The thrombospondin repeat containing protein MIG-21 controls a left-right asymmetric Wnt signaling response in migrating C. elegans neuroblasts

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of the C. elegans Q neuroblasts as a model to study how two initially equivalent cells are instructed to activate either β-catenin dependent or independent Wnt signaling pathways to control the migration of their descendants along the anteroposterior axis. We find that the specification of this difference in Wnt signaling response is dependent on the thrombospondin repeat containing protein MIG-21, which acts together with the netrin receptor UNC-40/DCC to control an initial left-right asymmetric polarization of the Q neuroblasts. Furthermore, we show that the direction of this polarization determines the threshold for Wnt/β-catenin signaling, with posterior polarization sensitizing for activation of this pathway. We conclude that MIG-21 and UNC-40 control the asymmetry in Wnt signaling response by restricting posterior polarization to one of the two Q neuroblasts.     

A Wnt signaling system that specifies two patterns of cell migration in C. elegans

In C. elegans, a bilateral pair of neuroblasts, QL and QR, give rise to cells that migrate in opposite directions along the anteroposterior (A/P) body axis. QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. We find that a Wnt family member, EGL-20, acts in a dose-dependent manner to specify these opposite migratory behaviors. High levels of EGL-20 promote posterior migration by activating a canonical Wnt signal transduction pathway, whereas low levels promote anterior migration by activating a separate, undefined pathway. We find that the two Q cells respond differently to EGL-20 because they have different response thresholds. Thus, in this system two distinct dose-dependent responses are specified not by graded levels of the Wnt signal, but instead by left-right asymmetrical differences in the cellular responsiveness to Wnt signaling.     

MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans.

The C. elegans Q neuroblasts and their descendants migrate along the anteroposterior (A/P) body axis to positions that are not associated with any obvious landmarks. We find that a novel protein, MIG-13, is required to position these cells correctly. MIG-13 is a transmembrane protein whose expression is restricted to the anterior and central body regions by Hox gene activity. MIG-13 functions non-cell autonomously within these regions to promote migration toward the anterior: loss of mig-13 activity shifts the Q descendants toward the posterior, whereas increasing the level of MIG-13 shifts them anteriorly in a dose-dependent manner. Our findings suggest that MIG-13 is a component of a global A/P migration system, and that the level of MIG-13 determines where along the body axis these migrating cells stop.     

Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development

Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.     

Establishment of left/right asymmetry in neuroblast migration by UNC-40/DCC, UNC-73/Trio and DPY-19 proteins in C. elegans

The bilateral C. elegans neuroblasts QL and QR are born in the same anterior/posterior (A/P) position, but polarize and migrate left/right asymmetrically: QL migrates toward the posterior and QR migrates toward the anterior. After their migrations, QL but not QR switches on the Hox gene mab-5. We find that the UNC-40/netrin receptor and a novel transmembrane protein DPY-19 are required to orient these cells correctly. In unc-40 or dpy-19 mutants, the Q cells polarize randomly; in fact, an individual Q cell polarizes in multiple directions over time. In addition, either cell can express MAB-5. Both UNC-40 and DPY-19, as well as the Trio/GTPase exchange factor homolog UNC-73, are required for full polarization and migration. Thus, these proteins appear to participate in a signaling system that orients and polarizes these migrating cells in a left/right asymmetrical fashion during development. The C. elegans netrin UNC-6, which guides many cells and axons along the dorsoventral axis, is not involved in Q cell polarization, suggesting that a different netrin-like ligand serves to polarize these cells along the anteroposterior axis.     

Multiple Wnts and frizzled receptors regulate anteriorly directed cell and growth cone migrations in Caenorhabditis elegans

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.     

Multiple Wnts redundantly control polarity orientation in Caenorhabditis elegans epithelial stem cells

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1-V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted--the stem cells are not polarized and divide symmetrically--suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals.     

Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.     

C. elegans POP-1/TCF functions in a canonical Wnt pathway that controls cell migration and in a noncanonical Wnt pathway that controls cell polarity

In Caenorhabditis elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a (beta)-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. I have found that this is the case for of a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. I have used a novel RNA-mediated interference technique to interfere specifically with pop-1 zygotic function and have determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans (beta)-catenin homologs appeared to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimicked the defects in egl-20/Wnt mutants and blocked the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities.     

Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans

Wnt signaling systems play important roles in the generation of cell and tissue polarity during development. We describe a Wnt signaling system that acts in a new way to orient the polarity of an epidermal cell division in C. elegans. In this system, the EGL-20/Wnt signal acts in a permissive fashion to polarize the asymmetric division of a cell called V5. EGL-20 regulates this polarization by counteracting lateral signals from neighboring cells that would otherwise reverse the polarity of the V5 cell division. Our findings indicate that this lateral signaling pathway also involves Wnt pathway components. Overexpression of EGL-20 disrupts both the asymmetry and polarity of lateral epidermal cell divisions all along the anteroposterior (A/P) body axis. Together our findings suggest that multiple, inter-related Wnt signaling systems may act together to polarize asymmetric cell divisions in this tissue.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

Netrins and Wnts Function Redundantly to Regulate Antero-Posterior and Dorso-Ventral Guidance in C. elegans

Guided migrations of cells and developing axons along the dorso-ventral (D/V) and antero-posterior (A/P) body axes govern tissue patterning and neuronal connections. In C. elegans, as in vertebrates, D/V and A/P graded distributions of UNC-6/Netrin and Wnts, respectively, provide instructive polarity information to guide cells and axons migrating along these axes. By means of a comprehensive genetic analysis, we found that simultaneous loss of Wnt and Netrin signaling components reveals previously unknown and unexpected redundant roles for Wnt and Netrin signaling pathways in both D/V and A/P guidance of migrating cells and axons in C. elegans, as well as in processes essential for organ function and viability. Thus, in addition to providing polarity information for migration along the axis of their gradation, Wnts and Netrin are each able to guide migrations orthogonal to the axis of their gradation. Netrin signaling not only functions redundantly with some Wnts, but also counterbalances the effects of others to guide A/P migrations, while the involvement of Wnt signaling in D/V guidance identifies Wnt signaling as one of the long sought mechanisms that functions in parallel to Netrin signaling to promote D/V guidance of cells and axons. These findings provide new avenues for deciphering how A/P and D/V guidance signals are integrated within the cell to establish polarity in multiple biological processes, and implicate broader roles for Netrin and Wnt signaling - roles that are currently masked due to prevalent redundancy.     

Heads or tails: Wnts and anterior-posterior patterning.

Cell-cell communication is critical during embryogenesis for organizing the vertebrate body plan. Members of the Wnt family of secreted signaling molecules possess axis-inducing and posteriorizing activity when overexpressed. Wnt signals are modulated extracellularly by a diverse group of secreted Wnt antagonists and cofactors. Recent work has revealed that inhibition of posteriorly localized Wnt signaling by anteriorly localized Wnt antagonists is critical for inducing the anterior structures, forebrain and heart, from neural ectoderm and mesoderm, respectively. This review centers on the role that Wnts and Wnt antagonists play in the patterning of the vertebrate anterior-posterior axis.     

An expression pattern screen for genes involved in the induction of the posterior nervous system of zebrafish

The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.     

Sequential antagonism of early and late Wnt-signaling by zebrafish colgate promotes dorsal and anterior fates

The establishment of the vertebrate body plan involves patterning of the ectoderm, mesoderm, and endoderm along the dorsoventral and antero-posterior axes. Interactions among numerous signaling molecules from several multigene families, including Wnts, have been implicated in regulating these processes. Here we provide evidence that the zebrafish colgate(b382) (col) mutation results in increased Wnt signaling that leads to defects in dorsal and anterior development. col mutants display early defects in dorsoventral patterning manifested by a decrease in the expression of dorsal shield-specific markers and ectopic expression of ventrolaterally expressed genes during gastrulation. In addition to these early patterning defects, col mutants display a striking regional posteriorization within the neuroectoderm, resulting in a reduction in anterior fates and an expansion of posterior fates within the forebrain and midbrain-hindbrain regions. We are able to correlate these phenotypes to the overactivation of Wnt signaling in col mutants. The early dorsal and anterior patterning phenotypes of the col mutant embryos are selectively rescued by inactivation of Wnt8 function by morpholino translational interference. In contrast, the regionalized neuroectoderm posterioriorization phenotype is selectively rescued by morpholino-mediated inactivation of Wnt8b. These results suggest that col-mediated antagonism of early and late Wnt-signaling activity during gastrulation is normally required sequentially for both early dorsoventral patterning and the specification and patterning of regional fates within the anterior neuroectoderm.     

Neurons Refine the Caenorhabditis elegans Body Plan by Directing Axial Patterning by Wnts

Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior–posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.