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Fig. 5 | BMC Biology

Fig. 5

From: The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Fig. 5

beta-Catenin dependent expression of HAS-7. ac ISH analysis of HAS-7 expression after ALP treatment shows a global upregulation after 24 h and a shift towards the developing ectopic organizers along the body column after 48 h as compared to DMSO-treated controls (d). At 0 h after ALP wash (a) no change of the HAS-7 expression pattern compared to untreated controls (compare d and Fig. 2c) was evident. Scale bars = 150 μm. e HAS-7 expression is globally upregulated in the gastric region of a transgenic actin::HyWnt3 animal with ectopic axis. Scale bar = 250 μm. Arrows indicate head structures. Representatives of 10 hydras examined. f Quantitative real-time PCR analysis of HAS-7 expression confirms the upregulated expression in actin::HyWnt3 animals compared to steady-state AEP animals (lower panel). Inhibition of beta-Catenin by siRNA knockdown reduces HAS-7 expression levels compared to siGFP treated controls (lower panel). Relative expression level is given in 2(-ΔΔCt). Results represent mean +/− S.D. from 3 independent experiments, analyzed by t tests. *p < 0.05. The individual data values are shown in Additional file 14. g, h No detectable binding of Hydra TCF to the HAS-7 promoter. g ChIP analysis of the Hydra magnipapillata HAS-7 promoter. Upper site: Topography of the HAS-7 5′-untranslated region (nt 1 to 4529). The ATG indicates the translation start site. The position of a canonical TCF binding motif (5′-CTTTGTT-3′) is indicated by a blue bar. The localization of the 165-bp DNA segment flanked by the specific ChIP primer pair is visualized with a grey bar. Lower site: ChIP analysis of the Hydra HAS-7 promoter region using chromatin from untreated whole hydra animals (ctrl), and from animals treated with ALP. A polyclonal antibody directed against Hydra TCF was used for precipitation, followed by PCR amplification of the indicated fragment from the HAS-7 regulatory region. Reactions with normal rabbit serum (NRS) or total chromatin (Input) were used as controls (n = 2). PCR products were resolved by agarose gel electrophoresis, and visualized by ethidium bromide staining. h ChIP analysis of the HmTSP promoter performed under the same conditions as in g and used as a positive control. Upper site: Topography of the 3,000-bp HmTSP promoter (nt − 2191 to + 809). Black boxes depict the first two exons of the HmTSP gene. The arrow indicates the transcription start site of the HmTSP mRNA, and ATG the translation start site. The position of the tested canonical TCF binding motif (5′-AACAAAG-3′) is indicated by a red bar. The localization and size of a 164-bp DNA segment flanked by the specific ChIP primer pair is visualized with a grey bar. Lower site: ChIP analysis as described under (g) (n = 2).

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