiple phosphorylation events. Here, we report that metastasis-associated kinase (MAK) is a novel regulator of Wnt signaling during morphogenetic movements, and eye and brain development in Xenopus embryos. Injected MAK RNA suppressed Wnt transcriptional reporters and activated Jun N-terminal kinase. Furthermore, MAK was recruited to the cell membrane by Frizzled 3, formed a complex with Dishevelled and phosphorylated Dsh in vitro. The regional brain markers Otx2, En2 and Gbx2 were affected in embryos with modulated MAK activity in a manner consistent with a role for MAK in midbrain-hindbrain boundary formation. Confirming the inhibitory role for this kinase in Wnt/β-catenin signaling, the midbrain patterning defects in embryos depleted of MAK were rescued by the simultaneous depletion ofβ -catenin. These findings indicate that MAK may function in different developmental processes as a switch between the canonical and non-canonical branches of Wnt signaling.

Lulu regulates Shroom-induced apical constriction during neural tube closure.

Apical constriction is an essential cell behavior during neural tube closure, but its underlying mechanisms are not fully understood. Lulu, or EPB4.1l5, is a FERM domain protein that has been implicated in apical constriction and actomyosin contractility in mouse embryos and cultured cells. Interference with the function of Lulu in Xenopus embryos by a specific antisense morpholino oligonucleotide or a carboxy-terminal fragment of Lulu impaired apical constriction during neural plate hinge formation. This effect was likely due to lack of actomyosin contractility in superficial neuroectodermal cells. By contrast, overexpression of Lulu RNA in embryonic ectoderm cells triggered ectopic apico-basal elongation and apical constriction, accompanied by the apical recruitment of F-actin. Depletion of endogenous Lulu disrupted the localization and activity of Shroom3, a PDZ-containing actin-binding protein that has also been implicated in apical constriction. Furthermore, Lulu and Shroom3 RNAs cooperated in triggering ectopic apical constriction in embryonic ectoderm. Our findings reveal that Lulu is essential for Shroom3-dependent apical constriction during vertebrate neural tube closure.

The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development.

The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors.