Transcriptional integration of Wnt and Nodal pathways in establishment of the Spemann organizer

Abstract

Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. Here, we demonstrate transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain.

Fig. 1

Nodal and Wnt effectors synergistically activate the Gsc promoter. One-cell stage embryos were injected with 50 pg of Sia, Twn or Xnr1 mRNAs, or a mixture of Sia (50 pg) and Xnr1 (50 pg) or Twn (50 pg) and Xnr1 (50 pg). At the two-cell stage plasmid encoding Gsc reporter (100 pg; diagramed at top) was injected with CMV-Renilla Luciferase (10 pg). Animal explants prepared at the blastula stage were assayed for luciferase activity at the midgastrula stage. Values shown are normalized to Renilla luciferase activity, and represent fold activation of reporter activity in the absence of injected mRNAs. The mean and standard error for three independent experiments are presented. ^* Indicates p value <0.05.

Fig. 2

Nodal and Wnt effectors synergistically activate organizer gene transcription. Analysis of Gsc (A), (B), Cer (C), (D) or Chd (E), (F) transcript expression in animal cap explants in response to injection of (A), (C), (E) 50 pg Sia, 50 pg Xnr1, or 50 pg Sia and 50 pg Xnr1 or (B), (D), (F) 50 pg Twn, 50 pg Xnr1, or 50 pg Twn and 50 pg Xnr1. Animal explants were analyzed by quantitative RT-PCR at the gastrula stage for the expression of Gsc, Chd or Cer normalized to Ef1α. Control represents uninjected animal explants and WE represents intact embryos. ^* Indicates p<0.05 as compared to the Sia, Twn and Xnr1 conditions. The mean and standard error for six independent experiments is presented. Identical reactions without reverse transcriptase served as negative control (data not shown).

Fig. 3

Wnt pathway effectors occupy organizer gene promoters. Genomic regions recovered by chromatin immunoprecipitation (ChIP) from embryos injected with 50 pg myc-Sia, 50 pg myc-Twn or 50 pg of a DNA-binding inactive Sia (myc-SiaQ191E) were evaluated by quantitative PCR (QPCR) for the (A) Gsc, (B) Cer, or (C) Chd promoters. Immunoprecipitation using anti-myc antibody was performed on uninjected embryos (Control). The mean fold enrichment (normalized to uninjected samples) and standard error for five independent experiments is presented. The white bars represent QPCR for genomic Xmlc2 as a control.

Fig. 4

Nodal pathway effectors occupy organizer gene promoters. (A), (C), (E) Genomic regions recovered by ChIP for myc-FoxH1, or myc-FoxH1 coexpressed with Xnr1 (myc FoxH1+Xnr1) were evaluated by QPCR for the (A) Gsc, (C) Cer, or (E) Chd promoters. Immunoprecipitation using anti-myc antibody was performed on uninjected embryos (Control). The data presented represent three independent experiments. (B), (D), (F) Genomic regions recovered by ChIP for endogenous Smad2/3 in uninjected embryos or embryos expressing Xnr1 mRNA (+Xnr1) were evaluated by QPCR for the (B) Gsc, (D) Cer, or (F) Chd promoters. Rabbit IGG added to uninjected embryo extract serves as a negative control (IGG). The mean fold enrichment (normalized to uninjected samples) and standard error for three independent experiments is presented. The white bars represent QPCR for genomic Xmlc2 as a control. ^* Indicates p value <0.05 as compared to uninjected embryos.

Fig. 5

Siamois/Twin occupancy at organizer promoters is enhanced by Nodal signaling. Genomic regions recovered by ChIP for 10 pg myc-Sia, 10 pg myc-Sia and 50 pg Xnr1, 10 pg myc-Twn, or 10 pg myc-Twn and 50 pg Xnr1 were evaluated by QPCR for the (A) Gsc, (B) Cer or (C) Chd promoters. The white bars represent QPCR for genomic EF1α as a control. The mean fold enrichment (normalized to uninjected samples) and standard error for eight independent experiments is presented.

Fig. 6

Smad2/3 occupancy at organizer promoters is enhanced by Siamois/Twn and Nodal. Genomic regions recovered from ChIP for endogenous Smad2/3 in uninjected embryos (Control), or embryos expressing 50 pg Sia, 50 pg Twn or 50 pg Xnr1, or combinations of 50 pg Sia and 50 pg Xnr1 or 50 pg Twn and 50 pg Xnr1 were evaluated by QPCR for the (A) Gsc, (B) Cer, or (C) Chd promoters. The white bars represent QPCR for genomic Xmlc2 as a control. Smad2/3 association with the promoters is significantly enhanced (^*p value <0.05) in the presence of Xnr1 as compared to uninjected embryos. Smad2/3 association with the promoters is further enhanced (^*p value <0.05) in the presence of Sia and Xnr1 or Twn and Xnr1 as compared to Sia, Twn, or Xnr1 alone. The mean fold enrichment (normalized to uninjected samples) and standard error for six independent experiments is presented.

Fig. 7

Recruitment of p300 to organizer gene promoters by Wnt and Nodal pathway effectors. (A)–(C) At the one-cell stage the animal pole was injected with Xnr1 (A), Sia (B) or Twn (C), either alone or together with full length E1A or E1AΔ2-36 as a negative control. Two-cell embryos were injected with the Gsc reporter (100 pg) and CMV-Renilla Luciferase (10 pg) plasmids. Animal explants prepared at the blastula stage were assayed for luciferase activity at the midgastrula stage. Values shown are normalized to Renilla luciferase activity, and represent fold activation of reporter activity in the absence of injected mRNAs. The mean and standard error for three independent experiments are presented. ^*Indicates p value <0.05 as compared to Xnr1, Sia or Twn activation of Gsc reporter. (D)–(F) Genomic regions recovered by ChIP for myc-p300 (4 ng plasmid injected) either alone or coexpressed with 150 pg GST-Sia or 150 pg GST-Twn or 50 pg Xnr1 were evaluated by QPCR for the (D) Gsc, (E) Cer, or (F) Chd promoters. The white bars represent QPCR for genomic Xmlc2 as a control. The mean fold enrichment (normalized to uninjected samples) and standard error for six independent experiments is presented.^*Indicates p<0.05 when compared to myc-p300 alone.