XB-ART-42789Dev Biol April 15, 2011; 352 (2): 367-81.
Siamois and Twin are redundant and essential in formation of the Spemann organizer.
The Spemann organizer is an essential signaling center in Xenopus germ layer patterning and axis formation. Organizer formation occurs in dorsal blastomeres receiving both maternal Wnt and zygotic Nodal signals. In response to stabilized βcatenin, dorsal blastomeres express the closely related transcriptional activators, Siamois (Sia) and Twin (Twn), members of the paired homeobox family. Sia and Twn induce organizer formation and expression of organizer-specific genes, including Goosecoid (Gsc). In spite of the similarity of Sia and Twn sequence and expression pattern, it is unclear whether these factors function equivalently in promoter binding and subsequent transcriptional activation, or if Sia and Twn are required for all aspects of organizer function. Here we report that Sia and Twn activate Gsc transcription by directly binding to a conserved P3 site within the Wnt-responsive proximal element of the Gsc promoter. Sia and Twn form homodimers and heterodimers by direct homeodomain interaction and dimer forms are indistinguishable in both DNA-binding and activation functions. Sequential chromatin immunoprecipitation reveals that the endogenous Gsc promoter can be occupied by either Sia or Twn homodimers or Sia-Twn heterodimers. Knockdown of Sia and Twn together, but not individually, results in a failure of organizer gene expression and a disruption of axis formation, consistent with a redundant role for Sia and Twn in organizer formation. Furthermore, simultaneous knockdown of Sia and Twn blocks axis induction in response to ectopic Wnt signaling, demonstrating an essential role for Sia and Twn in mediating the transcriptional response to the maternal Wnt pathway. The results demonstrate the functional redundancy of Sia and Twn and their essential role in direct transcriptional responses necessary for Spemann organizer formation.
PubMed ID: 21295564
PMC ID: PMC3065516
Article link: Dev Biol
Genes referenced: bag3 chrd.1 gsc myc myl1 nodal nodal1 sia1 sia2 tbxt wnt8a zic1
Morpholinos: sia1 MO2 sia2 MO2
Article Images: [+] show captions
|Fig.6. Siamois and Twin function redundantly in axial development and organizer formation. (A–L) At the 4-cell stage both dorsal blastomeres were injected with (B,D) a non-specific control morpholino oligonucleotide (NSMO, 50 ng), (E,G) a Sia-specific oligonucleotide (SiaMO, 25 ng), (F,H) a Twn-specific oligonucleotide (TwnMO, 25 ng), or (I,K) a lower dose combination of the Sia and Twn oligonucleotides (SiaMO + TwnMO, 25 ng + 25 ng) or (J,L) a higher dose combination of Sia and Twn oligonucleotides (SiaMO + TwnMO, 50 ng + 50 ng). (A–B,E–F,I–J) Whole embryo morphology (dorsal up and, anterior right) and (C–D,G–H,K–L) transverse histological sections (dorsal up) are shown at the tailbud stage. Dorsoanterior index (DAI) is indicated in the lower right corner for these representative embryos. Axial structures are indicated for the histological sections (n, notochord; sm, somitic muscle; nt, neural tube). Axial development was normal for embryos injected with the individual control, Sia or Twn MO, while coinjection of Sia and Twn MO resulted in severe axial defects, including loss of head structures, and reduction or loss of trunk and tail structures. Histological samples are presented for two examples of the double knockdown phenotype; (K) a partial loss of axial development with absence of notochord, somitic muscle crossing the midline, and mispatterning of the neural tube, and (L) a complete loss of axial development with no notochord, somitic muscle or neural tube. (M) Quantification of axial defects (DAI scores) observed for Control, NSMO, SiaMO, TwnMO and increasing doses of Sia+TwnMO. (N–L') Whole mount in situ hybridization analysis of gene expression at the early gastrula stage (stage 10.25). Embryos injected with 50 ng each of NSMO (S–W), SiaMO (X–B'), TwnMO (C'–G') or a combination of SiaMO and TwnMO (50 ng + 50 ng) (H'–L') were analyzed for organizer expression of Gsc (N,S,X,C',H') and Chordin (O,T,Y,D',I'), ventrolateral expression of Xwnt8 (P,U,Z,E',J'), panmesodermal expression of Xbra (Q,V,A',F',K'), and neural plate expression of Opal (R,W,B',G',L'). Shown are vegetal views with dorsal up (Gsc, Chordin, Xwnt8 and Xbra) and dorsal-vegetal views with dorsal up (Opal). Double knockdown of Sia and Twn together resulted in a reduction or loss of Gsc expression in 77% of embryos and a reduction or loss of Chordin expression in 100% of embryos.(A,C,N–R) Uninjected control embryos.|
|Fig.7. Siamois and Twin are required for Xwnt8 induction of ectopic axis formation. At the 4-cell stage both ventral blastomeres were injected with (C,D) a non-specific control morpholino oligonucleotide (NSMO, 50 ng), (E,F) a Sia-specific oligonucleotide (SiaMO, 25 ng), (G,H) a Twn-specific oligonucleotide (TwnMO, 25 ng), or (I,J) a combination of the Sia and Twn oligonucleotides (SiaMO + TwnMO, 25 ng + 25 ng). (B,D,F,H,J) At the 8-cell stage a single ventral blastomere was injected with Xwnt8 mRNA (5 pg). (A–J) Whole embryo morphology (dorsal up and, anterior right) is shown at the tailbud stage, with percentage of embryos displaying the representative phenotype and total embryos analyzed indicated in the lower right for each panel. (A) Uninjected control embryo.|
|Supplementary Figure. 3. Rescue of axial development in the Siamois-Twin double knockdown embryo. (B, D, F) At the 4-cell stage both dorsal blastomeres were injected with a combination of the Sia and Twn oligonucleotides (SiaMO + TwnMO, 25 ng + 25 ng). At the 8-cell stage a single dorsal blastomere was injected with 50 pg of (C, D) myc-Sia or (E, F) myc-Twn. myc-Sia and myc-Twn fully rescued axial development in double knockdown embryos (D, F), and resulted in mild dorsalization in control embryos (C, E). Whole embryo morphology (dorsal up and anterior right) is shown at the tailbud stage, with percentage of embryos displaying the representative phenotype and total embryos analyzed indicated in the lower right for each panel. (A) Uninjected control embryo.|
|Fig. 1. Siamois and Twin bind an identical conserved region within the Gsc proximal element. (A) Schematic of the Gsc promoter indicating sequence conservation within the proximal element (PE) across species. The P3 element and upstream half site are indicated by gray shading. DNase footprinting was performed on the Gsc promoter to identify regions protected by Sia and Twn (B,C). A double-stranded fragment of the Gsc promoter, radiolabeled on the top (B) or bottom (C) strand, was incubated with full-length Sia, Sia homeodomain (Sia HD), Twn homeodomain (TwnHD) or a mixture of the Sia and Twn homeodomains (S + T HD). Protected regions are indicated to the right of each autoradiogram (B,C) and summarized in schematic form (D). In addition to the major protected region containing the P3 site and the upstream half site, two minor protected regions (− 103 to − 93 and -15 bp to + 1 bp) were detected as well, but these did not contain apparent homeodomain binding sites and may be either non-specific or cryptic homeodomain binding sites. The region of protection for the top strand is overlined and for the bottom strand is underlined in (D), with the upstream half site and the P3 site indicated by gray shading. GA indicates a sequencing reaction run with purine terminators, providing a size ladder for DNAse cleavage products.|
|Fig. 2. The Gsc P3 element is required for stable binding and transactivation by Sia and Twn. (A) Sequence of oligonucleotide probes used in EMSA experiments with the P3 element and upstream half site indicated with gray shading. Mutated nucleotides are indicated with bold italics. (B) Increasing amounts of Sia protein was incubated with the indicated radiolabeled EMSA probes, and predicted monomer (M) and dimer (D) complexes were observed for the WT, 136 MT and 127 MT probes. Only the monomer complex was observed to form on the 2X MT probe and no complex formation was observed on the 3X MT probe (data not shown). (C) Assessment of the stability of the Sia–DNA complex. A constant amount of Sia protein was incubated with the indicated radiolabelled EMSA probe. Following a 20 min preincubation of Sia protein with radiolabelled probe (time 0), an excess of unlabelled WT competitor was added and complex formation was examined at the indicated times (5, 10, 20 and 30 min) following competitor addition. NP, no protein; F, free probe. (D) Requirement for the Gsc P3 element and upstream half site in Sia and Twn transactivation. At the one-cell stage Sia or Twn mRNA (100 pg) was injected into the animal pole and at the two-cell stage DNA for Gsc-Luciferase reporters (100 pg) containing the indicated forms of the Gsc promoter were injected together with DNA for 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 basal reporter activity in the absence of injected mRNAs. The mean and standard error for three independent experiments is presented.|
|Fig. 3. Siamois and Twin form homodimers and heterodimers through direct protein–protein interactions. (A) EMSA analysis of complex formation for Sia112–215 (lane 7), Twn HD (lane 2) or a combination of both proteins (lanes 3–6) bound to the WT Gsc probe. Twn HD concentration was constant (1 mM), while increasing concentrations of Sia112–215 were combined with Twn HD, as indicated at top in mM. Predicted complex formation indicated on right (M, monomer; D, dimer). (B) Protein interactions of purified Sia and Twn. GST pulldown analysis using purified full-length GST-Sia, His-Sia and His-Twn. Western blot analysis for His-tagged proteins indicating input proteins (lanes 1–2), lack of protein recovery with GST alone (lanes 3–4), and recovery of both His-Sia and His-Twn with GST-Sia (lanes 5–6). Protein size markers are indicated to the left in kD. (C–G) Crosslinking analysis of Sia and Twn dimerization. Homodimeric complex formation shown for Sia HD (C), Sia112–215 (D), Twn HD (E), and Sia112–215 and Sia HD (F), and heterodimeric complex formation for Sia112–215 and Twn HD (G). Predicted complex formation is indicated on the right (M, monomer; D, dimer; T, trimer), and protein size markers are indicated on the left in kD. Concentration of EGS (Ethylene Glycol-bis (succinic acid N-hydroxysuccinimide ester)) protein crosslinker (mM) is indicated at top. (H) Diagram of the Sia and Twn protein fragments used for the EMSA (A) and crosslinking (C–G) analyses.|
|Fig. 4. Siamois and Twin homodimers and heterodimers occupy the endogenous Gsc promoter. (A) Genomic regions recovered by chromatin immunoprecipitation for myc-Sia, myc-Twn or myc-SiaQ191E were evaluated by quantitative PCR (QPCR) for either the Gsc promoter or EF1α locus as control. The mean fold enrichment (normalized to uninjected samples) and standard error for three independent experiments are presented. (B) Genomic regions recovered by sequential chromatin immunoprecipitation were evaluated by QPCR for the Gsc promoter or Xmlc2 locus as control. Differentially tagged forms of Sia and Twn were coexpressed, samples were subjected to two rounds of immunoprecipitation, and recovered genomic sequences were analyzed by QPCR for each round. Coinjected mRNAs are indicated for myc-Sia, myc-Twn, GST-Sia and GST-Twn, and the order of the myc and GST immunoprecipitations are indicated as 1st IP and 2nd IP. As a control, a first immunoprecipitation with myc-Sia and a second immunoprecipitation with GST alone was also performed. Neither the Gsc nor Xmlc genomic regions were significantly recovered from the second immunoprecipitation. The mean fold enrichment (normalized to uninjected samples) and standard error for five independent experiments in presented.|
|Fig. 5. Siamois and Twin homodimers and heterodimers have indistinguishable transactivation and axis induction function in vivo. (A) At the one-cell stage the animal pole was injected with Sia, Twn or a mixture of both mRNAs at the indicated doses and at the two-cell stage with WT Gsc-Luciferase reporter (100 pg) and 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 basal reporter activity in the absence of injected mRNAs. The mean increase in luciferase activity and standard error for five independent experiments is presented. (B) At the 4-cell stage a single ventral blastomere was injected with Sia, Twn or a mixture of both mRNAs at the indicated doses. Embryos were scored for ectopic axis induction at the neurula stage. The partial axis class contained ectopic trunk structures extending anterior to the otic vesicle. The complete axis class contained trunk and head structures, including eyes and cement gland. The mean percentage and standard error for five independent experiments is presented. n, total embryos analyzed for each experimental condition.|
|Supplementary Fig. 1. The Siamois or Twin homeodomain is sufficient for DNA-binding and complex formation at the Gsc proximal element. Increasing amounts of purified Sia homeodomain (A) or Twn homeodomain (B) was incubated with the indicated radiolabeled EMSA probes. Probe sequences for wild-type and mutated forms of the Gsc proximal element are shown in Fig. 2A. Monomer (M) and dimer (D) complexes were observed for the WT, 136 MT and 127 MT probes. The monomer complex only was observed for the 2X MT probe and no complex formation was observed for 3X MT (data not shown). F, free probe.|
|Supplementary Fig. 2. Morpholino antisense oligonucleotides specifically block the translation and biological activity of Siamois and Twin. (A) In vitro translation reactions incubated with DNA constructs (1 μg) encoding native Sia or Twn, or myc-tagged forms of Sia or Twn, in the presence of oligonucleotides (100 ng) specific for Sia or Twn, or a non-specific control oligonucleotide (NS). Translation products were labeled with 35S-methionine, resolved by 12% SDS-PAGE, and visualized by autoradiography. Protein size markers are on the left. The Sia MO blocked translation of Sia, but not Twn. The Twn MO blocked translation of Twn, but not Sia. Neither oligonucleotide blocked translation of myc-Sia or myc-Twn, which have distinct upstream translation start sites. The NSMO oligonucleotide had no translation blocking activity for any of the proteins. (B–M) Inhibition of axis induction by Sia- or Twn-specific oligonucleotides. At the 4-cell stage both ventral blastomeres were injected with (E–G) a non-specific control morpholino oligonucleotide (NSMO, 25 ng), (H–J) a Sia-specific oligonucleotide (SiaMO, 25 ng), or (K–M) a Twn-specific oligonucleotide (TwnMO, 25 ng). At the 8-cell stage a single ventral blastomere was injected with 20 pg of (C,F,I,L) Sia, (D,G,J,M) Twn, (I, inset) myc-Sia, or (M, inset) myc-Twn mRNA. The Sia MO blocked axis induction by Sia (I), but not Twn (J). The Twn MO blocked axis induction by Twn (M), but not Sia (L). myc-Sia and myc-Twn were insensitive to the corresponding oligonucleotides and the NSMO oligonucleotide did not block axis induction for either Sia or Twn. Whole embryo morphology (dorsal up and anterior right) is shown at the tailbud stage, with percentage of embryos displaying the representative phenotype and total embryos analyzed indicated in the lower right for each panel. (B) Uninjected control embryo.|
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