Cha SW , McAdams M , Kormish J , Wylie C , Kofron M .

R01 HD045737 NICHD NIH HHS , R01HD45737 NICHD NIH HHS

	Figure 1. The spatial and temporal expression pattern of Foxi2 and its depletion at blastula stage. (A) RT-PCR to show relative expression levels of Foxi2 and VegT in either whole oocytes or dissected animal and vegetal halves. Foxi2 and VegT have reciprocal expression domains, with Foxi2 being highly enriched in the animal hemisphere. (B) In situ hybridization of tissue section at the late blastula stage to show distribution of Foxi2 mRNA. (C) RT-PCR analysis of developmental time course to show absence of early zygotic expression of Foxi2. (D) RT-PCR to show degree of depletion of Foxi2 mRNA by antisense oligo. (E�F) Degree of depletion of Foxi2 protein by mRNA depletion; a western blot of oocytes and early stages is shown in (E), and by immunostaining at the late blastula stage in (F). Scale bar in (F) = 50 �m. High magnification Foxi2 immunostaining images from Uninjected (F′) and Foxi2 depleted (F″) embryos. Scale bars in F′ and F″ = 20 �m. doi:10.1371/journal.pone.0041782.g001
	Figure 2. Expression of Foxi1e, as well as early ectodermal markers, requires Foxi2. (A�C) Show effect of Foxi2 depletion of Foxi1e mRNA levels at the late blastula and early gastrula stages (A), and on Foxi1e protein levels by western and by densitometric analysis of the western blot (A: middle panels) and immunostaining (B). (A) also shows by western blotting that the anti-Foxi1e antibody cross-reacts with Xenopus Foxi1e protein (lower panel). (C) Shows by RT-PCR the reduced levels of expression of ectodermal genes including Xlim5, E-cadherin, Sox2 and Cytokeratin in control and Foxi2-depleted embryos at the late blastula and early gastrula stages. (D) RT-PCR shows that expression levels of the mesodermal marker Xbra, endodermal marker Xsox17a and ectodermal marker, KLFn are unaffected by Foxi2 depletion. (E�F) show that reduction in expression of the ectoderm marker genes E-cadherin (E-cad), Foxi1e, FoxJ1, and grainyhead-like 3 (GHL3) caused by Foxi2 depletion are rescued by subsequent injection of Foxi2 mRNA (10 pg dose) in both whole embryos (E) and animal caps (F). Scale bar in (B) = 50 �m. doi:10.1371/journal.pone.0041782.g002
	Figure 3. The phenotype of Foxi2-depleted (Foxi2 KO7) embryos. (A) Foxi2-depleted embryos were normal at the blastula stage (left panel) but showed highly delayed and incomplete gastrulation and 37/45 embryos showed major defects at the tail bud stage compared with controls, of which 25/26 were normal (right panel). (C) In Nieuwkoop recombinant assays, Foxi2-depleted animal caps expressed reduced levels of ectodermal markers. (D) Animal caps from Foxi2-depleted embryos express mesodermal markers in response to signals from vegetal masses. doi:10.1371/journal.pone.0041782.g003
	Figure 4. Foxi2 protein directly binds to the Foxi1e promoter. (A) Luciferase activity in blastulae after injecting Xenopus tropicalis Foxi1e promoter-luciferase construct into either animal or vegetal blastomeres at 8-cell stage embryos. (B) Foxi1e promoter activity was measured in blastulae derived from control (uninj) or Foxi2-depleted (4 ng and 5 ng) oocytes. (C,D) Chromatin immunoprecipitation assay. (C) A schematic diagram of Xenopus tropicalis Foxi1e promoter shows potential Foxi2 binding sites and ChIP PCR primer amplicons. Real-time PCR shows significantly higher signal on primer pair −1982:−1734 after immunoprecipation by 6-myc-tagged Foxi2 protein. (D) A schematic diagram of Xenopus laevis Foxi1e promoter shows potential Foxi2 binding sites and ChIP PCR primer amplicons. Real-time PCR shows significantly higher signal on primer pair −1540:−1391 after immunoprecipation by myc-tagged Foxi2 protein. (E) Luciferase activity in blastulae after injecting either wild-type (WT) Xenopus tropicalis Foxi1e promoter constructs or a construct lacking the Foxi2 binding region (Deletion). While wild-type promoter showed its activity (WT Ani), the construct lacking the Foxi2 binding site mutated promoter showed a basal level of promoter activity (Deletion Ani). (F) Luciferase activity in blastulae after injecting Wild-type and Deletion Xenopus tropicalis Foxi1e promoter constructs into vegetal blastomeres. Foxi1e promoter construct lacking the Foxi2 binding site showed a basal level of promoter activity (Deletion Veg 8) while Foxi2 overexpression in vegetal blastomeres had no effect on this mutated construct (Deletion+Foxi2 Veg 8) while wild-type promoter showed increased luciferase activity upon overexpression of Foxi2 (WT+Foxi2 Veg 8). doi:10.1371/journal.pone.0041782.g004
	Figure 5. Spatial expression of Foxi1e is dependent on Foxi2. (A) The level of expression of Foxi1e is reduced in Foxi2-depleted animal caps (Foxi2 depleted caps) compared to the caps from control siblings (Uninj caps st.10) at the gastrula stage. Both dissociated superficial and deep cells from Foxi2-depleted embryos expressed reduced levels of Foxi1e mRNA. Note that the expression level of Foxi1e in dissociated superficial cells is much higher than in intact animal caps (Uninj superficial cells). (B) Maternal depletion of Dishevelled2 (Dvl2−) showed upregulation of Foxi1e expression. Double depletion of Foxi2 and Dvl2 (Foxi2−/Dvl2−) showed similar level of expression to Foxi2-depleted embryos (Foxi2−). (C) Maternal depletion of Vg1 (Vg1−) showed upregulation of Foxi1e expression. Double depletion of Foxi2 and Vg1 (Foxi2−/Vg1−) showed a level of expression similar to Foxi2-depleted embryos (Foxi2−). (D) Upper panel; Maternal depletion of transcription factor Mastermind1 (Mam1−) caused upregulation of Foxi1e expression. Double depletion of Foxi2 and Mastermind1 (Foxi2−/Mam1−) showed a similar level of expression as Foxi2 depleted embryos (Foxi2−). Lower panel; The overexpression of Supressor of Hairless (SuH) DNA binding mutant (SuH DBM) mRNA caused upregulation of Foxi1e expression. Foxi1e expression in Foxi2 depleted+1.2 ng of SuH DBM mRNA injected embryos showed similar level of expression as Foxi2 depleted embryos (Foxi2−). doi:10.1371/journal.pone.0041782.g005
	Figure S1. Foxi1e promoter construct is responsive to perturbations of Notch signaling but is not responsive to the influence of Nodal signaling. Either control (FL) or a construct lacking the Foxi2 binding region (Mut) Xenopus tropicalis Foxi1e promoter-luciferase constructs were injected into animal blastomeres of 8-cell stage embryos. When reporter-injected embryos are co-injected with mRNA encoding the Notch intracellular domain (NICD) to activate Notch signaling, there is a 5-fold reduction in luciferase activity of the wild-type promoter construct. Ectopic activation of Notch signaling also gives a 2.5-fold reduction in the activity of the promoter construct that lacks the Foxi2 binding domain (Mut). Foxi1e luciferase activity is unaffected by perturbing Nodal signaling via injection of Cerberus Short (CerS) mRNA. doi:10.1371/journal.pone.0041782.s001

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