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The reciprocal inhibition between two signaling centers, the Spemann organizer (dorsal mesoderm) and ventral region (mesoderm and ectoderm), collectively regulate the overall development of vertebrate embryos. Each center expresses key homeobox transcription factors (TFs) that directly control target gene transcription. Goosecoid (Gsc) is an organizer (dorsal mesoderm)-specific TF known to induce dorsal fate and inhibit ventral/ectodermal specification. Ventx1.1 (downstream of Bmp signaling) induces the epidermal lineage and inhibits dorsal organizer-specific genes from the ventral region. Chordin (Chrd) is an organizer-specific secreted Bmp antagonist whose expression is primarily activated by Gsc. Alternatively, chrd expression is repressed by Bmp/Ventx1.1 in the ventral/epidermal region. However, the regulatory mechanisms underlying the transcription mediated by Gsc and Ventx1.1 remain elusive. Here, we found that the chrd promoter contained two cis-acting response elements that responded negatively to Ventx1.1 and positively to Gsc. In the ventral/ectodermal region, Ventx1.1 was directly bound to the Ventx1.1 response element (VRE) and inhibited chrd transcription. In the organizer region, Gsc was bound to the Gsc response elements (GRE) to activate chrd transcription. The Gsc-mediated positive response on the chrd promoter completely depended on another adjacent Wnt response cis-acting element (WRE), which was the TCF7 (also known as Tcf1) binding element. Site-directed mutagenesis of VRE, GRE, or WRE completely abolished the repressive or activator activity of Ventx1.1 and Gsc, respectively. The ChIP-PCR results confirmed the direct binding of Ventx1.1 and Gsc/Tcf7 to VRE and GRE/WRE, respectively. These results demonstrated that chrd expression is oppositely modulated by homeobox TFs, Ventx1.1, and Gsc/Tcf7 during the embryonic patterning of Xenopus gastrula.
Figure 1. Ectopic expression of Gsc and Ventx1.1 differentially regulates the dorsal mesoderm (organizer) gene expression during gastrula. (A) The cloned chrd(-2250) promoter mapped into Pgl3 luc/eGFP vector. (B,C) The Xenopus embryos were injected with Gsc (B) and Ventx1.1 (C) (1 ng/embryo) at the one-cell stage. The reporter assay was performed at stage 11 in whole embryos (WEs). (C,D) To examine the endogenous expression, embryos were co-injected with Gsc and Ventx1.1 (1 ng/embryo) at the one-cell stage. RT-PCR was performed at stage 11 in Wes (D) and ACs (E). -RT referred to control reaction without reverse transcriptase.
Figure 2. The chrd(-2250) promoter region contains the positive and negative response elements for Gsc and Ventx1.1., respectively. (A) chrd(-2250) was injected with or without gsc (1 ng/embryo) and ventx1.1 (1 ng/embryo) at the one-cell stage. The luciferase reporter gene assay was performed at stage 11. (B) The quantification of eGFP relative fluorescence was analyzed with the embryos shown in (C). The chrd(-2250)eGFP was injected with or without gsc (1 ng/embryo) and ventx1.1 (1 ng/embryo) at the one-cell stage, and fluorescent analysis was performed at stage 10.5. ). * p ≤ 0.1, **** p ≤ 0.0001, and n.s. denotes nonsignificant values.
Figure 3. chrd promoter contains VRE. (A) Schematic representation of serially deleted chrd promoter constructs. (B) Relative luciferase activity of serially deleted chrd promoter constructs with or without Ventx1.1. (C) ChIP-sequencing coverage plot of Ventx1.1 within the chrd promoter region. (D) Schematic representations of site-directed mutagenesis and targeted nucleotides are italicized, underlined, and shown in red. (E) Relative luciferase activities of chrd(-2250), chrd(-2250)mVRE, chrd(-1473), and chrd(-1473)mVRE, with or without Ventx1.1. (F) The location of designed primers for VRE and the internal negative control (C). (G) Chromatin immunoprecipitation (ChIP) assay was performed to test the occupancy of Ventx1.1-Flag to VRE within the promoter region of chrd. ** p ≤ 0.01, **** p ≤ 0.0001, and n.s. denotes nonsignificant values.
Figure 4. Site-directed mutagenesis of GRE1 and WRE within the chrd promoter completely abolishes Gsc-mediated transcriptional activation. (A) Relative luciferase activity of serially deleted chrd promoter constructs with or without Gsc. (B) Chrd promoter (-2250 to -2214 bps)with highlighted GRE1 and WRE (dotted boxes). The bottom arrowhead indicates the sequences of chrd(-2250) and chrd(-2239) reporter constructs. (C) Schematic representation of the site-directed mutagenesis scheme; targeted nucleotides are shown in italics and red. (D) Relative luciferase activity of chrd(-2250)mGRE1 and chrd(-2250)mWRE constructs with or without Gsc. **** p ≤ 0.0001, and n.s. denotes nonsignificant values.
Figure 5. Tcf7 binds to WRE to activate chrd transcription. (A) Relative luciferase activity of chrd(-2250) promoter constructs with Gsc and Tcf7. (B) Relative luciferase activity of chrd(-2250)mGRE1 and chrd(-2250)mWRE constructs with Tcf7. (C) Schematic representation of designing the ChIP-PCR primer for GRE, WRE, and the internal negative control. (D) Chromatin immunoprecipitation (ChIP) assay was performed to test the occupancy of Gsc-Flag and Tcf7-Myc to GRE/WRE within the promoter region of chrd. * p ≤ 0.1, **** p ≤ 0.0001, and n.s. denotes nonsignificant values.
Figure 6. A putative model of Ventx1.1 and Gsc/Tcf7-mediated regulation of chrd transcription. A schematic representation of Ventx1.1-mediated negative regulation of chrd transcription by direct binding to VRE within the chrd promoter region during gastrula for ectoderm specification in Xenopus embryos. Whereas in the organizer (or dorsal mesoderm) region, Gsc interacts with GRE/WRE for activation. Tcf7 specifically binds to the WRE and activates chrd transcription.
Figure S1. Serially deleted chrd promoter constructs injected with and without gsc mRNA at the one-cell stage. The relative luciferase activity was measured at stage 11.
Figure S2. Identification of GRE1 within the chrd promoter. (A and B) The systematic representation of two targeted point mutations (namely GRE1 and GRE2, shown in the dotted box) was generated by site-directed mutagenesis in the upstream 11 bp (from -2250 to -2239) within the chrd promoter. (C) The mutated chrd(-2250)mGRE1, chrd(-2250)mGRE2, and wild-type chrd(-2250) promoter constructs were then injected with and without gsc mRNA at the one-cell stage. The relative luciferase activity was measured at stage 11.
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