The Zic4 positively regulated by Sp5 and Wnt/β-catenin
Zic4 appears positively regulated by Sp5 and Wnt/β
-catenin signaling in homeostatic conditions, as Zic4 expression is notably reduced in the tentacle zone after Sp5 or β-catenin knockdown by RNA interference (RNAi) (Fig. 1, D and E, and fig. S3A). To test Zic4 regulation in conditions where organizer activity gets reestablished in multiple spots (34), we performed reaggregation experiments from tissues knocked down for β-catenin (Fig. 1F and fig. S3B). In control conditions, Hydra reaggregates can regenerate and present axes that form tentacles emerging 5 days after the second electroporation (EP2). When β-catenin is down-regulated, the levels of Zic4 are reduced about twofold and the aggregates cannot form tentacles in the manner control animals do. The Wnt/β-catenin signaling pathway can also be ubiquitously activated by incubating the animals in alsterpaullone (ALP), a treatment that efficiently promotes stabilization of β-catenin in Hydra (35). After a 2-day treatment, we observed a dual regulation for Zic4, first a transient down-regulation in the apical region accompanied by an overall up-regulation along the body column, followed 2 days later by the appearance of an “Octopus” phenotype, characterized by multiple Zic4-expressing rings, each ring corresponding to the base of an ectopic tentacle (Fig. 1G and fig. S4). This dynamic expression pattern of Zic4 both in the tentacle zone and along the body column when Wnt/β-catenin signaling is up-regulated indicates that Zic4 is downstream of Wnt/β-catenin signaling.
The Zic4 promoter is positively regulated by Wnt/β-catenin and Sp5
To test whether Wnt/β-catenin regulates Zic4 directly, we characterized a 3505–base pair (bp) fragment upstream to the Zic4 transcription start site and produced a Zic4-3505:GFP transgenic line that drives green fluorescent protein (GFP) fluorescence in the tentacle zone mimicking the Zic4 expression pattern (Fig. 1H). Upon ALP treatment, the expression of GFP is enhanced in the upper body column, indicating a positive regulation of the Zic4-3505 promoter via canonical Wnt signaling (Fig. 1H). This regulation is possibly direct as the 3505-bp Zic4 upstream sequences contain four consensus T cell factor/lymphoid enhancer factor (TCF/LEF) binding sites (Fig. 2A). When expressed in HEK293T cells, the Zic4-3505:luciferase construct is up-regulated when human β-catenin, Wnt3, LRP6, or full-length Sp5 are overexpressed (Fig. 2, B and C). We produced a series of deletion mutants of the Zic4-3505 promoter (Fig. 2D) and noted a two-step reduction in the levels of luciferase in HEK293T cells in the presence of active β-catenin when the most upstream TCF-BS1 is deleted and then when the most proximal TCF-BS4 is additionally removed (Fig. 2E). In the presence of Hydra Sp5, we recorded the most significant reductions of luciferase activity when three regions are removed: (i) the most upstream 500 bp (−3505 to −2999), (ii) the −1985 to −1478 region, and (iii) the −978 to −463 region that contains TCF-BS4 and Sp5-BS5 (Fig. 2F). These data demonstrate a positive regulation by Wnt/β-catenin signaling and Sp5 on Zic4 expression, likely direct in the mammalian HEK293T cells. Overall, we concluded that Zic4 is an excellent candidate for a transcription factor directly regulated by Wnt/β-catenin and Sp5, during both homeostasis and regeneration in Hydra.
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