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The circadian gene Clock is required for the correct early expression of the head specific gene Otx2.
Morgan R
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The circadian cycle is a universal molecular mechanism for imposing cyclical control on cellular processes. Here we have examined the role of one of the crucial circadian genes, Clock, in early Xenopus development. We show that a dominant negative version of Clock can block the function of the endogenous Clock gene. Doing so during early development reduces Otx2 expression in a highly specific manner and results in anterior defects. Together with previous work (Green et al. (2001) Mech. Dev. 105-110), these results suggest that a positive regulatory loop exists between Clock and Otx2.
Fig. 1. CLδQ-GFP acts as a dominant negative inhibitor of endogenous Xclk activity. (A) The CLOCK/BMAL1 dimer binds the E-box consensus sequence in the reporter construct and activates transcription of the luciferase reporter gene. CLδQ-GFP can still bind BMAL1 and the heterodimer can still bind the E-box sequence, although without activating transcription. It thus acts in a dominate negative fashion. (B) Reporter gene activity in embryos from untreated eggs or eggs injected with CLδQ-GFP and / or Clk RNA as shown. The error bars shown are the standard deviations from the mean. The numbers in each sample are shown in the figure (n).
Fig. 2. Inhibiting endogenous Xclk activity disrupts anterior development. The heads of tailbud stage embryos which have developed from fertilised eggs which were either untreated (A) or injected with 400 pg of CLδQ-GFP RNA (B), or with 400 pg of CLδQ-GFP RNA and 1 ng of Clock RNA (C). The embryos wereallowed to develop to the tailbud stage. The proportion of embryos in each sample showing normal head morphology are shown in (D); error bars represent the standard deviation from the mean of three separate experiments.
Fig. 3. CLδQ-GFP blocks Otx2 expression. (A) RT-PCR analysis of genes expressed in discreet positions along the anteroposterior axis, from embryos injected with CLδQ-GFP or CLδQ-GFP and Clock RNAs as shown. The colour bars represent the expression domains of each of the genes shown. FB, forebrain; MB, midbrain; HB, hindbrain; SC, spinal chord. ef1α is included as a loading control. (B-D) In situ analysis of Otx2 and Hoxb-4 expression in the heads of tailbud stage embryos from eggs that were either uninjected (B), injected with 400 pg CLδQ-GFP RNA (C), or injected with 1 ng Xclk RNA and 400 pg of CLδQ-GFP (D). Otx2 expression in the midbrain is indicated by the blue line, and the anterior limit of Hoxb-4 expression is marked by the green line. e, eye.
Fig. 4. (Left) RT-PCR analysis of Otx2 and ef1α expression in neurula stage embryos. Fertilised eggs were injected with either CLδQ-GFP RNA, CLK-GR RNA, or both, and allowed to develop to the neurula stage. NIC, non-injected control. Before RNA extraction the embryos were treated with dexamethasone (dex) and cycloheximide (chx), either alone or in combination, as shown. ef1α was included as a loading control.
Fig. 5. (Right) Putative interactions between the circadian cycle and the wnt signal transduction pathway. The blue arrows indicate positive interactions and the red lines indicate inhibitory interactions. The boxed components are proteins and those in ovals are genetic loci. PER, Period; CKI, Caesin Kinase I; DVL, Dishevelled.
