Localization of Wnt3 in the Hydra Head Organizer

The Hydra, belonging to the phylum Cnidaria, is considered a model organism for studying metazoan axis formation. The Wnt signaling pathways have been a central focus of genetic and molecular testing as they demonstrate conserved expression patterns for primary axis formation in bilaterians and prebilaterians. Nakamura et al. performed  experiments that allowed them to identify two deciding factors of Wnt3 localization to the Hydra head organizer. They include autoregulation by mediating inputs from Wnt/B-catenin pathways to activate HyWnt3 transcription and parallel repression of the autoregulatory element [1].

Video of Hydra Vulgaris Using Dark Field Polarized Microscopy


Body Patterning

Cnidarians and bilaterians have a common form of a developmental blastopore structure from which Wnt gene expression originates. The Wnt pathways play directing roles in establishing primary body axes [2]. They Hydra has a single body axis with a head at the oral end and a foot at the aboral end. There is an arch-shaped hypostome structure containing the mouth at the apical portion of the head. The lower portion of the head consists of tentacles. The head organizer of the Hydra is located at the tip of the hypostome.

Wnt/B-catenin signaling has been suggested responsible for autocatalytic activators and inhibitors with long distance acting potential [3]. While the Hydra Wnt (HyWnt) genes are more localized in expression to the organizer region, the Hydra transcriptional factors (HyTcf) and nuclear HyB-catenin decrease in a gradient like manner from most concentrated in the hypostome to  less concentrated along the oral-aboral axis. The Wnt3 ligand has been termed the “master ligand” in head organizer formation and regeneration [4]. While it has been understood that its transcription is influenced by HyTcf and HyB-catenin, the regulation of the ligand in the head organizer is still not fully understood. In the following experiments by Nakmura et al., this localization process was further investigated through experimentation with transgenes, revealing new components to these pathways.

Upstream Promoter Sequence

Figure 1: Expression of HyWnt3 mRNA (A) and HyWnt3FL-EGFP transgene (B) in the adult Hydra. Nakamura et al.

Nakamura et al. analyzed a 2,149 bp fragment, named HyWnt3FL,  in attempt to find the HyWnt3 cis-regulatory element. Using an EGFP reporter gene expressing the entire fragment, expression was shown in distal tip of the hypostome (Figure 1). After an alsterpaullone treatment, which inhibits GSK-3B activity that normally degrades B-catenin in Hydra, additional spots of the reporter gene were visualized within the body column. This suports the role of HyB-catenin in inducing Wnt3 expression.

To investigate the specific regions of the fragment responsible for activation and repression, a series of deletion experiments with the transgenes was performed. Significant reduction in expression was observed with deletion of the -1,201 to -604 bp region, while no expression was detected with deletion of -842 to -406 bp. These regions

Figure 2: EGFP expression in hypostome with -985 construct. Nakamura et al.

Figure 2: EFGP expression in hypostome with HyWnt3act. Nakamura et al.

together, therefore, spanned the sequences necessary for HyWnt3 expression. A -985 to -406 region demonstrated reporter expression specifically in the head organizer, signifying this region’s central role in HyWnt3 hypostome expression (Figure 2D). When a -985 to -386 bp fragment, named HyWnt3act, was expressed alone in the Hydra, reporter expression was shown in the upper and lower portin of the head (Figure 2E). Considering the HyWnt3FL fragment was restricted to the upper tip, the authors assumed there had to have been some sequence located downstream the -386 region necessary to restrict or repress HyWnt3 expression. Similarly HyWnt3act was expressed only in ectodermal cells while HyWnt3FL was expressed in ectodermal and endodermal cells, signifying the -386 downstream sequence again necessary for complete and proper expression.

Figure 2: EGFP expression in hypostome with Act-Wnt3prox. Nakamura et al.

Testing for Repression

To test the proposed repressive action of the -406 bp downstream fragment, named HyWnt3prox, the sequence was fused with the HyActin 5′ promoter sequence, typically active throughout the organism outside the head organizer region. Expression was found only in the apical tip of the hypostome, similar to the HyWnt3FL fragment expression (Figure 2K). This repressive function of HyWnt3prox was deemed enough to repress activity outside of the head organizer region, so named HyWnt3rep.

Transcription Factor Inputs and Additional Regulators

Figure 3: EMSA reveals recombinant His-tagged XTcf-3 protein binding to TCF sites 1, 2 and 3. Nakamura et al.

Nakamura et al. also considered the prospect of a positive feedback loop for HyWnt3 expression as a result of the Wnt/B-catenin pathway. The authors identified three possible T cell-specific factor (TCF) binding sites. Using EMSA, the three sites were shown to have specific binding affinity for His-tagged Xenopus Tcf-3 (Figure 3A).

Figure 3: EGFP expression after mutating all TCF sites (C), site 1 (D), site 2 (E), and site 3 (F). Nakamura et al.

To test the effects of TCF binding to the HyWnt3act enhancer region, the authors mutated the sites simultaneously and indepenently. Sites 2 and 3 were found to be essential for HyWnt3 expression while site 1 was not (Figure 3 C-F). Surprisingly, deletion of the site 1 sequence resulted in no reporter expression of Wnt3.  This suggested there is additional regulation and interaction involving the site 1 location that is necessary for proper Wnt3 expression. One such interaction, the authors mused, could be with the CREB/CRE transcriptional elements active during head regeneration in the Hydra as coactivators for Wnt3 expression [5].

Nakamura et al. wanted to test if HyB-catenin recruitment to the previously identified binding sites could be majorly influencing Wnt3 transcription. In using HyB-catenin-EGFP transgenes, the authors saw most expression in the hypostome. Alsterpaullone treatment revealed increased expression throughout the body. Chip analysis after alsterpaullone treatment revealed the previously isolated sites 2 and 3 with high expresion of HyB-catenin-EGFP while site 1  revealed none, as might have been expected (Figure 4E). Theses results are highly indicative of HyB-catenin and TCF site interaction in the regulation of Wnt3 [1].

Figure 4: Enrichment of sites 2 and 3 in ChiP of asterpaullone-treated HyB-catenin-EGFP transgenic animals compared to no enrichment of site 1 . Nakamura et al.

Finally, the authors addressed how HyB-catenin might be involved in stimulating Wnt3 expression. They targeted the repression of HyWnt3rep within the organizer region.  When Act-Wntprox transgenic animals underwent an alsterpaullone treatment, HyWnt3 signals were produced evenly throughout the body, however EGFP expression was revealed in a spotted pattern, similar to the HyWnt3FL experiment. As the HyWnt3prox alone does not show any expression, it is suggested that only at very high levels of Wnt singalling is HyWnt3rep repressed itself. The authors propose the HyActin 5′ sequence is “de-repressed”, thereby becoming active in the head. WntB-catenin activity, therefore, is believed to stimulate the repression of HyWnt3rep in the head organizer. As this type of regulation could not involve the TCF binding sequences that are part of the HyWnt3 sequence itself, other factors must be at play.

Model for transcriptional regulation of head organizer-specific HyWnt3 expression. Nakamura et al.

Conclusion and Critiques:

The experiments by Nakamura et al. significantly contributed to developmental biologists’ understanding of the intricate steps involved in regulating HyWnt3 expression. Using the Hydra as a model organism for metazoans, the signaling pathways for body axis formation are being uncovered one experiment at a time. Nakamura et al. have made great headway in providing an outline for autoregulation and repression feedback loops, as shown in the model above.

The authors were very thorough in following up with each discovery in the pathway to see how significant a role each factor may have in regulation. Examples include determining TCF sites 2 and 3, but not site 1, to interact with B-catenin and ruling out the possibility of the Wnt3 binding sequence having a direct role in regulating the extent of repression. This is where the studies end, however, leaving question as to how and what extent site 1 is involved in the activation process. The authors go on to briefly discuss multiple possibilites for long range inhibitors and the likelihood of input from additional transcription factors. However, their exact identities remain unknown and the questions are left open-ended. One proposed inhibitor included Hydra Dickkopf1/2/4 which was considered only briefly as it inhibits the ligand receptor and not transcriptional interactions. Though identified Wnt regulators have not been found that span all metazoan lineages, simlar transcriptional control mechanisms may be well conserved across phyla. Future research may pick up from here to answer more questions on Wnt3 transcriptional control, leading to further potential for  inducing regeneration in metazoans.

Resources:

1. Nakamura, Y. et al. “Autoregualtory and repressive inputs localize Hydra Wnt3 to the head organizer.” Proceedings of the National Academy of Sciences. vol. 108 no. 22: 9137- 9142. 2011.
Nakamura link

2. Petersen C.P., Reddien PW. “Wnt signalling and the polarity of the primary body axis.” Cell. 139: 1056-1066. 2009.
Petersen link

3. Gierer, A., Meinhardt, H., “A theory of biological pattern formation.” Kybernetik. 12:30-39. 1972.
Gierer link

4. Lengfeld, T., et al. “Multiple Wnts are involved in Hydra head organizer formation and regenration.” Dev Biol. 330: 186- 199. 2009.
Lengfeld link

5. Galliot, B. et al. “The cAMP response element binding protein is invovled in hydra regeneration.” Development. 121: 1205-1216. 1995.
Galliot link

Last updated 4/23/13 by Emily Aquadro

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