The planar polarity gene strabismus regulates convergent extension movements in Xenopus

The signaling mechanisms that specify, guide and coordinate cell behavior during embryonic morphogenesis are poorly understood. We report that a Xenopus homolog of the Drosophila planar cell polarity gene strabismus (stbm) participates in the regulation of convergent extension, a critical morphogenetic process required for the elongation of dorsal structures in vertebrate embryos. Overexpression of Xstbm, which is expressed broadly in early development and subsequently in the nervous system, causes severely shortened trunk structures; a similar phenotype results from inhibiting Xstbm translation using a morpholino antisense oligo. Experiments with Keller explants further demonstrate that Xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue. The specification of dorsal tissues is not affected. The Xstbm phenotype resembles those obtained with several other molecules with roles in planar polarity signaling, including Dishevelled and Frizzled-7 and -8. Unlike these proteins, however, Stbm has little effect on conventional Wnt/beta-catenin signaling in either frog or fly assays. Thus our results strongly support the emerging hypothesis that a vertebrate analog of the planar polarity pathway governs convergent extension movements.     

Xenopus fibrillin regulates directed convergence and extension

Fibrillin-based human diseases such as Marfan syndrome and congenital contractural arachnodactyly implicate fibrillins in the function and homeostasis of multiple adult tissues. Fibrillins are also expressed in embryos, but no early developmental role has been described for these proteins. We use three independent methods to reveal a role for Xenopus fibrillin (XF) at gastrulation. First, expressing truncated forms of XF in the embryo leads to failure of gastrulation concomitant with a dominant-negative effect on native fibrillin fibril assembly. Expressing truncated XF also inhibits normal progression of the patterned, polarized cell motility that drives convergence and extension at gastrulation and perturbs directed extension in cultured explants of dorsal mesoderm. Second, injection of a synthetic peptide encoding a cell-binding domain of XF into midgastrula embryos causes acute failure of gastrulation associated with defective fibrillin fibril assembly. These injections also reveal a critical role for this peptide in the fibril assembly process. Third, morpholino-mediated knockdown of translation of XF in the embryo also perturbs normal gastrulation and directed extension. Together, these data show that native Xenopus fibrillin is essential for the process of directed convergent extension in presumptive notochord at gastrulation.     

Calpain2 protease: A new member of the Wnt/Ca pathway modulating convergent extension movements in Xenopus

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca²⁺ pathway and Dishevelled can modulate convergent extension movements.     

Xenopus p21-activated kinase 5 regulates blastomeres' adhesive properties during convergent extension movements

The p21-activated kinase (PAK) proteins regulate many cellular events including cell cycle progression, cell death and survival, and cytoskeleton rearrangements. We previously identified X-PAK5 that binds the actin and microtubule networks, and could potentially regulate their coordinated dynamics during cell motility. In this study, we investigated the functional importance of this kinase during gastrulation in Xenopus. X-PAK5 is mainly expressed in regions of the embryo that undergo extensive cell movements during gastrula such as the animal hemisphere and the marginal zone. Expression of a kinase-dead mutant inhibits convergent extension movements in whole embryos and in activin-treated animal cap by modifying behavior of cells. This phenotype is rescued in embryo by adding back X-PAK5 catalytic activity. The active kinase decreases cell adhesiveness when expressed in animal hemisphere and inhibits the calcium-dependent reassociation of cells, while dead X-PAK5 kinase localizes to cell-cell junctions and increases cell adhesion. In addition, endogenous X-PAK5 colocalizes with adherens junction proteins and its activity is regulated by extracellular calcium. Taken together, our results suggest that X-PAK5 regulates convergent extension movements in vivo by modulating the calcium-mediated cell-cell adhesion.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

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.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis

During amphibian development, non-canonical Wnt signals regulate the polarity of intercalating dorsal mesoderm cells during convergent extension. Cells of the overlying posterior neural ectoderm engage in similar morphogenetic cell movements. Important differences have been discerned in the cell behaviors associated with neural and mesodermal cell intercalation, raising the possibility that different mechanisms may control intercalations in these two tissues. In this report, targeted expression of mutants of Xenopus Dishevelled (Xdsh) to neural or mesodermal tissues elicited different defects that were consistent with inhibition of either neural or mesodermal convergent extension. Expression of mutant Xdsh also inhibited elongation of neural tissues in vitro in Keller sandwich explants and in vivo in neural plate grafts. Targeted expression of other Wnt signaling antagonists also inhibited neural convergent extension in whole embryos. In situ hybridization indicated that these defects were not due to changes in cell fate. Examination of embryonic phenotypes after inhibition of convergent extension in different tissues reveals a primary role for mesodermal convergent extension in axial elongation, and a role for neural convergent extension as an equalizing force to produce a straight axis. This study demonstrates that non-canonical Wnt signaling is a common mechanism controlling convergent extension in two very different tissues in the Xenopus embryo and may reflect a general conservation of control mechanisms in vertebrate convergent extension.     

Planar cell polarity genes and neural tube closure

Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of closure results in neural tube defects (NTDs), common and clinically severe congenital malformations whose molecular mechanisms remain poorly understood. On the other hand, it is increasingly well established that common molecular mechanisms are employed to regulate morphogenesis of multicellular organisms. For example, signaling triggered by polypeptide growth factors is highly conserved among species and utilized in multiple developmental processes. Recent studies have revealed that the Drosophila planar cell polarity (PCP) pathway, which directs position and direction of wing hairs on the surface of the fly wing, is well conserved, and orthologs of several genes encoding components of the pathway are also found in vertebrates. Interestingly, in vertebrates, this signaling pathway appears to be co-opted to regulate "convergent extension" cell movements during gastrulation. Disruption of vertebrate PCP genes in Xenopus laevis or zebrafish causes severe gastrulation defects or the shortening of the trunk, as well as mediolateral expansion of somites. In Xenopus, in which the neural tube closes by elevation and fusion of neural folds, inhibition of convergent extension can also prevent neural tube closure causing a "spina bifida-like" appearance. Furthermore, several of the genes involved in the PCP pathway have recently been shown to be required for neural tube closure in the mouse, since mutation of these genes causes NTDs. Therefore, understanding the mechanisms underlying the establishment of cell polarity in Drosophila may provide important clues to the molecular basis of NTDs.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus

Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. We have identified a second Xenopus Wnt11-Related gene (Wnt11-R) that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube.     

Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm

It is known the interactions between the neural plate and epidermis generate neural crest (NC), but it is unknown why the NC develops only at the lateral border of the neural plate and not in the anterior fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC from the anterior region. We identify prechordal mesoderm as the tissue that inhibits NC in the anterior territory and show that the Wnt/beta-catenin antagonist Dkk1, secreted by this tissue, is sufficient to mimic this NC inhibition. We show that Dkk1 is required for preventing the formation of NC in the anterior neural folds as loss-of-function experiments using a Dkk1 blocking antibody in Xenopus as well as the analysis of Dkk1-null mouse embryos transform the anterior neural fold into NC. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior patterning of the neural plate, or placodal specification. Finally, we show that the NC cells induced at the anterior neural fold are able to migrate and differentiate as normal NC. These results demonstrate that anterior regions of the embryo lack NC because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1.     

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.     

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.     

Formation, architecture and polarity of female germline cyst in Xenopus

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis. We found that the germline cyst in Xenopus contains 16 cells that are similar in general architecture and molecular composition to the cyst in Drosophila. Nest cells are connected by cytoplasmic bridges that contain ring canal-like structures. The nest cells contain a structure similar to the Drosophila fusome that that is probably involved in anchoring of the centrioles and organization of the primary mitochondrial cloud (PMC) around the centriole. We also find that in contrast to other organisms, in Xenopus, apoptosis is a rare event within the developing ovary. Our studies indicate that the processes responsible for the formation of female germline cysts and the establishment of germ cell polarity are highly conserved between invertebrates and vertebrates. The dissimilarities between Drosophila and Xenopus and the uniqueness of each system probably evolved through modifications of the same fundamental design of the germline cyst.     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.     

Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.