Kofron M et al. (2004), New roles for FoxH1 in patterning the early emb...

XB-ART-2932

Development 2004 Oct 01;13120:5065-78. doi: 10.1242/dev.01396.

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New roles for FoxH1 in patterning the early embryo.

Kofron M , Puck H , Standley H , Wylie C , Old R , Whitman M , Heasman J .

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FoxH1 (Fast1) was first characterized as the transcriptional partner for Smad proteins. Together with Smad2/4, it forms the activin response factor (ARF) that binds to the Mix.2 promoter in Xenopus embryos. Foxh1 is expressed maternally in Xenopus. Depletion of maternal Foxh1 mRNA results in abnormalities of head and dorsal axis formation. We show that FoxH1 is required, together with XTcf3/beta catenin, to activate the zygotic expression of the nodal gene, Xnr3 in a Smad2-independent manner. In contrast, maternal FoxH1 acts as an inhibitor of Xnr5 and 6 transcription, preventing their upregulation on the ventral side of the embryo, by the maternal T-box transcription factor VegT. We conclude that maternal FoxH1 has essential, context-dependent roles in regulating the pattern of zygotic gene expression in the early embryo.

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Species referenced: Xenopus
Genes referenced: actl6a cer1 chrd ctnnb1 dkk1 fgf8 foxh1 foxh1.2 gal.1 gs17 gsc lhx1 mixer nodal nodal1 nodal2 nodal3.1 nodal3.2 nodal5 nodal5.2 nodal5.4 nodal6 odc1 sia1 smad2 sox17a tbxt tcf3 vegt ventx2.2
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	Fig. 5. FoxH1 regulates the correct spatio-temporal expression of Xnr5 and 6. (A) The relative expression level of Xnr5 in control versus FoxH1-depleted embryos at the stages shown assayed by real-time RT-PCR and normalized to ODC. The upregulation of Xnr5 in FoxH1-depleted embryos is rescued by the injection of 15 or 30 pg Foxh1 mRNA before maturation. (B) Xnr5 begins to be upregulated in FoxH1-depleted embryos (O) compared to controls (U) at the late blastula stage (stage 9). (C) In situ hybridization of hemisected control (top row) and FoxH1-depleted late blastulae (bottom row) for Xnr5 and Xnr6, showing the nuclear location and higher levels of expression caused by FoxH1 depletion. (D) The relative expression of Xnr5 and 6 in wild-type (U) versus FoxH1-depleted (O) explants at the early gastrula stage. ac: animal cap; eq: equatorial explant; vm: vegetal mass; we: one wild-type embryo. RNA was pooled from ten caps, or four vegetal or equatorial explants in each case. Xnr5 and 6 are both restricted to vegetal cells and are both more abundantly expressed in FoxH1-depleted explants compared to controls. (E) The relative expression levels of Xnrs 3, 5 and 6 in control (U) and FoxH1-depleted (O) embryos dissected into dorsal and ventral halves at the late blastula and early gastrula stages. Four ventral or dorsal half embryos were pooled for each RNA sample. U, uninjected; O, antisense oligo injected; D, dorsal halves; V, ventral halves. At both stages, Xnr5 and 6 are expressed at a higher level in ventral halves of FoxH1-depleted embryos than in control ventral halves. The accuracy of the dissection is shown by the expression of Xnr3 restricted to the dorsal halves. (F) Nodal signaling was inhibited in control and FoxH1-depleted oocytes by the injection of 500 pg of CerS mRNA into oocytes. Embryos were frozen at the late blastula and early gastrula stages and assayed for Xnr5 and Xnr6 mRNA expression. Xnr5 and 6 mRNA are expressed at higher levels in control embryos in which nodal signaling is blocked by CerS.
	Fig. 1. Antisense depletion of maternal FoxH1: 4 ng FoxH1 antisense oligo injected into oocytes causes a depletion of FoxH1 mRNA in oocytes that is maintained through early embryogenesis. (A,B) Control and FoxH1-depleted oocytes and embryos derived from the same batch of oocytes were cultured to the stages shown, frozen, and assayed by real-time RT-PCR. Expression levels were normalized to ODC. (A) No wave of zygotic transcription of Foxh1 is seen in control or FoxH1-depleted embryos at the gastrula and neurula stages. (B) The related family member Fast3 is a zygotic transcript, expressed in control and FoxH1-depleted embryos, at the same time as the marker of the mid-blastula transition, GS17. Oocytes and embryos were cultured, frozen, and assayed by real-time RT-PCR. Expression levels were normalized to ornithine decarboxylase (ODC). (C) ARE-luciferase activity is induced in animal caps by activin protein (10 ng/ml), and this induction is severely inhibited in FoxH1-depleted animal caps at the early gastrula stage. (D) ARE-luciferase activity is induced by endogenous nodal signaling in the vegetal (DNA Veg), or equatorial region (DNA EQ) of control embryos at the early gastrula stage. This activation is significantly reduced in FoxH1-depleted explants. (E) FoxH1 antisense oligo causes dose-responsive effects on head and axis formation. Oocytes injected with 2.5 and 3 ng of oligo develop as embryos with a headless phenotype. (F) The morphology of a FoxH1-depleted embryo at the swimming tadpole stage compared to control. (G) In histological sections, headless, FoxH1-depleted embryos at the late tailbud stage embryos have abnormal dorsal axes, lacking notochords and with somites fused across the midline (arrow). (H) Tailbud stage wild-type and FoxH1 embryos showing Xgal labeled progeny of one ventral cell injected at the four-cell stage, at the equator. Although FoxH1 depletion reduces the length of the embryo, the progeny of the ventral cell are in the same posterior and trunk locations as the control.
	Fig. 2. FoxH1 regulates Xnr3 gene expression in a specific fashion. (A) Embryos derived from oligo injected and control uninjected oocytes were frozen at 2-hour intervals during the blastula and gastrula stages, and assayed by real-time RT-PCR for the relative expression of organizer genes Xnr3, siamois, chordin and goosecoid. Expression levels were normalized to ODC. (B) 15 pg of Foxh1 mRNA injected into the vegetal area of wild-type embryos causes increased activation of ARE-luciferase reporter compared to control levels, while higher doses inhibit the activation of ARE-luciferase reporter. (C) The reintroduction of 15 or 30 pg of Foxh1 mRNA into FoxH1-depleted oocytes before maturation rescues the expression of Xnr3 and chordin mRNA at the early gastrula stage. (D) The reintroduction of 30 pg of Foxh1 mRNA into FoxH1-depleted oocytes before maturation rescues the headless phenotype.
	Fig. 3. Partial depletion of maternal XTcf3 and FoxH1 together causes a complete loss of Xnr3 expression. (A) Oocytes were injected with 6 ng of XTcf3 oligo, 2.5 ng of FoxH1 oligo or both, cultured for 58 hours and then fertilized by the host transfer method. Embryos derived from these oocytes were frozen at two-hourly intervals through the late blastula and gastrula stages and analysed by real-time RT-PCR for the expression of Xnr3 and goosecoid mRNAs. Double-depleted embryos have complete loss of expression of Xnr3 mRNA throughout the gastrula stages. In comparison, goosecoid mRNA was reduced but not eliminated in double-depleted embryos. (B) Sibling embryos to those analyzed in (A) were allowed to develop to the tailbud stage. Phenotypically, partially XTcf3-depleted embryos have an anteriorized phenotype (TCF3 depleted), Partial FoxH1-depleted embryos have reduced or absent anterior structures and double-depleted embryos have more severe axial defects, than those caused by the depletion of XTcf3 or FoxH1 alone. (C) Nodal signaling was inhibited in control and FoxH1-depleted oocytes by the injection of 500 pg of CerS mRNA into oocytes. Embryos were frozen at the late blastula and early gastrula stages and assayed for Xnr3 and Xnr1 mRNA expression. Xnr3 continues to be expressed in control embryos in which nodal signaling is blocked by CerS. In contrast, Xnr1 expression is dependent on nodal signaling. (D) Western blot analysis with anti-phospho-Smad2 (pSmad2) antibody of wild-type embryos and embryos injected as oocytes with 500 pg CerS mRNA and analysed at two stages during gastrulation. CerS blocks nodal signaling and completely prevents Smad2 phosphorylation (as well as Smad2* phosphorylation; a second phosphorylated form). The blot was reprobed for actin as a loading control.
	Fig. 4. FoxH1 depletion does not prevent animal caps from responding to activin or Xnr1. (A) Groups of ten control or FoxH1-depleted animal caps were dissected at the mid-blastula stage, treated with activin (1 Î¼g/ml) for 4 hours and frozen at the early gastrula stage and assayed for expression of nodal target genes including Mix.2, Fgf8, Xbra, goosecoid, chordin, Xbra and Xnr1. In the same experiment, Xnr1 mRNA (2 pg), was injected into wild-type or FoxH1-depleted embryos at the two-cell stage, and caps dissected as above. Expression levels were compared with one wild-type embryo at the early gastrula stage (control we). No significant changes in activin and nodal target gene expression were seen in FoxH1-depleted caps compared to control caps. (B,C,D) In one experiment, the degree of reduction of ARE-luciferase activity (B), and the level of induction of nodal-response genes in sibling animal caps (C) was measured at the gastrula stage, and the phenotype of sibling embryos was examined (D) at the tailbud stage. (B) FoxH1-depleted caps are unable to activate ARE-luciferase in response to activin (1 Î¼g/ml). (C) Sibling FoxH1-depleted caps respond normally to Xnr1 mRNA (2 pg) injected at the two-cell stage by expressing Mix.2, chordin, Fgf8, Xbra, Xnr2 and goosecoid as measured by real time RT-PCR. (D) Sibling embryos (right) develop with a headless phenotype compared to controls (three embryos on the left). (E) The expression of mesodermal and endodermal early zygotic genes analysed by real-time RT-PCR in a temporal series of control and FoxH1-depleted embryos, frozen at the blastula and gastrula stages. All of these results were repeated in a second experiment. Most endodermal and mesodermal genes show some reduction of expression. Xnr5 and 6 showed increased expression compared to control levels.
	Fig. 6. Maternal FoxH1, XTcf3 and VegT regulate Xnr5 and 6. (A) The relative expression level of Xnr5 and 6 in wild-type, FoxH1-depleted, XTcf3-depleted and FoxH1/XTcf3-depleted embryos at the stages shown assayed by real-time RT-PCR and normalized to ODC. Both FoxH1 and XTcf3 depletion enhances the expression of Xnr5 and 6 compared to controls, but the double depletion does not have an additive effect. (B) The relative expression level of Xnr3 and 5 in wild-type, FoxH1-depleted, VegT-depleted and FoxH1/VegT-depleted embryos at the stages shown assayed by real-time RT-PCR and normalized to ODC. Xnr5 over-expression in FoxH1-depleted embryos is dependent on maternal VegT. In comparison, Xnr3 expression is not dependent on VegT.
	Fig. 7. The misregulation of Xnr3 and Xnr5 in FoxH1-depleted embryos contributes to the later abnormalities. (A) Control and FoxH1-depleted embryos injected with 150 pg Xnr3 mRNA in one dorsal animal cell at the eight-cell stage. Xnr3 overexpression causes convergence extension defects in control embryos and partially rescues head formation in FoxH1-depleted embryos. (B) The relative expression levels of Xnr5 and Xbra mRNA in wild-type early gastrulae injected with 0, 600, 60, 6 and 0.6 pg of Xnr5 mRNA into one ventral cell at the four-cell stage. (C) The phenotype of sibling embryos of those frozen in (B).