As mentioned earlier, Wnt genes are extremely important in the developmental biology of many organism including vertebrates. These genes have remained in play for a long time and have developed specific roles in different organism. To investigate the role of Wnt3 and Wnt8 in Achaearanea tepidariorum, orthologs of these genes were created. Surprisingly, Wnt3, although exists in many organisms such as cnidarian’s, cloning an ortholog in chelicerates was a failed attempt as it most likely was lost in the linage. No protostomes have been discovered with the Wnt3 gene. These orthologs were then used to test various developmental features using parental RNAi knockouts. The Wnt8 ortholog (At-Wnt8), however, was successful.
At-Wnt8 were cloned via degenerate PCR techniques from the embryonic cDNA using specific primers. The sequences of the full coding region of At-Wnt8 were obtained by RACE PCR. The embryos were fixed and in situ hybridizations were preformed by DIG (Roche)-labeled probes. Phosphohistone H3 and Caspase 3 antibodies were used to assay cell division and death respectively. Parental RNAi was carried out by injecting dsRNA via the Wnt8 ortholog coding region (McGregor, 2008).
First expressed in the anterior region then continuously expressed in the posterior domain and the germ band growth zones, At-Wnt8 showed a consistent role in its presence. To determine its role, parental RNAi was preformed (At-Wnt8pRNAi). The phenotype of the results after the knockout was then divided in to three categories.
Class I, the weakest affect, resulted in an increased distance between the L3 and L4 limbs. The majority of the developmental issues occurred with the limb buds and the opisthosoma. This is evident in figure 2a, 2b, 2e, and 2f.
Class II had phenotype’s that were a bit more severe. As you can see (figures 2c, 2g, and 2j), the L3 and L4 buds along with other parts are more distant from each other and variable in size with a decreased in number compared to the wild type (2a). In class I and Class II embryo’s were observed with two or more germ bands in the abdomen; however, Class II in some cases showed fused limbs and a narrower L4 bud along the D-V axis. Lastly Class III phenotype’s, the most severe cases, resulted in failure to develop all opisthosomal segments and L3 and L4 were also further apart to the A-P axis (figure 2d, 2h). L4 buds were fused sometimes and ectopic clusters of disordered cell patters were present posterior to the germband.
Supplemental data figure S3: http://download.cell.com/current-biology/mmcs/journals/0960 9822/PIIS0960982208011238.mmc1.pdf
Clearly it is evident the Wnt8 is necessary for limb development (specifically L3/L4) and abdomen development as knockdown of this gene cause abnormalities with the respective development. The resulting ectopic cell internalization causing abdomen fragmentation and or complete absence of it also indicates the role of Wnt8 in establishing a growth zone.
So how does this absence of growth zone result?
It was found that the issue was not a difference in cell division or cell death, but what is more probable is that fewer cells are committing to the growth zone fate. The delta expression in normal embryos initiates at the center of the germ disc; however, in At-Wnt8pRNAi the Dl expressions continues through the opening germ disc and expands in to a larger domain (Figure 3B) causing Dl expression through stages 6, 7, and 8 of the embryo’s (Figure 3D).
As seen in figure 3E and 3F, expression of hairy also persists in the At-Wnt8pRNAi knockout as it is most likely regulated by Delta/Notch. This explains that the over expression of Dl and his associated with smaller growth zones and misregulation’s with L3/L4 limb buds.
Twi and cad are also regulated by Dl and h expression. We now know Wnt8 is required for clearing Dl and h expression during development; however, this is not only to prevent their over expression but it is necessary for twi repression and cad activation in growth zone establishment (Mcgregor, 2008).
The lack of clearing of Dl and h can be seen in figure 3F is associated with the expansion of L3 and L4 segments and a smaller growth zone in the At-Wnt8pRNAi embryo’s. The loss of the posterior segments was believed to be caused by the over expression of twi (mesodermal) and therefore the lack of expression of cad since clearing of twi is required for cad (ectodermal) activation as just mentioned. Embryos with little or no clearing of Dl only a few cad expressing cell (Figure 3H) clusters were observed which seemingly took on a growth zone. This leads to thinking that there is a threshold of activity of the Wnt8 ortholog required for growth zone cells.
Wnt8 establishes and maintains growth zones
Similar experimentations were done in mice and zebra fish two model organisms. Wnt3a and Wnt8 are not cyclical; however there are downstream components of Wnt such as Axin2 which is cyclically expressed in mice and vital on delta-notch dependant segmentation clock; however, these pathways oscillate in the presence of ß-catenin which means Wnt may not be the primary instructions for the segmentation clock. A similar study in zebrafish stem cells explains how Wnt8 acts as a precursor to the stem cell population rather than directly regulating it. In spiders this signifies that Wnt8 plays a pessimistic role and maintains a pool of cells for segmentation (McGregor, 2008)
Inhibiting of Wnt8 or Wnt3a in Xenopus, zebrafish, and mouse resulted in truncated embryos due to fusion of segments. In class II and class III knockouts, L4 buds were fused with missing ventral parts as visible in figures 2C, 2D, and 2J. Similar to its role in vertebrates, this suggests that Wnt8 is responsible for dorso-ventral patterning along with anterior-posterior patterning (McGregor, 2008).
Wnt8 Part of an Ancient Regulatory Network?
Taking in to account that Delta/Notch signaling is involved in posterior development in spiders and the results from this paper showing that Wnt8 is required to clear Dl and h expression in the posterior to repress twi, activate cad, and thus establish the growth zone; this hints that a regulatory genetic network for posterior specification. “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” (McGregor, 2008). Drosophila, for example, had lost this development mode during evolution.
Wnt8 is required for maintaining undifferentiated cells for the development of the opisthosoma and regulating posterior growth zone. This is relevant for both spiders and vertebrates via the same ligand, Wnt8. Using Delta/Notch, and cad, Wnt8 also signifies as part of an ancient regulatory network responsible for abdominal development and segmentation.
In general, the authors of this paper did an ideal job in comparing the operation of the targeted gene to various model organism and the Vertebrate phyla in general, providing sufficient support to indicate that the tested results were legitimate and applicable to further testing in humans. Another major factor the authors took into account were the co-existing factors such as TCF and ß-catenin which have been implicated in posterior patterning specifically in insects (which generally have a fair amount of segmentation). Though discussing the specific role of Wnt8 in D-V and A-P patterning and segmentation, one thing that was neglected was what the implications are, such as to control this growth zone establishment and potentially apply this in human testing to activate or inhibit this and what effects it could yield. Furthermore, what are the potential interactions of other ligands, as Wnt genes are quite active and various regions for many developmental reasons.
Pechmann, M., A. P. McGregor, et al. (2009). “Dynamic gene expression is required for anterior regionalization in a spider.” Proc Natl Acad Sci U S A 106(5): 1468-1472.
McGregor, A. P., M. Pechmann, et al. (2008). “Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider.” Curr Biol 18(20): 1619-1623.
Oda, H., O. Nishimura, et al. (2007). “Progressive activation of Delta-Notch signaling from around the blastopore is required to set up a functional caudal lobe in the spider Achaearanea tepidariorum.” Development 134(12): 2195-2205.