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 Toll10b 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 Toll10b embryos corroborates the results of microarray analysis, i. e., lower expression in the dorsal–/– and increased expression in the Toll10b 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).
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).
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