Role of PKA as a negative regulator of PCP signaling pathway during Xenopus gastrulation movements

Convergent extension (CE) movements in gastrulation are essential for the establishment of the body axis during early vertebrate development. Although the precise molecular mechanisms of CE movements are not clearly understood, noncanonical Wnt pathway is known to be important for the control of CE movements. Here, we present evidence that PKA is implicated in noncanonical Wnt pathway. Overexpression and specific depletion of PKA inhibit CE movements. PKA depletion also disrupts cell morphology, protrusive activity, and cortical actin formation in dorsal mesodermal cells. Moreover, PKA activity is negatively regulated by major components of planar cell polarity (PCP) pathway. In line with this, overexpression of PKA can rescue the inhibition of CE movements caused by overexpression of these molecules. We also demonstrate that this regulation of PKA activity is dependent upon Galphai signaling. As a negative component of PCP signaling, PKA inhibits not only the activation of RhoA and JNK but also the Dsh-Daam1-RhoA complex formation which is essential for the regulation of RhoA activity. Together, our study suggests a molecular pathway from Wnt/Dsh/PKA signaling to Rho activation in PCP signaling.     

The mitochondrial-apoptotic pathway is triggered in Xenopus mesoderm cells deprived of PDGF receptor signaling during gastrulation

Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation.     

PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis

We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin β1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated α5β1 integrin and cytoskeleton strength during cell movement.     

Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation

We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension.     

Pnas4 is a novel regulator for convergence and extension during vertebrate gastrulation

Recent studies show that human Pnas4 might be tumor associated, while its function remains unknown. Here, we investigate the developmental function of Pnas4 using zebrafish as a model system. Knocking down Pnas4 causes gastrulation defects with a shorter and broader axis, as well as a posteriorly mis-positioned prechordal plate, due to the defective convergence and extension movement. Conversely, over-expression of Pnas4 mRNA leads to an elongated body axis. We further demonstrate that Pnas4 is required cell-autonomously for dorsal convergence but not for anterior migration. In addition, genetic interaction assays indicate that Pnas4 might act in parallel with non-canonical Wnt signal in the regulation of cell movement. Our data suggest that Pnas4 is a key regulator of cell movement during gastrulation.     

An SEM study of cellular morphology,contact, and arrangement,as related to gastrulation inXenopus laevis

Summary Cellular morphology, contact, and arrangement in the late blastula and in various stages of gastrulation ofXenopus were examined by SEM of specimens dissected after fixation or fractured in amyl acetate. The prospective ectoderm of the blastocoel roof consists of several layers of interdigitating cells connected by numerous small protrusions which may function in the decrease in number of cell layers observed during ectodermal epiboly. During gastrulation, prospective mesoderm is regionally differentiated by cellular morphology and arrangement into preinvolution mesoderm, the mesodermal involution zone, and involuted mesoderm. The involuted anterodorsal (head), lateral, and ventral mesoderm consists of a stream of loosely-packed, irregularly shaped cells having large extensions of the cell body attached locally to other cells by small protrusions. Involuted posterodorsal mesoderm (chordamesoderm) consists of elongated cells arranged in palisade fashion and connected by similar protrusions. Involuted mesodermal cells in all regions are attached to the overlying prospective ectodermal cells by numerous small protrusions along the entire interface between the two cell layers. Suprablastoporal endodermal cells involute as an epithelial sheet, changing in shape in the process, to form the roof of the archenteron. Bottle cell morphology, arrangement, and position with respect to the mesodermal cell stream is described. Evidence presented here and elsewhere suggests that involution of mesoderm and of the archenteron roof inXenopus is dependent primarily upon the relative movement of the mesodermal cell stream and of the overlying ectoderm.     

FGF signal regulates gastrulation cell movements and morphology through its target NRH

We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH.     

Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation

Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin protein Daam1 and an effector molecule XProfilin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProfilin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProfilin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1 regulates blastopore closure, overexpression or depletion of XProfilin2 specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1. Together, our studies demonstrate that XProfilin2 and XProfilin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.     

Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos

Wnt signaling plays important roles in development and disease. The X-chromosomal Porcupine homolog gene (Porcn) encodes an evolutionary conserved member of the membrane bound O-acyl transferase (MBOAT) superfamily that has been shown to be required for the palmitoylation and secretion of Wnt3a, a mechanism that has been suggested to be conserved for all mammalian Wnt ligands. PORCN mutations in humans cause Focal Dermal Hypoplasia (FDH), a disorder causing developmental defects in heterozygous females and embryonic lethality in hemizygous males. In this study, Porcn mutant mouse embryonic stem (ES) cells were used to analyze the role of Porcn in mammalian embryonic development. In vitro, we show an exclusive requirement for Porcn in Wnt secreting cells and further, that any of the four Porcn isoforms is sufficient to allow for the secretion of functional Wnt3a. Embryos generated by aggregation of Porcn mutant ES cells with wildtype embryos fail to complete gastrulation in vivo, but remain in an epiblast-like state, similar to Wnt3 and Gpr177/Wls mutants. Consistent with this phenotype, in vitro differentiated mutant ES cells fail to generate endoderm and mesoderm derivatives. Taken together, these data confirm the importance of Porcn for Wnt secretion and gastrulation and suggest that disruption of early development underlies the male lethality of human PORCN mutants.     

Essential role of MARCKS in cortical actin dynamics during gastrulation movements

Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements.     

Xenopus paraxial protocadherin has signaling functions and is involved in tissue separation

Protocadherins have homophilic adhesion properties and mediate selective cell-cell adhesion and cell sorting. Knockdown of paraxial protocadherin (PAPC) function in the Xenopus embryo impairs tissue separation, a process that regulates separation of cells of ectodermal and mesodermal origin during gastrulation. We show that PAPC can modulate the activity of the Rho GTPase and c-jun N-terminal kinase, two regulators of the cytoskeletal architecture and effectors of the planar cell polarity pathway. This novel signaling function of PAPC is essential for the regulation of tissue separation. In addition, PAPC can interact with the Xenopus Frizzled 7 receptor, and both proteins contribute to the development of separation behavior by activating Rho and protein kinase Calpha.     

Recruitment of Cdc42 through the GAP domain of RLIP participates in remodeling of the actin cytoskeleton and is involved in Xenopus gastrulation

The transduction pathways that branch out of fibroblast growth factor signaling are essential for the induction of the mesoderm and the specification of the vertebrate body plan. One of these pathways is thought to control remodeling of the actin cytoskeleton through the Ral binding protein (RLIP also known as RalBP1), an effector of the small G protein Ral. RLIP contains a region of homology with the GTPase-activating protein (GAP) domain involved in the regulation of GTPases of the Rho family. We demonstrate here that the GAP domain of RLIP is responsible for the stability of the actin cytoskeleton in Xenopus laevis embryos. We also demonstrate that the complete N-terminal domain of RLIP containing the μ2 binding domain (μ2BD) and the GAP domain induces disruption of the actin cytoskeleton when targeted to the plasma membrane. Neither domain, however, has any effect on the actin cytoskeleton when individually targeted to the plasma membrane. We also determined that Cdc42-GDP, but neither Rac-GDP nor Rho-GDP, rescues the effect of expression of the membrane-localized Xenopus RLIP on the actin cytoskeleton. We show that the GAP domain of RLIP interacts in vivo with Cdc42-GTP and Cdc42-GDP. Finally, a single mutation (K244A) in the GAP sequence prevented embryos from gastrulating. These results demonstrate that to participate in the control of the actin cytoskeleton, RLIP needs its complete N-terminal region coding for the μ2BD and the GAP domain. We suggest that RLIP, by coordinating two complementary mechanisms, the endocytosis of clathrin-coated pits and the remodeling of cortical actin, participates in the gastrulation process.     

Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation

Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.     

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.     

Ultrastructural changes during early development of retinal ganglion cells in Xenopus

Summary All cells in the optic vesicle of Xenopus embryos from stages 27 to 31 have the same ultrastructure. They are elongated and appear to extend from the internal to the external surfaces of the optic vesicle. They are bound together by terminal bars at the internal (lumen) margin, have microvilli and a cilium on the internal margin, and are covered with a basement membrane on the external margin. Their cytoplasm contains abundant free ribosomes, polysomes, mitochondria, yolk and lipid inclusions, and sparse endoplasmic reticulum.Although other studies have shown that retinal ganglion cells originate at stages 29–30 and have their central connections determined before stage 31, these events could not be correlated with any ultrastructural changes. The first sign of differentiation in retinal cells was an increase in endoplasmic reticulum and Golgi apparatus at stage 32. Microtubules and microfilaments appeared at stage 33 in association with the first axonal outgrowth from retinal ganglion cells. Cytodifferentiation proceeded gradually until large areas of Nissl substance had developed by stage 35. At larval stage 48 the ganglion cells resembled those in the adult.The authors wish to thank Marija Duda for her excellent technical assistance during this investigation.Supported by Public Health Service Predoctoral Fellowship No. 5 FO 1 GM37746-02 and Postdoctoral Fellowship 1 F2 NB37,746-01.Supported by Grant GB8315 from the National Science Foundation.     

The polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation with 14-3-3 and aPKC

Par (partitioning-defective) genes were originally identified in Caenorhabditis elegans as determinants of anterior/posterior polarity. However, neither their function in vertebrate development nor their action mechanism has been fully addressed. Here we show that two members of Par proteins, 14-3-3 (Par-5) and atypical PKC (aPKC), regulate the serine/threonine kinase Par-1 to control Xenopus gastrulation. We find first that Xenopus Par-1 (xPar-1) is essential for gastrulation but not for cell fate specification during early embryonic development. We then find that xPar-1 binds to 14-3-3 in an aPKC-dependent manner. Our analyses identify two aPKC phosphorylation sites in xPar-1, which are essential for 14-3-3 binding and for proper gastrulation movements. The aPKC phosphorylation-dependent binding of xPar-1 to 14-3-3 does not markedly affect the kinase activity of xPar-1, but induces relocation of xPar-1 from the plasma membranes to the cytoplasm. Finally, we show that Xenopus aPKC and its binding partner Xenopus Par-6 are also essential for gastrulation. Thus, our results identify a requirement of Par proteins for Xenopus gastrulation and reveal a novel interrelationship within Par proteins that may provide a general mechanism for spatial control of Par-1.