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).

GENE STRUCTURE

cDNA clone length - 1163

Bases in 5' UTR - 60

Exons - 1

Bases in 3' UTR - 173

PROTEIN STRUCTURE

Amino Acids - 309

Structural Domains

The predicted amino acid sequence of CG8458, corresponding to WndD, has the closest homology to cephalochordate Wnt8, chicken Wnt8C, and zebrafish Wnt8. Sequence alignment and pair-wise comparison show that CG8458 (FlyBase name: Wnt8) is a distal member of this subfamily. The average identity between CG8458 and other Wnt8 molecules is approximately 27%, while the identity among other members is higher than 50%. Nonetheless, 20 out of the 22 characteristic cysteine residues of Wnt proteins are conserved in CG8458. FlyBase has named this gene Drosophila Wnt8. However, a recent report suggests that this gene may not be an ortholog of vertebrate Wnt8 but instead an orphan Wnt gene (Kusserow, 2005). Based on functional analysis, the name Drosophila wntD was used for the annotated gene CG8458, and the encoded protein WntD (Wnt inhibitor of Dorsal) (Ganguly, 2005).

date revised: 25 October 2005

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