March 1, 2008;
Long- and short-range signals control the dynamic expression of an animal hemisphere-specific gene in Xenopus.
Little is known of the control of gene expression in the animal hemisphere
of the Xenopus embryo
. Here we show that expression of FoxI1e
, a gene essential for normal ectoderm
formation, is expressed regionally within the animal hemisphere
, in a highly dynamic fashion. In situ hybridization shows that FoxI1e
is expressed in a wave-like fashion that is initiated on the dorsal side of the animal hemisphere
, extends across to the ventral
side by the mid-gastrula stage
, and is then turned off in the dorsal ectoderm
, the neural plate
, at the neurula stage
. It is confined to the inner layers of cells in the animal cap
, and is expressed in a mosaic fashion throughout. We show that this dynamic pattern of expression is controlled by both short- and long-range signals. Notch
signaling controls both the mosaic, and dorsal/ventral
changes in expression, and is controlled, in turn, by Vg1
signaling from the vegetal mass. FoxI1e
expression is also regulated by nodal
signaling downstream of VegT
. Canonical Wnt signaling contributes only to late changes in the FoxI1e
expression pattern. These results provide new insights into the roles of vegetally localized mRNAs in controlling zygotic genes expressed in the animal hemisphere
by long-range signaling. They also provide novel insights into the role of Notch
signaling at the earliest stages of vertebrate development.
Notch signaling pathway
[+] show captions
References [+] :
Fig. 1. FoxI1e is expressed in a dorsal to ventral wave in the blastula and gastrula stages. (A) In situ hybridization for FoxI1e shows initial staining at stage 9.5 on one side of the embryo, and then spreading across the embryo. Its expression is always mosaic. (B) Embryos injected in the two dorsal, animal blastomeres at the 4-cell stage with 50 pg of β-Gal mRNA were stained with Red-Gal before in situ hybridization, showing the initial expression is on the dorsal side of the embryo. (C) Embryos were dissected into dorsal and ventral halves at stage 10 and frozen for real-time PCR. FoxI1e expression is enriched on the dorsal side at stage 10. Results are normalized to ODC expression levels. (D) Stage 11 embryos were stained for FoxI1e and sectioned. Staining with Wheat Germ Agglutinin defines a small population of FoxI1e-positive cells between the sensorial and epithelial layers of the ectoderm. Scale bars represent 200 μm, unless otherwise noted.
Fig. 3. VegT and Nodal signaling act at long-range to affect FoxI1e expression in the animal cap. (A) In situ hybridization in VegT-depleted and CerS-injected embryos shows FoxI1e is most upregulated in the animal cap rather than the vegetal mass at stage 10. Dorsal is the to the right in the bisected uninjected embryo. The VegT and CerS embryos did not dorsal axes. (B) Vegetal masses stripped of all mesoderm contamination dissected from control and CerS-injected embryos confirm that the vast majority of increase in FoxI1e expression is derived from non-endodermal tissue. Scale bars represent 200 μm.
Fig. 4. Vg1 is a long-range inhibitor of FoxI1e expression. (A, B) Depletion of Vg1 results in a 5-fold increase in FoxI1e expression at stage 10, resulting largely from an increase in expression in non-endodermal tissues. (C) Co-depletion of VegT and Vg1 does not increase the expression of FoxI1e in the vegetal mass over either one alone, indicating the presence of an unidentified inhibitor in the vegetal mass, or the absence of an activator. Dorsal is to the right in the bisected embryos shown. Scale bars represent 200 μm.
Fig. 5. Notch signaling is responsible for the initial dorsal restriction and mosaic expression of FoxI1e. (A) Maternal Xotch mRNA is depleted to 20–25% with 15 ng of thioate-modified DNA oligo. The level of Xotch remains low in the blastula and gastrula. (B) FoxI1e expression is 2- to 3-fold upregulated by depletion of Xotch. In situ hybridization shows an expansion of FoxI1e expression (C). (D) Injection of the constitutively active Notch Intracellular Domain (NICD) causes downregulation of FoxI1e relative to controls, and injection of the dominant negative construct Su(H)-DBM causes an upregulation of FoxI1e (E). (F) NICD upregulates the Notch target ESR-1, and Su(H)-DBM downregulates it. (G) In situ hybridization for FoxI1e comparing control and Su(H)-DBM injected embryos at stage 9.5 indicates that more, and in the most severe cases, all of the sensorial-layer animal cap cells express FoxI1e. Dorsal is to the right in all embryos shown. Scale bars represent 200 μm.
Fig. 6. Maternal Vg1 activates Notch signaling in the blastula to control FoxI1e expression. (A) Vg1-depleted embryos were injected with 50 or 500 pg of NICD mRNA at the 2-cell stage. NICD rescued the increase in FoxI1e expression caused by Vg1 depletion. (B) These results were confirmed by in situ hybridization for FoxI1e at stage 10, which shows an upregulation of FoxI1e in Vg1-depleted embryos, and a reversal of this upregulation by subsequent injection with NICD. The control embryo is oriented with dorsal to the right. Depletion of Vg1 results in a delay of gastrulation, and so the orientations of both the Vg1-depleted and the NICD-rescued embryos are indeterminate. Scale bars represent 200 μm. (C) Real-time PCR at stage 10 shows that the Notch target ESR-1 is downregulated in Vg1-depleted embryos relative to controls, indicating that Notch signaling depends on Vg1 at this stage. (D) 200 pg of Vg1 mRNA was unable to rescue the increase in FoxI1e expression induced by loss of Notch signaling by injection of 500 pg Su(H)-DBM mRNA.
Fig. 2. Wnt-dependent dorsal axis formation controls late, but not early, FoxI1e expression. Embryos injected with 40 ng β-Cat MO had reduced levels of direct targets Siamois and Xnr3 at stage 10 (A), and embryos injected with 50 pg β-Cat mRNA had increased levels (B). In control explants, the level of FoxI1e was higher in dorsal halves than ventral halves (C). The total level and distribution of FoxI1e was unchanged by β-Cat MO or mRNA (D–F). (G) Levels of FoxI1e mRNA at stages 9.5, 10, and 14 (compared to ODC mRNA levels at each stage) in embryos injected with either 100 pg BMP4 mRNA (upper panels), or 10, 40, or 160 pg of noggin mRNA (lower panels). Neither BMP4 overexpression, nor inhibition using Noggin, consistently affected the level of FoxI1e expression at the late blastula stage (stage 9.5). By the early gastrula stage (stage 10), BMP4 overexpression increased FoxI1e expression, but Noggin still had little effect. Expression of FoxI1e in the early neurula (stage 14) was increased by BMP4 overexpression and completely ablated by Noggin. This indicates that the early expression of FoxI1e is BMP-independent, but that the restriction of FoxI1e to the epidermis at neurulation is BMP-dependent. (H) Overall level of FoxI1e expression at stage 14 is unaffected by β-Cat MO. Embryos injected with 40 ng β-Cat MO at the 2-cell stage were injected with β-Gal at the 32-cell stage in the A1 blastomere. Red-gal staining, and FoxI1e in situ hybridization were carried out at stage 14. The anterior (A), posterior (P), dorsal (D), and ventral (V) regions of the embryo are marked in the control embryo, with the derivatives of the dorsal animal blastomere (A1), marked by the yellow arrowhead in the neural plate. Embryos lacking β-Cat (right panel) lack axes altogether. The red-gal positive cells (arrowed) mark the derivatives of the A1 blastomere. FoxI1e expression in β-Cat-depleted embryos persists in that clone of cells, indicating the β-Cat dependence of restriction of FoxI1e from the prospective CNS. The reddish cast toward the posterior end of the uninjected embryo is residual maternal pigment, not affected by bleaching. Scale bars represent 200 μm.
Abe, Activin-like signaling activates Notch signaling during mesodermal induction. 2004, Pubmed