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Figure 1
Axis formation is affected by siamois fusions. (A) Schematic of the siamois fusion constructs. A C-terminal region of siamois (residues 122â246), containing the homeodomain (HD), was fused to the engrailed repressor (residues 1â298) (Eng-Sia) or the VP16 activator (residues 410â490) (VP16-Sia). At the four-cell stage, one ventral (BâE, JâM) or two dorsal (FâI, NâQ) blastomeres were injected with 30 pg of β-galactosidase (B, F, J, N), siamois (C, G, K, O), VP16-Sia (D, H, L, P), or Eng-Sia (E, I, M, Q) mRNA. (JâQ) Transverse sections. See Table 1 for quantitation.
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Figure 2
Rescue of axial defects resulting from Eng-Sia injection. At the four-cell stage, both dorsal blastomeres were injected with 30 pg of Eng-Sia (A–D) in combination with 1 ng of β-galactosidase (A), 5 pg of Xwnt8 (B), 1 ng of β-catenin (C), or 100 pg of siamois (D) mRNA. (E) As a control, both dorsal blastomeres were injected with 1 ng of β-galactosidase mRNA. See Table 2 for quantitation. In a parallel experiment, ventral injection with Xwnt8, β-catenin, or siamois mRNA induced axis formation (data not shown).
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Figure 3
Organizer formation is affected by siamois fusions. At the two-cell stage, both blastomeres were injected with 30 pg of β-galactosidase, siamois, VP16-Sia, or Eng-Sia mRNA, and embryos were harvested at stage 10.25 (early gastrula). (A–D) Vegetal view of injected embryos analyzed by in situ hybridization for goosecoid expression. (A) β-Galactosidase had no effect on the organizer-specific expression of goosecoid (n = 24). Siamois (B) and VP16-Sia (C) resulted in expansion of goosecoid expression in 79% (n = 24) and 88% (n = 24) of embryos, respectively. (D) Eng-Sia resulted in a reduction or loss of goosecoid expression in 83% (n = 24) of embryos. (E) Embryos were harvested at the gastrula stage and processed for reverse transcription–PCR analysis of the organizer genes noggin (Nog), Xnr3, chordin (Chd), follistatin (Xfs), and cerberus (Cer), the ventrolateral gene Xwnt8, the panmesodermal gene brachyury (Xbra), endogenous siamois (Sia), and the ubiquitous EF1α. Injection of siamois (lane 3) or VP16-Sia (lane 4) inhibited Xwnt8 expression and enhanced expression of noggin and chordin, without affecting Xnr3, follistatin, cerberus, siamois, or brachyury expression. Eng-Sia (lane 5) inhibited expression of noggin, Xnr3, chordin, follistatin, and cerberus, enhanced Xwnt8 expression, and had no effect on siamois or brachyury. EF1α is a control for RNA recovery and loading. β-Galactosidase-injected embryos (lane 2) served as a positive control, while an identical reaction without reverse transcriptase controlled for PCR contamination (lane 1).
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Figure 4
Eng-Sia inhibits axis induction by the wnt pathway. At the four-cell stage, one ventral blastomere was injected with 30 pg of β-galactosidase (A–F) or Eng-Sia (G–L) in combination with 5 pg of Xwnt8 (B, H), 1 ng of β-catenin (C, I), 30 pg of siamois (D, J), 200 pg of noggin (E, K), or 200 pg of chordin (F, L) mRNA. Axis induction was observed in response to Xwnt8, β-catenin, siamois, noggin, and chordin. Eng-Sia blocked axis induction by Xwnt8, β-catenin, and siamois but not by noggin and chordin. Neither β-galactosidase nor Eng-Sia induced axis formation. See Table 4 for quantitation. In a parallel experiment, dorsal injection of Eng-Sia inhibited axis formation (data not shown).
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Figure 5
Siamois activates the goosecoid promoter via a wnt-responsive regulatory element. A 50-bp wnt-responsive proximal element (PE) is located between bases −155 and −105 of the goosecoid promoter (46). Luciferase reporter constructs containing 155 bp of promoter sequence (including PE) or the 104-bp minimal promoter were tested for responsiveness to Xwnt8, siamois, Eng-Sia, or a mixture of mRNAs. At the four-cell stage, one ventral blastomere was injected with 200 pg of the −155 or −104 reporter plasmid in combination with 50 pg of β-galactosidase, Xwnt8, siamois, or Eng-Sia mRNA, or mixtures of Eng-Sia and Xwnt8 or siamois mRNAs. At the gastrula stage, extract was prepared and luciferase activity was measured. Basal activity of uninjected embryos was subtracted for all values, averages were determined for duplicate samples, and values were normalized to the activity of the −104 reporter coinjected with β-galactosidase. The data presented are representative of three experiments.
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