InteractiveFly: GeneBrief

wnt inhibitor of Dorsal: Biological Overview | Regulation | Developmental Biology | Effects of Overexpression and Deletion | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The maternal Toll signaling pathway sets up a nuclear gradient of the transcription factor Dorsal in the early Drosophila embryo. Dorsal activates twist and snail, and the Dorsal/Twist/Snail network activates and represses other zygotic genes to form the correct expression patterns along the dorsoventral axis. An essential function of this patterning is to promote ventral cell invagination during mesoderm formation, but how the downstream genes regulate ventral invagination is not yet known. wntD (FlyBase name: Wnt8) is shown to be a member of the Wnt family. The expression of wntD is activated by Dorsal and Twist, but the expression is much reduced in the ventral cells through repression by Snail. Overexpression of WntD in the early embryo inhibits ventral invagination, suggesting that the de-repressed WntD in snail mutant embryos may contribute to inhibiting ventral invagination. The overexpressed WntD inhibits invagination by antagonizing Dorsal nuclear localization, as well as twist and snail expression. Consistent with the early expression of WntD at the poles in wild-type embryos, loss of WntD leads to posterior expansion of nuclear Dorsal and snail expression, demonstrating that physiological levels of WntD can also attenuate Dorsal nuclear localization. The de-repressed WntD in snail mutant embryos contributes to the premature loss of snail expression, probably by inhibiting Dorsal. Thus, these results together demonstrate that WntD is regulated by the Dorsal/Twist/Snail network, and is an inhibitor of Dorsal nuclear localization and function. The closest homologs of Drosophila WntD, vertebrate Wnt8 proteins, regulate mesoderm patterning, neural crest cell induction, neuroectoderm patterning, and axis formation (Hoppler, 1998; Lekven, 2001; Lewis, 2004; Popperl, 1997). These vertebrate Wnt8 proteins may transmit the signal through the canonical pathway, but the exact mechanism remains unclear. So far, the downstream mediators of Drosophila WntD signaling are not known (Ganguly, 2005).

A second study (Gordon, 2005) confirms and extends Ganguly (2005) by inducing a mutation in wntD by homologous replacement. The Gordon study shows that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal, during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo. By engineering a gene knockout, this study shows that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal (Gordon, 2005).

Mesoderm is the middle germ layer formed during gastrulation. In Drosophila, the mesoderm arises from the invagination of the ventral cells of the blastoderm. The mesoderm provides the precursor cells for muscles, hemocytes, lymph glands, the somatic gonad and the heart. The maternal Toll signaling pathway has a crucial role in establishing the ventral cell fate and thus mesoderm formation (Ganguly, 2005).

Toll is a single-pass transmembrane receptor and is activated by a series of upstream serine proteases that process the ligand Spätzle. The activated Toll recruits the cytoplasmic components MyD88, Tube and Pelle to regulate the nuclear transport of the transcription factor Dorsal. Dorsal, an NF-kappaB homolog, is normally retained in the cytoplasm by Cactus, an IkappaB homolog. Toll signaling causes the phosphorylation and degradation of Cactus, thereby allowing Dorsal to enter the nucleus and regulate gene expression. These signaling components are ubiquitously distributed, but the pathway is activated only in the ventral side of the embryo. Thus, activation of Toll by the diffusible Spätzle leads to the formation of a nuclear gradient of Dorsal, with the highest concentration in ventral nuclei (Ganguly, 2005 and references therein).

A single gradient of nuclear Dorsal can generate multiple patterns of zygotic gene expression along the dorsoventral axis. Dorsal acts as both a transcriptional repressor and activator. For example, zerknüllt and decapentaplegic are repressed by Dorsal and therefore can be expressed only in the dorsal side of the embryo where the dorsal ectoderm is formed. Meanwhile, Dorsal activates other zygotic genes, such as twist, snail, rhomboid, short gastrulation, lethal of scute and single-minded (sim). Depending on the affinity of the Dorsal-binding sites and on the presence of co-activator sites on their promoters, these target genes are activated by different thresholds of the Dorsal gradient, and thus have ventral expression with variable lateral limits (Ganguly, 2005 and references therein).

High levels of nuclear Dorsal activate the expression of twist and snail, and the Dorsal/Twist/Snail network regulates ventral cell invagination to form the mesoderm. In dorsal, twist or snail mutants, no ventral invagination occurs and no mesodermal tissues are formed. Twist is a basic helix-loop-helix transcription factor and acts as a co-activator for Dorsal to optimally activate other zygotic target genes, including snail. Snail contains five zinc fingers and functions as a transcriptional repressor. A model for this gene regulatory network in promoting mesoderm formation is that Dorsal/Twist activates multiple zygotic genes that are expressed in the ventral region with different lateral limits. These target genes may promote the ventral (mesodermal) cell fate or the lateral (neuroectodermal) cell fate. Snail specifically represses those genes that are not compatible with mesoderm formation. Consistent with this model, many genes, including rhomboid, sim, lethal of scute, short gastrulation, crumbs, Delta and Enhancer of split, are repressed by Snail in the ventral region and their expression is, therefore, restricted to the lateral regions. In snail mutant embryos, these genes are de-repressed into the ventral region. However, it has not been demonstrated that any of these Snail target genes can directly inhibit ventral invagination and mesoderm formation (Ganguly, 2005 and references therein).

To identify novel components in the dorsoventral pathway, a microarray assay was carried out using embryos derived from gain-of-function and loss-of-function mutants of the Toll pathway. Among the novel genes identified, the expression and function of wntD was analyzed because the Wnt family of secreted proteins regulates patterning, cell polarity and cell movements. The results show that wntD is activated by Dorsal and Twist but repressed by Snail. Increased expression of WntD in wild-type early embryos inhibits ventral invagination. Thus, wntD is the first Snail target gene shown to have an interfering function in mesoderm invagination. The overexpressed WntD blocks invagination by inhibiting Dorsal nuclear localization. Loss-of-function analyses also show that physiological levels of WntD can attenuate Dorsal nuclear localization and function. Therefore, wntD is a novel downstream gene of the Dorsal/Twist/Snail network and can feed back to inhibit Dorsal (Ganguly, 2005).

The dynamic pattern of wntD expression in the early embryo is a combined result of activation by Dorsal/Twist and repression by Snail. Overexpressed WntD negatively regulates Dorsal nuclear localization, leading to an inhibition of ventral cell invagination. Physiological levels of WntD can also negatively regulate Dorsal, since loss of WntD leads to detectable expansion of both Dorsal nuclear localization and snail expression in the posterior regions. Furthermore, de-repressed WntD expression in the ventral region of snail mutant embryos can also attenuate Dorsal function. However, the loss of WntD could not rescue the invagination defect of the snail mutant embryo, suggesting that in the snail mutant embryo there are other de-repressed genes that can interfere with ventral invagination (Ganguly, 2005).

The wntD loss-of-function phenotype correlates with the expression of wntD at the poles of pre-cellular blastoderms. wntD is also expressed a bit later in the mesectoderm, and weakly in the mesoderm. Because WntD can inhibit Dorsal, one speculation is that WntD in the early mesectoderm may help to establish the sharp snail expression at the mesectoderm-neuroectoderm boundary. However, no changes were detected in the Dorsal protein gradient or snail pattern in the trunk regions of the Df(3R)l26c embryos. It is speculated that the timing of early expression of wntD, which may have additional input from the Torso pathway at the poles, is important for the feedback inhibition of Dorsal. By the time of cellularization, the Dorsal protein gradient is well established. This well-established Dorsal gradient activates the wntD gene in the trunk regions, but the subsequently translated WntD protein may not be capable of exerting a strong negative-feedback effect on the already formed Dorsal gradient. This timing argument is supported by the results of WntD-overexpression experiments. The use of maternal nanos-Gal4 caused a strong inhibition of Dorsal nuclear localization and of ventral invagination, whereas the use of zygotic promoters did not result in a significant phenotype (Ganguly, 2005).

Snail acts as a transcriptional repressor for at least 10 genes in the ventral region where mesoderm arises. In snail mutant embryos, all of these target genes are de-repressed in the ventral cells, concomitant with severe ventral invagination defects. However, no direct evidence has been reported on whether these de-repressed genes interfere with invagination. This study showed for the first time that a target gene of Snail, namely wntD, can block ventral invagination when overexpressed. If de-repressed WntD is solely responsible for inhibiting ventral invagination, it would be expected that, in the snail;Df(3R)l26c double-mutant embryos, ventral invagination would appear again. No rescue of ventral invagination was detected in the double-mutant embryos, suggesting that wntD is not the only de-repressed target gene that inhibits invagination. Nonetheless, the de-repressed WntD can attenuate Dorsal function, and may contribute to the ventral invagination defect (Ganguly, 2005).

Previous reports have shown that overexpression of String/Cdc25 leads to early mitosis in the ventral cells and a block in ventral invagination. The zygotic transcription of string in the ventral region is activated by the Dorsal/Twist/Snail network. Meanwhile, the String protein is kept at a low level in the ventral cells by Tribbles through protein degradation, and this process requires the positive input of Snail. Therefore, in the snail;Df(3R)l26c double-mutant embryos, the ventral cells should have increased String protein, as well as many other de-repressed gene products. Perhaps the cumulative effect contributed by many of these snail target genes causes the severe invagination defect observed in the snail mutant embryo; the simultaneous deletion of wntD and other interfering genes may be required to suppress the ventral invagination phenotype in snail mutants (Ganguly, 2005).

WntD may inhibit a component in the Toll pathway, or a component in the nuclear import/export pathway, leading to the cytoplasmic localization of Dorsal. However, the downstream mediators of Drosophila WntD signaling are not known. Being the closest homologs of Drosophila WntD, vertebrate Wnt8 proteins regulate mesoderm patterning, neural crest cell induction, neuroectoderm patterning, and axis formation (Hoppler, 1998; Lekven, 2001; Lewis, 2004; Popperl, 1997). These vertebrate Wnt8 proteins may transmit the signal through the canonical pathway, but the exact mechanism remains unclear (Lekven, 2001; Lewis, 2004; Momoi, 2003). Drosophila embryos were examined that lacked maternal and zygotic functions of both Frizzled 1 and Frizzled 2 but no obvious defects were observed in Dorsal or snail expression. A similar experiment using a dishevelled null mutant also did not reveal any such defects. Furthermore, overexpression of Dishevelled or dominant-negative Gsk3 did not cause a detectable change in dorsoventral patterning. These results suggest that Drosophila WntD may use other components for signaling. Wnt molecules employ multiple receptors and pathways to regulate various processes. For example, Drosophila Wnt5 interacts with the receptor tyrosine kinase Derailed to regulate axon guidance. There are seven Wnt proteins and five Frizzled receptors in Drosophila, and WntD showed detectable affinity towards Frizzled 4 in cell culture assays (Wu, 2002), but the in vivo relevance of this interaction is not clear. It is important to elucidate how Drosophila WntD transmits its signal. Equally important is to find out whether WntD interacts with the Toll pathway, and whether the interaction also occurs in processes such as the immune response and cancer progression in other organisms (Ganguly, 2005).

Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes gastrulation patterning

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, was followed in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. It is concluded that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos (Rahimi, 2019).

The early Drosophila embryo provides extreme challenges for the generation and maintenance of extracellular morphogen gradients. Most notably, the peri-vitelline fluid surrounding the embryo facilitates rapid diffusion of molecules. In addition, the alteration in the surface of the plasma membrane at every nuclear division provides an active mixing force. Thus, analysis of the early morphogen gradients operating in this environment, including ventral Spz/Toll activation and the subsequent BMP gradient patterning the dorsal aspect, should consider this highly dynamic environment. In the case of the Toll pathway, the active Spz ligand is generated by proteolytic processing within the extra-embryonic peri-vitelline fluid in a broad ventral region, defined by the activation domain of the Easter (Ea) protease. The generation of a sharp Spz activation gradient within this broad ventral domain of processing takes place by diffusion-based shuttling. Previous work demonstrated that the pro-domain of Spz plays an instructive role in delivering the active, cleaved ligand towards the ventral midline. While a variety of experiments and computational analyses indicated the utilization of a 'self-organized shuttling' mechanism in this context, it was imperative to visualize the actual dynamics of the process (Rahimi, 2019).

It was possible to infer the dynamics of the extracellular Spz gradient by following the kinetics of Dl-GFP nuclear accumulation in individual live embryos during the final syncytial nuclear division cycles and the early phase of NC 14. Nuclear levels of Dl are not a direct readout of the extracellular gradient, since accumulation of Dl in the nuclei is re-initiated at the onset of every nuclear cycle. Nevertheless, it is possible to infer key features of the extracellular Spz gradient from this dynamic behavior. Using this approach hallmarks of ligand shuttling were identified, most notably the lateral overshoot and the presence of two lateral peaks which converge to a central ventral peak. This convergence takes place within a timeframe of minutes, and repeats at every nuclear cycle. Since new protein molecules of the extracellular components are continually translated, the ongoing activity of the shuttling process is vital. Therefore, shuttling is important not only for generating the gradient, but also for maintaining it, in the face of rapid diffusion and mixing within the peri-vitelline fluid. Importantly, by ~10-15 minutes into NC 14, when the robust induction of transcription of the cardinal zygotic Dl-target genes twt and sna ensues, the nuclear gradient of Dl is sharp and a single ventral peak is resolved (Rahimi, 2019).

Having described the dynamics of Dl-nuclear entry and gradient formation, an experimental approach was used in order to examine regulatory processes affecting Toll signaling. The Wnt family ligand WntD provides an essential buffering system to variations in Toll signaling between embryos. wntD is an early zygotic gene that is expressed initially at the posterior-ventral region of the embryo, and its expression levels depend on the magnitude of Toll signaling. Although WntD is produced locally, the rapid secretion and diffusion of the protein in the peri-vitelline space generates a uniform attenuation of Toll signaling throughout the embryo surface. The activity of WntD leads in different embryos to convergence of the variable global Toll activation gradient to a similar pattern, which is dictated by the fixed final signaling level that shuts off wntD expression. This paradigm, termed 'distal pinning', is achieved in this case by an induction-contraction mechanism (Rahimi, 2019).

Secreted WntD is recruited to the plasma membrane by binding to its receptor Fz4. Epistasis assays have indicated that WntD exerts its inhibitory effect on Toll signaling by associating with the extracellular domain of Toll, thereby reducing the number of Toll receptors that are available for binding Spz. Bearing the cardinal features of shuttling in mind, this mode of inhibition implies that the effect of WntD would be global and non-autonomous, and will actually change the shape of the gradient, making it sharper. The observed dynamics of Dl-GPF in wntD mutant embryos indeed confirms this prediction. The shuttling process is driven by competition between the inhibitory Spz pro-domain and the Toll receptor for binding free, active Spz. Binding to the pro-domain is favored in the lateral part of the embryo, where its concentration is higher, while in more ventral regions binding to Toll takes over. Since WntD impinges on the extracellular properties of the Toll receptor, the active ligand is deposited in more ventral regions, where the concentration of the pro-domain is lower. Thus, WntD does not simply reduce the overall profile of Toll activation, but actually re-directs the ligand from the lateral regions to the ventral domain. Previous work has shown that accumulation of excess ligand in the peak by shuttling is an effective mechanism to buffer noise. Since activation in this region is already maximal, the excess ligand will not alter the resulting cell fates (Rahimi, 2019).

The rapid timing of processes in the early embryo and the short duration of interphases between nuclear divisions raises the question of whether it is actually possible to produce sufficient levels of WntD that will drive the morphogen profile to the desired equilibrium. When monitoring wntD transcription directly utilizing the MS2 system, most, if not all embryos were shown to express wntD indicating that Toll signaling overshoots in most embryos. Furthermore, within single embryos the number of nuclei expressing wntD was reduced between NCs 12 and 13, and completely terminated by NC 14, implying that WntD impinges on the Toll gradient and its own expression by this time. The intronless arrangement of the wntD gene and the rapid secretion of the protein, which does not require post-translational modifications (Herr et al., 2012), may facilitate the process (Rahimi, 2019).

The ventral cohort of zygotic target genes including twi and sna is induced by the Toll activation gradient, and the threshold for their induction corresponds to ~50% of maximal Dl-nuclear localization. Within the ventral domain, nuclei exhibit a similar level of sna transcription. These genes are triggered at NC 14 after the Dl gradient is stabilized and a distinct activation peak generated (Rahimi, 2019).

Are there zygotic target genes that respond to the dynamics of Dl nuclear targeting, before it stabilizes? This appears to be the case for T48, which encodes a transmembrane protein that facilitates recruitment of RhoGEF2 and ultimately Rho and actomyosin, to mediate apical constriction of invaginating mesodermal cells. Graded distribution of myosin II was shown to be critical for proper invagination of these cells to form the ventral furrow (Rahimi, 2019).

This study provides evidence that the graded distribution of T48 mRNA results from the dynamics of Dl-nuclear re-entry at NC 14. The ventral-most cells reach the threshold of T48 induction earlier than more lateral cells, and hence will express the gene longer. Integration of the length of expression along the D-V axis then leads to a gradient of cytoplasmic T48 mRNA accumulation. This example represents a unique case, where graded morphogen activation instructs the generation of a gradient of target-gene expression. The strict dependence on the timing of transcription initiation provides another mechanism to generate differences between adjacent nuclei along the D-V axis (Rahimi, 2019).

In conclusion, this work has utilized live imaging of Toll pathway activation, to identify and characterize the hallmarks of ligand shuttling. This process is rapid and takes place continuously throughout the final nuclear division cycles, to generate and maintain a sharpactivation gradient in the diffusible environment of the peri-vitelline fluid. WntD impinges on Spz shuttling, and is responsible not only for buffering variability between embryos, but also for generating a sharp activation peak. This peak is utilized to induce a graded expression of a zygotic target gene that is essential for executing processes that drive gastrulation. Thus, diffusion-based ligand shuttling, coupled with a dynamic readout, establishes a refined pattern within the environment of early embryos (Rahimi, 2019).

REGULATION

Transcriptional Regulation

To understand the regulation of wntD, its expression was examined in various genetic mutants. No signal was observed in embryos derived from dorsal^–/– mothers, demonstrating that the expression in both the trunk and the poles is absolutely dependent on Dorsal. In embryos derived from Toll^10b mothers, the expression of wntD is expanded into the dorsal side but the overall staining is not stronger than wild type, probably as a result of both activation by Dorsal and repression by Snail. In conclusion, the mRNA staining in dorsal^–/– and Toll^10b embryos corroborates the results of microarray analysis, i. e., lower expression in the dorsal^–/– and increased expression in the Toll^10b embryos (Ganguly, 2005).

In snail homozygous mutant embryos, a higher level of wntD expression was present throughout the ventral region but mesectodermal expression was not obvious. In some heterozygous embryos there was normal mesectodermal staining but higher ventral expression of wntD. Gene expression in the mesectoderm is regulated by a complex interaction between the Notch pathway and Snail, such that the mesectodermal expression of sim also requires the positive input of Snail. The mesectodermal expression of wntD in both wild-type and snail mutant embryos is similar to that of sim, suggesting that wntD and sim are regulated by a similar mechanism. More importantly, the results demonstrate that Snail also represses wntD expression in the ventral cells (Ganguly, 2005).

In twist mutant embryos, wntD shows a narrower version of the wild-type pattern, centered on the ventral midline. The Snail pattern is significantly reduced in twist mutant embryos. Therefore, the narrower Wnt8 pattern in twist mutants can be explained by the reduced expression of the repressor Snail. In twist snail double-mutant embryos, the expression of wntD was weak and only present in the ventral-most cells. It is speculated that high levels of Dorsal are sufficient to activate this weak expression of wntD in the ventral nuclei. However, the overall ventral staining of wntD in the double mutant is much weaker than that in snail mutants, suggesting that wntD is weakly activated by Dorsal and strongly activated by Dorsal/Twist cooperation, as has been shown for other target genes of the dorsoventral pathway. A stronger activation by the Dorsal/Twist combination may also explain the detectable expression of wntD in the ventral cells of wild-type embryos despite the repression by Snail. Within 1.6 kb of the 5' flanking sequence of wntD, there are seven sites that are similar to the Snail-binding consensus and five sites that are similar to Dorsal-binding consensus. However, the demonstration of whether wntD is a direct target requires further evidence (Ganguly, 2005).

wntD expression in the neuroectoderm depends on Delta. In zygotic Delta mutant embryos, the early wntD pattern is largely unaffected but the late pattern during germ-band extension is reduced and subsequently lost. Early embryos contain a significant maternal load of Delta gene products. As a result, the expression of target genes such as sim remains unaffected until later stages. The regulation of wntD by Delta in the neuroectoderm may depend on a similar mechanism (Ganguly, 2005).

Lipid-independent secretion of a Drosophila WntD protein

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study it was found that WntD (also known as Wnt8), a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, a novel labeling method and mass spectrometry were used to ask whether WntD undergoes lipid modification, and it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. The results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins (Ching, 2008).

WntD is secreted at extremely high levels in cell culture, and its secretion and function are independent of Porcupine and Wntless in vivo. WntD secretion does, however, maintain a requirement for Rab1, an early component of the secretory pathway that regulates the transport of vesicles from the ER to the cis-Golgi compartment. While Porcupine is involved in lipid modification in the ER, Wntless is thought to escort proteins such as Wingless between the trans-Golgi network and the plasma membrane. These results suggest that WntD might be sorted to an alternative secretory route, possibly at the level of the trans-Golgi network, a major sorting site for intracellular trafficking in the secretory pathway (Ching, 2008).

These observations raise interesting questions about the role of post-translational lipid modification in regulating Wnt secretion. It has been suggested that lipid attachments may function to target modified Wnt proteins such as Wingless to specific intracellular membrane subdomains that direct the trafficking of the protein through the secretory pathway along a particular route that could, for example, lead to packaging of the protein into secretory vesicles after exit from the trans-Golgi network. Perhaps WntD is targeted to a different membrane subdomain due to its lack of lipid modification. Consequently it is sorted to an alternative secretory route, independent of carrier proteins such as Wntless, leading to robust levels of secretion. It is interesting to consider the possible biological role of an alternative mode of secretion for WntD. WntD is known to act in the adult Drosophila innate immune response by inhibiting the Toll/Dorsal-mediated antimicrobial response. It is possible that this unique function requires that it be distributed and act systemically in rapid response to infection, aided in part by robust levels of secretion and a greater range of action for this non-lipid-modified Wnt protein (Ching, 2008).

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. This study demonstrates that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, it was shown that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, the results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo (Helman, 2012).

Specification of body axes in all metazoans is initiated by a small number of inductive signals that must be integrated in time and space to control complex and unique patterns of gene expression. It is therefore of utmost importance to unravel the mechanisms underlying crosstalk between different signaling cues that concur during early development. This study has elucidated a novel signal integration mechanism that coordinates RTK signaling pathways with the Dl nuclear gradient, and thus with terminal and DV patterning of the Drosophila embryo (Helman, 2012).

Previous work had identified an input by Torso signaling into specific transcriptional effects of Dl. The current results establish a general mechanism, which involves RTK-dependent control of the nuclear Dl gradient itself, and thus affects a large group of Dl targets. This regulatory input is based on RTK-dependent derepression of wntD, a Dl target that encodes a feedback inhibitor of the Dl gradient. Thus, Dl activates wntD effectively only when accompanied by RTK signaling, enabling region-specific negative-feedback control of the nuclear Dl gradient. In the absence of RTK signaling, wntD is not expressed and the levels of nuclear Dl are elevated. Consequently, Dl target genes are ectopically expressed, both at the poles and along the DV axis (Helman, 2012).

Torso RTK signaling depends on maternal cues and is independent of the Dl gradient. Thus, it can be viewed as a gating signal that operates only at the embryonic poles, where it controls Dl-dependent gene regulation. However, the activity of the EGFR RTK pathway later on in development crucially depends on Dl, which induces the neuroectodermal expression of rhomboid, a gene encoding a serine protease required for processing of the EGFR ligand Spitz. In this case, EGFR-dependent induction of WntD represents a negative feedback loop that reduces nuclear levels of Dl laterally and, consequently, limits the expression of multiple Dl targets along the DV axis (Helman, 2012).

It should be noted that the regulatory interactions that have been characterized do not preclude the existence of other mechanisms modulating nuclear Dl concentration or activity. For example, the progressive dilution or degradation of maternal components involved in Toll receptor activation upstream of Dl should cause reduced Dl nuclear accumulation and retraction of its targets as development proceeds. It is also possible that Torso- or EGFR-induced repressors block transcription of Dl target genes directly. Accordingly, the ectopic sna expression observed in embryos mutant for components of the Torso pathway such as DSor and trunk probably reflects both loss of WntD activity on Dl and loss of Hkb-mediated repression of sna. In this context, it is interesting to note that sna expression expands and colocalizes with Hkb at the poles of wntD mutants; perhaps repression of sna by Hkb is not sufficient to override increased Dl activation in this genetic background. Thus, the Torso pathway probably employs more than one mechanism to exclude Dl target expression from the termini. Furthermore, the existence of such additional regulatory mechanisms could explain why wntD mutants do not have a clear developmental phenotype, despite the broad effects on Dl-dependent gene expression patterns caused by the genetic removal of wntD. It is proposec that corrective mechanisms are present, which make the terminal and DV systems robust with respect to removal of the WntD-based feedback, such as RTK-induced repressors. Understanding the basis of this robustness will require additional studies (Helman, 2012).

This work shows that RTK-dependent relief of Gro- and Cic-mediated repression is essential for transcriptional activation of wntD by Dl. Correspondingly, in the absence of cic or gro, the early expression of wntD expands ventrally throughout the domain of nuclear Dl. The early onset of this derepression, and the presence of at least one conserved Cic-binding site in the proximal upstream region of wntD, indicate that repression of wntD may be direct. Interestingly, it is thought that Gro and Cic are also involved in assisting Dl-mediated repression of other targets such as dpp and zen, as gro and cic mutant embryos show derepression of those targets in ventral regions. However, as ectopic wntD expression in these mutants leads to reduced nuclear localization of Dl along the ventral region, it is conceivable that decreased Dl activity also contributes to the derepression of dpp and zen (Helman, 2012).

In conclusion, the data presented in this study demonstrate RTK-dependent control of nuclear Dl via wntD, based on multiple regulatory inputs, including negative gating, feed-forward loops and negative feedback control. Together, these mechanisms provide additional combinatorial tiers of spatiotemporal regulation to Dl target gene expression. Future studies will show whether other signal transduction cascades and/or additional developmental cues also impinge on the Dl morphogen gradient (Helman, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

Microarray chips (Affymetrix) were used to study the gene expression profiles of gastrulating embryos derived from wild-type, dorsal^–/– and Toll^10b flies. Toll^10b codes for a gain-of-function Toll receptor. Many known target genes, such as twist, snail, short gastrulation, tinman and mef2, showed lower expression in the dorsal^–/– sample and increased expression in the Toll^10b sample, as predicted. Among the novel targets, the annotated gene CG8458 was selected for further study because it encodes a member of the Wnt family, and Wnt proteins have been implicated in controlling cell polarity and cell movement in many organisms (Ganguly, 2005).

In situ analysis reveals that wntD mRNA is expressed in a dynamic pattern in the early embryo. There is no detectable maternally deposited RNA and the earliest zygotic expression is present at the anterior and posterior poles of early stage 4 embryos. Soon after, wntD is expressed in a few patches of ventral cells. This low level of expression remains in the ventral cells throughout the blastoderm stage. Meanwhile, expression arises in two lines of cells abutting the mesoderm. These two lines of staining coincide with the mesectoderm, the precursor of ventral midline cells. The expression of wntD in the mesectoderm persists during germ band extension and gradually disappears. De novo expression appears around stage 8 in the ventral neuroectodermal cells adjacent to the midline. This expression continues in the neuroectoderm through stages 9 and 10, and is reduced to an undetectable level by stage 11. No expression was detected in other stages of embryonic development. The expression pattern of wntD is largely different from other Drosophila Wnt genes (Ganguly, 2005).

Double in situ hybridization to determine the location of the two lateral lines of wntD expression. Previous reports demonstrate that sim is expressed in the mesectoderm and is strongly repressed by Snail in ventral cells. Embryos that contained both wntD and sim in situ probes showed essentially the same lateral pattern as embryos that contained either probe, demonstrating that the two patterns overlap. Double in situ hybridization also showed that, similar to sim, the lateral expression of wntD is at the border of the snail pattern. Thus, it is concluded that the two lateral lines of wntD expression are in the mesectoderm (Ganguly, 2005).

EFFECTS OF MUTATION

Drosophila WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo

The dorsal-ventral (D-V) axis of the Drosophila embryo is initially patterned by a ventral-to-dorsal nuclear gradient of Dorsal protein activity under the control of Spatzle/Toll signalling. Toll activates Dorsal primarily through the degradation of Cactus, thereby freeing Dorsal to enter the nucleus and activate or repress target genes. The transcriptional profile that is regulated by Dorsal defines the spatial organization of tissues in the embryo, with ventral-most cells becoming mesoderm, flanked by the mesectoderm and neuroectoderm in more lateral regions, and gut primordia at the poles (Gordon, 2005).

The gene wntD was identified as a member of the Drosophila Wnt family based on a genomic search for Wnt-related genes (synonyms CG8458 and wnt8; Llimargas, 2001). Examination of wntD RNA in situ revealed that the first detectable expression is seen at the ventral poles of the blastoderm embryo, followed by sequential ventral-to-dorsal expression in the presumptive mesoderm, mesectoderm and neuroectoderm. Embryos derived from mothers carrying a dominant, activated allele of Toll, express wntD RNA more broadly and at higher levels than wild type. This demonstrates that wntD expression is induced by Toll signalling. Examination of WntD protein distribution shows that WntD is secreted and travels multiple cell diameters away from producing cells, suggesting that WntD is capable of signalling at a distance (Gordon, 2005).

To uncover the signalling activity of WntD in the embryo, WntD was ectopically expressed in the female germline, producing blastoderm stage embryos that contain high levels of WntD protein. These embryos lacked detectable nuclear Dorsal; although total cellular levels of Dorsal protein remained unchanged. Consequently, the mesodermal Dorsal target gene Twist was not expressed, and the embryos produced only dorsal cuticle. Furthermore, the observed defects were specific to dorsal-ventral patterning, since the anterior-posterior patterning gene hunchback was unaffected by WntD (Gordon, 2005).

In order to determine the point of intersection between WntD activity and the Toll/Dorsal pathway, flies that overexpress WntD and carry strong hypomorphic alleles of cactus were constructed. Although maternal cactus mutants exhibit a ventralized phenotype, those also overexpressing WntD are dorsalized, and indistinguishable from embryos overexpressing WntD alone. These data demonstrate that WntD (a secreted growth factor) is capable of producing a signal that blocks Dorsal nuclear translocation downstream of, or in parallel to, Cactus. It has been shown previously that Dorsal undergoes Toll-dependent and -independent phosphorylation, and that Dorsal nuclear localization can be regulated independently of Cactus (Drier, 2000; Gordon, 2005)

That WntD is a member of the Wnt family of growth factors raises the question of whether it signals through the well-characterized Frizzled/Armadillo (beta-catenin) pathway. It is suggested that WntD does not, based on two lines of evidence: (1) germline clones of axin, a negative regulator of Armadillo, do not produce dorsalized embryos, and (2) overexpression of WntD in tissues sensitive to Armadillo signalling does not have any detectable effect. These observations however, do not rule out the possibility that WntD signals through a Frizzled receptor in an Armadillo-independent manner (Gordon, 2005).

In order to investigate the role of endogenous WntD, a loss-of-function mutation was constructed using 'ends-out' gene targeting. The modified wntD locus produced no detectable protein, as assayed by Western blot. Analysis of flies homozygous for either of two knockout alleles (labelled wntD^KO1 or wntD^KO2) revealed that wntD is not essential for viability or fertility (Gordon, 2005).

Despite their viability, wntD mutant embryos show an expansion of nuclear Dorsal into the pole regions where endogenous WntD is first detected. This indicates that the earliest role of WntD in the embryo is to restrict the field of Dorsal activation, thereby ensuring the establishment of the proper boundary between the developing ventral and terminal domains; Dorsal, along with A-P positional information, induces transcription of wntD at the ventral poles of the embryo, and WntD in turn feeds back to repress Dorsal nuclear translocation, and prevent improper spread of the ventral domain. This mechanism stands in contrast to another characterized mode of Dorsal pathway repression at the embryonic termini -- that of signalling from the Torso (Tor) receptor tyrosine kinase. In the case of Torso, signalling at the poles of the embryo selectively interferes with the ability of Dorsal to repress the expression of specific target genes, although exerting only a minor effect on those genes activated by Dorsal. These data suggest that Torso signalling affects the activity of nuclear Dorsal, whereas WntD signalling affects Dorsal's nuclear translocation (Gordon, 2005).

In addition to its role in D-V patterning, it has been well established that Toll/NF-kappaB signalling has a more evolutionarily conserved role in regulating the innate immune system. During the immune response Toll induces the nuclear translocation of two NF-kappaB family members: Dorsal and Dorsal-related immunity factor (Dif). Genetic analysis has suggested that Dif, although dispensable for development, is the major transcription factor involved in the Toll-mediated immune response. In addition to Dorsal and Dif, the fly immune response also uses a third NF-kappaB related protein, Relish, which is activated on signalling by PGRP-LC (Choe, 2002) and Imd (Hedengren, 1999). Together, these pathways regulate the expression of hundreds of genes after microbial infection (Gordon, 2005).

In light of the interaction between WntD and Dorsal in the embryo, it was asked if WntD could have a role later in the fly's life as a repressor of Toll/Dorsal-mediated immunity. Polymerase chain reaction with reverse transcription (RT-PCR) was used to confirm expression of endogenous wntD RNA in adults. wntD mutant adults appear normal, with the exception that at low frequency (1%-2%) sites of ectopic melanization have been observed, most notably on the wing hinge. This is consistent with a role for WntD in maintaining low basal levels of Toll/Dorsal signalling; other mutations that hyper-activate Toll show increased levels of phenoloxidase-driven melanization. Furthermore, Dorsal has been shown to be an essential component of the melanization response in larvae (Gordon, 2005).

To investigate the role of WntD after septic injury, wntD and control flies were injected with a dilute culture of the gram-positive bacterium Micrococcus luteus, and the induction of antimicrobial peptide (AMP) transcripts were monitored over time using quantitative RT-PCR. It was observed that some, but not all, AMPs showed aberrant expression in wntD mutants. The AMP diptericin is most severely affected, with wntD flies displaying dramatically elevated basal levels of expression (approximately 15-fold), and significantly higher mRNA levels following infection. In contrast, Drosomycin mRNA levels were not significantly different from controls in either uninfected or infected wntD mutants. A third AMP, defensin, showed an intermediate pattern of expression, with elevated mRNA levels in wntD mutants at some time points (Gordon, 2005).

These results pose an apparent paradox, since previous experiments have characterized diptericin as a target of IMD/Relish, and drosomycin as a target of Toll signalling. Drosomycin expression is reported to be primarily regulated by Dif in adult flies, and appears to be unaffected by increased Dorsal activity. Thus, the results for Drosomycin are consistent with past work. The diptericin result initially appears puzzling, but existing data demonstrate that the signal transduction pathways regulating immunity are not as specific as initially described. For example, Relish is required for diptericin induction in response to infections in vivo, but constitutive activation of Toll signalling results in elevated levels of diptericin in adult flies. Furthermore, Dorsal is sufficient to activate the diptericin promoter in vitro. The simplest explanation for these observations is that diptericin transcription can be induced by Toll/Dorsal signalling. Taken together, these data support a model in which WntD signalling specifically represses Toll/Dorsal, and not Toll/Dif signalling (Gordon, 2005).

Given a role for WntD in the regulation of antimicrobial gene transcription, attempts were made to determine whether wntD mutants were immunocompromised. To test this, wntD and control adults were infected with the gram-positive, lethal pathogen Listeria monocytogenes. In response to infection, wntD mutants exhibit significantly higher levels of mortality when compared with parental lines. Importantly, this phenotype is suppressed by the introduction of dorsal mutations, with close to full suppression in the absence of both copies of dorsal and partial suppression in flies heterozygous for a dorsal mutation. These genetic interactions are consistent with the assertion that WntD specifically regulates Dorsal, and not other mediators of immunity. Recent reports have demonstrated that a fly's response to bacterial challenge includes factors that are damaging to the host, and that increased Toll signalling can render flies more susceptible to viral infection. It is therefore proposed that it is the deleterious hyper-activation of specific Dorsal target genes that is responsible for the increased mortality seen in wntD mutants. Furthermore, the susceptibility of wntD mutants to a lethal infection suggests a reason for the positive selection of wntD during evolution; immune responses have a cost, and their appropriate downregulation would be expected to provide flies with a selective advantage. Although wntD flies appear healthy in a lab environment, it is easy to imagine that under the more stressful, and septic, conditions in the wild, flies lacking wntD would suffer the perils of a hyperactive immune system (Gordon, 2005).

This study has presented evidence that WntD, a Wnt family member, produces a signal that blocks the nuclear translocation of Dorsal. Furthermore, WntD is a target of Toll/Dorsal signalling, and creates a negative feedback loop to repress Dorsal activation. wntD is not required for viability under lab conditions, but wntD mutants show defects in embryonic Dorsal regulation, and in the adult innate immune system. Since the WntD signal in the embryo is not mediated by Armadillo, it is supposed that the immune function of WntD is also Armadillo-independent, although immune defects have been observed in flies expressing a dominant-negative form of the Aramdillo partner DTCF. Further characterization of signalling events bridging WntD and Dorsal could yield valuable insight into the regulation of the therapeutically important NF-kappaB family of proteins (Gordon, 2005).

Increased expression of WntD blocks presumptive mesoderm invagination

An essential biological function of the Dorsal/Twist/Snail network is to promote invagination of the ventral cells to form the mesoderm. Although the repressor function of Snail is required for ventral invagination, none of the known target genes normally repressed by Snail has been directly implicated in disrupting ventral invagination. Because wntD is repressed by Snail, wntD expression was increased in wild-type embryos in an attempt to phenocopy the defects in snail mutant embryos. The maternal nanos-Gal4 line was used to direct the ubiquitous expression of UAS-wntD in early embryos. It was found that approximately 50% of these embryos at the gastrulation stage had observable defects in ventral invagination. Approximately one quarter of these defective embryos had completely lost the ventral furrow, and the others showed varying degrees of invagination with the anterior regions always being worse than the posterior regions. The ventral invagination defect is not a result of general problems in cell shape changes or cell movements because cephalic furrow formation and germ-band extension occurs normally in these embryos. Tissue sectioning confirmed the phenotype that the mesoderm is largely missing in gastrulating embryos (Ganguly, 2005).

The nanos-Gal4 female flies deposit maternally the Gal4 gene products, which direct the UAS-dependent WntD expression ubiquitously in pre-blastoderm stage embryos. The rhomboid-Gal4 driver was tested; this rhomboid promoter contains mutations in its Snail-binding sites and directs zygotic Gal4 expression in the ventral half of the blastoderm. In these experiments, approximately 5% of embryos at gastrulation stage show slightly defective invagination. The rhomboid promoter, as well as other ventral zygotic promoters, is activated by Dorsal. Thus, the expression of WntD by zygotic promoters may be too late to induce a substantial phenotype. This speculation is consistent with the proposed mechanism of feedback inhibition of Dorsal by WntD (Ganguly, 2005).

WntD blocks invagination by disrupting the expression of mesoderm determinants

The Wnt family of secreted proteins regulates cell fate, cell polarity, cytoskeleton and cell movement. To elucidate the mechanism that underlies the invagination defect induced by WntD, various markers of cell fate and cell shape were examined. It was surprising to find that twist and snail expression becomes highly abnormal in the nanos-Gal4-driven WntD-expressing embryos. The twist pattern was narrower than 12-cell widths along the circumference, compared with 20-cell widths in wild-type embryos. The snail expression pattern was even more severely affected. A total of 93% (n=147) of WntD-expressing embryos at the blastoderm and gastrulation stages showed abnormality in the snail expression pattern. The abnormality was variable and ranged from a few cells narrower to an almost complete disappearance of the pattern. The anterior expression was always more severely affected, and the posterior expression was affected to various extents in different embryos. The phenotype was quantitated by counting the width of the snail expression domains at 50% egg-length. In WntD-overexpression embryos that were assigned to have a phenotype, the snail pattern varied from zero to 11 cells, with an average width of seven cells. For wild-type embryos, the width of the snail domain is 13 to 17, with an average of 15 cells. Thus, all the embryos that were assigned to have a phenotype showed quantitative defects (Ganguly, 2005). sim and rhomboid are normally repressed by Snail in the presumptive mesoderm. In WntD-overexpression embryos the sim pattern disappears in the anterior region and the lateral rows of staining come closer in the posterior region. Snail represses sim expression in the ventral cells but the expression and positioning of sim in the presumptive mesectoderm also requires Snail. Therefore, the abnormal sim pattern follows exactly the reduced snail expression. rhomboid shows similar narrowing of the pattern, consistent with the model that Snail is a simple repressor of rhomboid. In conclusion, increased WntD expression causes highly reduced twist and snail expression, leading to abnormal expression of other genes in ventral cells. Even though increased WntD expression may also cause other defects, the reduced twist and snail expression is probably sufficient to account for the loss of invagination (Ganguly, 2005).

Negative regulation of Dorsal nuclear localization by WntD

The direct activator of twist and snail expression in the blastoderm is Dorsal. Therefore, the distribution of the Dorsal protein was examined. In wild-type blastoderm and gastrulating embryos, Dorsal shows the characteristic ventral nuclear pattern. By contrast, WntD overexpression causes low-level staining around the periphery of the whole embryo, and high-resolution imaging showed that the ventral cells have Dorsal proteins predominantly in the cytoplasm. This phenotype is similar to that of embryos derived from a gastrulation-defective mutant, which causes no activation of the Toll pathway. Embryos derived from the opposite Toll^10b gain-of-function mutant show nuclear staining all around the embryo. The phenotype induced by WntD overexpression is different from that in the Dorsal protein null mutant, which essentially showed no staining. These results together suggest that the overexpressed WntD inhibits Dorsal nuclear localization (Ganguly, 2005).

Specific mutants of wntD are not yet available. Therefore, a few deficiency strains were examined by staining for wntD mRNA expression in the embryo; it was confirmed that Df(3R)l26c, which has the 87E1-87F11 region deleted, has uncovered wntD. This deficiency was used to assess whether endogenous WntD regulates Dorsal. In wild-type blastoderm, the Dorsal nuclear gradient extends into the neuroectoderm and the posterior end. Before the onset of gastrulation, the posterior Dorsal staining is normally retracted. However, in embryos derived from the Df(3R)l26c strain, Dorsal nuclear staining expands in the posterior region. Because the earliest wntD expression is at the anterior and posterior regions, the loss of wntD in the deficiency could be the underlying reason for the posterior expansion of Dorsal in these embryos (Ganguly, 2005).

WntD attenuates the function of Dorsal

The posterior expression of snail in wild-type embryos is retracted and shows a sharp pattern before the onset of gastrulation. snail expression in Df(3R)l26c mutant embryos, however, expands into the posterior region. Double staining shows that, in wild-type embryos, the posterior gene huckebein is complementary to the snail pattern. No change was detected in the huckebein pattern in the Df(3R)l26c mutant embryos. Using huckebein expression as a position marker, it was found that the snail pattern expands into the posterior region so that it overlaps with that of huckebein in the deficiency mutant embryo. Quantitation by using the snail pattern revealed that 24% (n=55) of all gastrulating embryos from Df(3R)l26c heterozygous parents showed the posterior expansion. Based on Mendelian ratios, this result represents an almost full penetrance. Thus, there is a posterior expansion of snail expression that correlates with the posterior expansion of nuclear Dorsal. Subtle broadening of the snail pattern was also observed in the anterior region, suggesting that there is an increase of nuclear Dorsal in the anterior region, but the increase was not detectable by immunofluorescence staining (Ganguly, 2005).

Because Df(3R)l26c removes a number of genes in addition to wntD, a genetic rescue experiment was performed to confirm the involvement of wntD. A transgenic line that contained a wntD genomic fragment was generated that showed all the normal expression patterns of wntD in the early embryo. When this genomic construct was crossed into the Df(3R)l26c mutant, the posterior and anterior expansion phenotype of snail was completely rescued. Posterior Dorsal expansion was not observed in any of the embryos derived from the wntD-rescued Df(3R)l26c strain. The rescue experiment demonstrates that the deletion of wntD in the deficiency strain is responsible for the observed Dorsal and snail expression phenotypes. RNA interference of wntD was performed by injecting double-stranded RNA into wild-type pre-blastoderm stage embryos. Approximately 10% of these injected embryos at late blastoderm stage had a mild posterior expansion of snail, and none of the embryos injected with buffer alone showed such a phenotype. This result further supports the idea that loss of WntD causes posterior expansion of snail expression (Ganguly, 2005).

Whether the de-repressed WntD expression in the ventral cells of snail mutant embryos can inhibit Dorsal function was examined. A previous report demonstrated that in mutant embryos that produced non-functional Snail proteins, the expression of snail mRNA disappears prematurely. This premature loss of snail mRNA expression could be due to the inhibition of Dorsal function by the de-repressed WntD. It was surmised that the removal of wntD in a snail mutant should lead to enhanced Dorsal function. Thus, a snail;Df(3R)l26c double-mutant strain was established. The double homozygous embryos were identified by the lack of wntD mRNA staining and the lack of a ventral furrow. The snail mRNA pattern in snail mutant embryos exhibited a fuzzy border around the onset of gastrulation. By contrast, double-mutant embryos showed a snail pattern with sharp borders, similar to that observed in wild-type or Df(3R)l26c embryos. The developmental stages of these embryos were very similar based on the position of the pole cells, the degree of cellularization, and the cephalic furrow formation, supporting the argument that the genetic defect is the cause for the change of snail pattern. By mid-germ band extension, the snail mRNA staining became very weak in snail mutant embryos, but in double-mutant embryos the snail mRNA level was better sustained. The establishment and maintenance of the sharp snail pattern requires Dorsal. The Df(3R)l26c deficiency strain has many genes deleted and the effect cannot be attributed directly to the loss of wntD, but the result is consistent with the speculation that deleting wntD in the snail mutant embryo allows Dorsal to function more efficiently in activating target genes (Ganguly, 2005).

EVOLUTIONARY HOMOLOGS

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration. In bilaterians, there are at least 12 subfamilies of Wnt genes. Wnt3 and Wnt8 are required for somitogenesis in vertebrates and are thought to be involved in posterior specification in deuterostomes in general. Although TCF and β-catenin have been implicated in the posterior patterning of some short-germ insects, the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. This study investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. These results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, it is proposed that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes (McGregor, 2008).

The posterior truncation phenotypes resulting from pRNAi against Wnt8 in the spider are at least superficially similar to those observed when Wnt8 and/or Wnt3 are perturbed in vertebrate embryos. Removal or blocking Wnt8 and/or Wnt3 in Xenopus, zebrafish, and mouse results in truncated embryos with only a few anterior somites and no tail bud. Although analysis of TCF and β-catenin in Oncopeltus and Gryllus, respectively, indicated that Wnt signaling might be involved in the development of the growth zone and posterior segments in arthropods, the current data show that in fact the same ligand, Wnt8, is employed in posterior development in both vertebrates and arthropods (McGregor, 2008).

In class II and III At-Wnt8^pRNAi embryos exhibiting fused L4 limb buds, it also appeared that the most ventral part of this segment is missing. This phenotype shows similarities to the phenotype when short-gastrulation is knocked down in this spider. It suggests that, in addition to A-P patterning, At-Wnt8 is involved in D-V patterning in the spider, a role Wnt8 genes also perform in vertebrates (McGregor, 2008).

There is evidence that Wnt signaling acts upstream of Delta/Notch in vertebrate somitogenesis. Although the expression of Wnt3a and Wnt8 is not cyclical during somitogenesis in vertebrates, some downstream components of Wnt signaling, such as Axin2, are cyclically expressed in mice and possibly are integral to the Delta/Notch-dependent segmentation clock. However, recent experiments have shown that Axin2 and components of the Delta/Notch pathway continue to oscillate in the presence of stabilized β-catenin, which suggests that in mice, Wnt signaling may be permissive for the segmentation clock rather than instructive. Similarly, in zebrafish it is thought that Wnt8 may act to maintain a precursor population of stem cells in the PSM and tailbud rather than directly regulate the segmentation clock. It is proposed that the same ligand, Wnt8, could play a similar permissive role for segmentation in the growth zone of the spider by establishing and possibly maintaining a pool of cells that develop into the opisthosomal segments. When At-Wnt8 activity is reduced, cells are ectopically used in L3/L4 or internalized, depleting the putative growth-zone pool. This depletion manifests as a smaller opisthosoma, separated clusters of cells that give rise to separate irregular germbands, or even no opisthosoma (McGregor, 2008).

It was previously shown that Delta/Notch signaling is also involved in posterior development in the spiders Cupiennius. These new results reveal that in the spider, Wnt8 is required for the clearing of Dl and h expression in the posterior and that this is necessary for repression of twi, activation of cad, and establishment of the growth zone (McGregor, 2008).

The involvement of Wnt8, Delta/Notch signaling, and cad in the posterior development of other arthropods has also been directly demonstrated by functional analysis or inferred from expression patterns, and in vertebrates, Wnt3a and Wnt8 probably act upstream of Delta/Notch and cad during somitogenesis. Taken together, this suggests that a regulatory genetic network for posterior specification including Wnt8, Delta/Notch signaling, and cad could have been present in the last common ancestor of protostomes and deuterostomes, but has subsequently been modified in some lineages. For example, in Drosophila, Delta/Notch signaling is not involved in segmentation, and although the Drosophila Wnt8 ortholog, WntD, is required for D-V patterning, it is not involved in posterior development. Segments arise almost simultaneously in Drosophila, rather than sequentially from a growth zone, so this may suggest that the role of Wnt8 in posterior development was not required for this mode of development and therefore was lost during the evolution of these insects (McGregor, 2008).

These results suggest that Wnt8 regulates formation of the posterior growth zone and then maintains a pool of undifferentiated cells in this tissue required for development of the opisthosoma. Wnt signaling thus regulates the establishment and maintenance of an undifferentiated pool of posterior cells in both vertebrates and spiders and in fact the same Wnt ligand, Wnt8, is used in both phyla. Therefore, Wnt8 could be part of an ancient genetic regulatory network, also including Dl, Notch, h, and cad, that was used for posterior specification in the last common ancestor of deuterostomes and protostomes (McGregor, 2008).

Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum

wingless/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved. Wnt signaling is also implicated in posterior patterning and germband elongation, but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions. Tribolium contains additional Wnt family genes that are also expressed in the growth zone. After depleting Tc-WntD/8, a small percentage of embryos were found lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods and vertebrates (Bolognesi, 2008).

Wnt8s and the establishment of the dorsoventral axis

Chicken Wnt8 transcripts are detected prior to overt gastrulation when they are found in the epiblast of the posterior marginal zone overlying Koller's sickle, a location and timing of expression that is consistent with a role in axis induction. However, such precocious localized expression has not been detected in other vertebrates. Therefore, in the mouse, as in Xenopus, it is unlikely that Wnt8 is the natural inducer of the primary signaling center responsible for axis formation. Transgenic mouse embryos expressing CWnt8C under the control of the human ß-actin promoter exhibit duplicated axes or a severely dorsalized phenotype. Although the transgene is introduced into fertilized eggs, all duplications occur within a single amnion and, therefore, arise from the production of more than one primitive streak at the time of gastrulation. Morphological examination and the expression of diagnostic markers in transgenic embryos suggest that ectopic Cwnt8C expression produces only incomplete axis duplication: axes are always fused anteriorly, there is a reduction in tissue rostral to the anterior limit of the notochord, and no duplicated expression domain of the forebrain marker Hesx1 is observed. Anterior truncations are evident in dorsalized transgenic embryos containing a single axis. These results are discussed in the light of the effects of ectopic Xwnt8 in Xenopus embryos, where its early expression leads to complete axis duplication but expression after the mid-blastula transition causes anterior truncation. It is proposed that while ectopic Cwnt8C in the mouse embryo can duplicate the primitive streak and node this only produces incomplete axis duplication because specification of the anterior aspect of the axis, as opposed to maintenance of anterior character, is established by interaction with anterior primitive endoderm rather than primitive streak derivatives, for example, the node, the prechordal plate and notochord. These results do not necessarily contradict experiments in amphibians where organizer grafts generate complete secondary axes. Instead, they point to a different topography between the mouse and frog. In the mouse, due to the cylindrical nature of the mouse embryo, the classical organizer associated with the primitive streak and the endoderm happen to be on opposite sides of the conceptus, while in Xenopus the deep endomesoderm of the dorsal half of the embryo immediately abuts the dorsal blastopore lip organiser (Popperl, 1997).

Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar functions of these molecularly very different classes of signaling molecules prompted a study of their mutual regulation, and their roles in mesoderm patterning were closely compared. Wnt-8 and BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4 signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8 overexpression cannot compensate for an inhibition of BMP-2/-4 function, but rather BMP-4 overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4 appears to generally inhibit the expression of dorsal genes, Xenopus Wnt-8 inhibits the expression of only the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal fate, these results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm into the most dorsal sector from which the notochord develops and the dorsolateral sector from where the somites differentiate (Hoppler, 1998).

wnt8a is essential for normal patterning during vertebrate embryonic development, and either gain or loss-of-function gene dysregulation results in severe axis malformations. The zebrafish wnt8a locus is structured such that transcripts may possess two regulatory 3' untranslated regions (UTRs), raising the possibility of post-transcriptional regulation as an important mode of wnt8a signaling control. To determine whether both UTRs contribute to post-transcriptional wnt8a gene regulation, each UTR (UTR1 and UTR2) was tested in transient and transgenic reporter assays. Both UTRs suppress EGFP reporter expression in cis, with UTR2 exhibiting a more pronounced effect. UTR2 contains a 6 base sequence necessary for UTR2 regulatory function that is complementary to the seed of the microRNA, miR-430. A target protector morpholino that overlaps the seed complement stabilizes both reporter mRNAs and wnt8a mRNAs, and produces phenotypic abnormalities consistent with wnt8a gain-of-function. In rescue assays, specific functions can be attributed to each of the two wnt8a proteins encoded by the locus. An interplay of wnt8a.1 and wnt8a.2 regulates neural and mesodermal patterning and morphogenesis as well as patterning between brain subdivisions. Thus, post-transcriptional control of wnt8a is essential to fine tune the balance of the signaling outputs of the complex wnt8a locus (Wylie, 2013).

Transcriptional regulation of Wnt8s

Formation of the vertebrate body plan is controlled by discrete head and trunk organizers that establish the anteroposterior pattern of the body axis. The Goosecoid (Gsc) homeodomain protein is expressed in all vertebrate organizers and has been implicated in the activity of SpemannÕs organizer in Xenopus. The role of Gsc in organizer function was examined by fusing defined transcriptional regulatory domains to the Gsc homeodomain. Like native Gsc, ventral injection of an Engrailed repressor fusion (Eng-Gsc) induces a partial axis, while a VP16 activator fusion (VP16-Gsc) does not, indicating that Gsc functions as a transcriptional repressor in axis induction. Dorsal injection of VP16-Gsc results in loss of head structures anterior to the hindbrain, while axial structures are unaffected, suggesting a requirement for Gsc function in head formation. The anterior truncation caused by VP16-Gsc is fully rescued by Frzb, a secreted Wnt inhibitor, indicating that activation of ectopic Wnt signaling is responsible, at least in part, for the anterior defects. Supporting this idea, Xwnt8 expression is activated by VP16-Gsc in animal explants and the dorsal marginal zone, and repressed by Gsc in Activin-treated animal explants and the ventral marginal zone. Furthermore, expression of Gsc throughout the marginal zone inhibits trunk formation, identical to the effects of Frzb and other Xwnt8 inhibitors. A region of the Xwnt8 promoter containing four consensus homeodomain-binding sites has been identified and this region mediates repression by Gsc and activation by VP16-Gsc, consistent with direct transcriptional regulation of Xwnt8 by Gsc. Therefore, Gsc promotes head organizer activity by direct repression of Xwnt8 in SpemannÕs organizer and this activity is essential for anterior development (Yao, 2001).

There is growing evidence that Gli proteins participate in the mediation of Hedgehog and FGF signaling in neural and mesodermal development. However, little is known about which genes act downstream of Gli proteins. The regulation of members of the Wnt family by Gli proteins in different contexts is shown in this study. These findings indicate that Gli2 regulates Wnt8 expression in the ventral marginal zone of the early frog embryo: activating Gli2 constructs induce ectopic Wnt8 expression in animal cap explants, whereas repressor forms inhibit its endogenous expression in the marginal zone. Using truncated Frizzled and dominant-negative Wnt constructs, the requirement of at least two Wnt proteins, Wnt8 and Wnt11, for Gli2/3-induced posterior mesodermal development is shown. Blocking Wnt signals, however, inhibits Gli2/3-induced morphogenesis, but not mesodermal specification. Gli2/3 may therefore normally coordinate the action of these two Wnt proteins, which regulate distinct downstream pathways. In addition, the finding that Gli1 consistently induces a distinct set of Wnt genes in animal cap explants and in skin tumors suggests that Wnt regulation by Gli proteins is general. Such a mechanism may link signals that induce Gli activity, such as FGFs and Hedgehogs, with Wnt function (Mullor, 2001).

A gene encoding Wnt8, a ligand that activates the ß-catenin/Tcf system, is expressed in the same prospective endomesodermal cells in which the autonomous maternal system initially causes ß-catenin nuclearization. This observation implies an autoreinforcing Tcf control loop, which is set up within the endomesodermal domain once this is defined. This loop is necessary, for if it is blocked by introduction of a negatively acting form of the Wnt8 ligand, so is endomesoderm specification. The inferred Wnt8 loop conforms to the 'community effect' concept of Gurdon, i.e., a requirement for intercellular signaling within a field of cells in a given state of specification that is necessary for the maintenance and the further developmental progression of that state (Davidson, 2002).

Mutation of Wnt8s

In vertebrates, wnt8 has been implicated in the early patterning of the mesoderm. Sequencing of the wnt8 locus reveals a second wnt8 coding region approximately 800 bp downstream and in tandem to the first (the two coding regions are referred to as ORF1 and ORF2). ORF1 is the gene reported previously described as wnt8, while translation of ORF2 reveals that it has the potential to encode a distinct full-length Wnt8 protein. A comparison of the predicted translation products from ORF1 and ORF2 shows that they are approximately 70% identical, with the most divergence in amino acid sequence at the amino and carboxy termini. To determine directly the embryonic requirements for wnt8, a chromosomal deficiency was generated in zebrafish that removes the bicistronic wnt8 locus. Homozygous mutants exhibit pronounced defects in dorso-ventral mesoderm patterning and in the antero-posterior neural pattern. Despite differences in their signaling activities, either coding region of the bicistronic RNA can rescue the deficiency phenotype. Specific interference of wnt8 translation by morpholino antisense oligomers phenocopies the deficiency. Interference with wnt8 translation in ntl and spt mutants produces embryos lacking trunk and tail. These data demonstrate that the zebrafish wnt8 locus is required during gastrulation to pattern both the mesoderm and the neural ectoderm properly (Lekven, 2001).

Only a very small number of eukaryotic cellular genes are known to encode multicistronic mRNAs, of which c-myc is one. Further experiments are required to determine the significance of the bicistronic transcript from wnt8 in zebrafish and whether this genomic structure exists in other species, but regulatory control through internal ribosome entry sites in the 5' UTRs of a number of developmental regulatory genes has been shown. Precise regulation of wnt8 expression via its 3' UTR is critical for its proper function during development of Xenopus; thus, additional levels of control of wnt8 expression could be essential in ensuring its proper function. Considering that several transcripts are produced from this locus, precise regulation of each transcript may be essential to modulate carefully wnt8 signaling during embryogenesis (Lekven, 2001).

Wnt8 signaling

Wnt/ß-catenin signaling regulates many aspects of early vertebrate development, including patterning of the mesoderm and neurectoderm during gastrulation. In zebrafish, Wnt signaling overcomes basal repression in the prospective caudal neurectoderm by Tcf homologs that act as inhibitors of Wnt target genes. The vertebrate homolog of Drosophila nemo, nemo-like kinase (Nlk), can phosphorylate Tcf/Lef proteins and inhibit the DNA-binding ability of ß-catenin/Tcf complexes, thereby blocking activation of Wnt targets. By contrast, mutations in a C. elegans homolog show that Nlk is required to activate Wnt targets that are constitutively repressed by Tcf. Overexpressed zebrafish nlk, in concert with wnt8, can downregulate two tcf3 homologs, tcf3a and tcf3b, that repress Wnt targets during neurectodermal patterning. Inhibition of nlk using morpholino oligos reveals essential roles in regulating ventrolateral mesoderm formation in conjunction with wnt8, and in patterning of the midbrain, possibly functioning with wnt8b. In both instances, nlk appears to function as a positive regulator of Wnt signaling. Additionally, nlk strongly enhances convergent/extension phenotypes associated with wnt11/silberblick, suggesting a role in modulating cell movements as well as cell fate (Thorpe, 2004).

These results support a role for nlk in the activation of Wnt targets during zebrafish embryogenesis. Overexpressed nlk downregulates two tcf3 homologs, tcf3a and tcf3b, that repress activation of Wnt target genes during neural patterning. This functional interaction with Tcf3 homologs requires wnt8 signaling, and thus probably ß-catenin, consistent with previous data indicating that Nlk specifically interferes with the DNA-binding ability of ß-catenin/Tcf complexes, not that of Tcf alone. Interference with endogenous nlk function reveals important roles in two processes that are regulated by canonical Wnts, mesoderm patterning by wnt8, and patterning of midbrain and forebrain by wnt8b. Since loss of nlk enhances or phenocopies loss of function of these two Wnts, it is concluded that nlk functions as an activator of some canonical Wnt targets in zebrafish. nlk also interacts, directly or indirectly, with non-canonical Wnt pathways, since inhibition of nlk strongly enhances convergent extension phenotypes associated with loss of wnt11 function. A role was uncovered for an unusual wnt8 homolog, wnt8 ORF2, in regulating cell movements during gastrulation (Thorpe, 2004).

Wnt8s and anterior neuroectoderm patterning

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. A zebrafish frizzled8a (fz8a) gene is expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. The role of Fz8a-mediated Wnt8b signaling in anterior brain patterning has been investigated in zebrafish. In zebrafish embryos Wnt signaling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation. Fz8a plays an important role in mediating anterior brain patterning. Wnt8b and Fz8a functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos. Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signaling. It is suggested that a gradient of Fz8a-mediated Wnt8b signaling may play a crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula (Kim, 2002).

The data suggest that LiCl treatment at the late gastrula stage (90% epiboly) acts as an artificial Wnt signal activator, thus significantly increasing fkd5 and pax6 expression in the posterior diencephalon. However, eng2 expression is not dramatically increased, although Wnt signaling is highly activated by LiCl treatment at the late gastrula stage. Nevertheless, injections of wnt8b-MO and fz8a-MO morpholinos, which might cause partial reductions of Wnt8b and Fz8a, reduced eng2 expression in the midbrain more sharply compared with decreased expressions of fkd5 and pax6 in the posterior diencephalon. These results indicate that eng2 in the midbrain is highly sensitive to a decrease of Wnt8b signal activity but less sensitive to an excess of Wnt signal, whereas fkd5 and pax6 in the posterior diencephalon is highly sensitive to an excess of Wnt signal but less sensitive to a decrease of Wnt8b signal. These observations indicate that patterning of the midbrain needs a higher threshold of Wnt8b activity, while that of the posterior diencephalon may require relatively lower Wnt8b thresholds (Kim, 2002).

To explain a gradient of Fz8a-mediated Wnt8b signal activity required for the proper patterning of the anterior neuroectoderm (posterior diencephalon and midbrain), a model is proposed that can generate a sharp gradient of Fz8a-mediated Wnt8b signaling activity, with a peak at the midbrain. First, at the 90% epiboly stage, adjacent expression domains for fz8a and wnt8b partially overlap in the putative midbrain. At the same time, a small amount of Wnt8b, possibly stabilized by binding to Fz8a, might further diffuse towards the presumptive posterior diencephalon from midbrain. Therefore, low Wnt8b signal activity and high Wnt8b signal activity might be imposed on the posterior diencephalon and midbrain region, respectively. Subsequently, at late gastrula stage, two overlapping expression domains are separated by the repression of fz8a expression caused by Wnt8b thus generating a decreasing gradient of Fz8a receptor towards the caudal anterior neuroectoderm. Thus a gradient of Fz8a-mediated Wnt8b signal activity becomes sharper at late gastrula stage. Consequently, a gradient of pax6 expression in the diencephalon from posterior to anterior can be established by low level of Wnt8b activity, while eng2 expression in the midbrain can be regulated by high level of Wnt8b activity. This hypothesis that pax6 and eng2 expression requires lower and higher level of Wnt signaling, respectively, has also been evidenced in chick gastrula (Kim, 2002).

Wnt8s and the posteriorization of neural tissue

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. The requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue has been analysed in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants); these embryos show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MOwnt8) translational interference, abrogates formation of spinal cord and posterior brain fates dose-dependently, without blocking ventrolateral mesoderm formation. MOwnt8 also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. These findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS (Erter, 2001).

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, known to pattern ventro-lateral mesoderm, is the neural posteriorizing factor that acts upon neuroectoderm, Frizzled 8c and Frizzled 9 were first assigned to be functional receptors for Wnt8. Transplanted non-axial mesoderm was then transplanted 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 induces 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 ß-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. It is thus concluded that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis (Momoi, 2003).

Wnt8s and morphogenesis of the posterior body

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides leads to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure -- a more severe phenotype than with inhibition of each Wnt alone -- indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediate Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not Bmps or Cyclops, is strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos display complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression is down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling is required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish (Shimizu, 2004).

Tail formation in vertebrates involves the specification of a population of multipotent precursors, the tailbud, which will give rise to all of the posterior structures of the embryo. Wnts are signaling proteins that are candidates for promoting tail outgrowth in zebrafish, although which Wnts are involved, what genes they regulate, and whether Wnts are required for initiation or maintenance steps in tail formation has not been resolved. Both wnt3a and wnt8 are shown to be expressed in the zebrafish tailbud. Simultaneous inhibition of both wnt3a and wnt8 using morpholino oligonucleotides can completely block tail formation. In embryos injected with wnt3a and wnt8 morpholinos, expression of genes in undifferentiated presomitic mesoderm is initiated, but not maintained. To identify genes that might function downstream of Wnts in tail formation, a DNA microarray screen was conducted, revealing that sp5l, a member of the Sp1 family of zinc-finger transcription factors, is activated by Wnt signaling. Moreover, sp5l expression in the developing tail is dependent on both wnt3a and wnt8 function. Supporting a role for sp5l in tail formation, it was found that inhibition of sp5l strongly enhances the effects of wnt3a inhibition, and overexpression of sp5l RNA is able to completely restore normal tail development in wnt3a morphants. These data place sp5l downstream of wnt3a and wnt8 in a Wnt/ß-catenin signaling pathway that controls tail development in zebrafish (Thorpe, 2005).

Antagonism of Wnt8s

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which has been called sizzled, for 'secreted frizzled') encodes a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (Szl) is capable of inhibiting Xwnt8 as assayed by (1) a dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts, and in particular of Xwnt8, plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone (Salic, 1997).

Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis

Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. This study demonstrates that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/beta -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, the affinity of Noggin4 for Wnt8 in living embryos was directly estimated and it was determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. These results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development (Eroshkin, 2016).

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Search PubMed for articles about Drosophila wnt inhibitor of Dorsal

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date revised: 15 April 2020

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