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.