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Biochem Biophys Res Commun
2004 Feb 27;3151:100-6. doi: 10.1016/j.bbrc.2004.01.019.
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Inhibition of FGF signaling causes expansion of the endoderm in Xenopus.
Cha SW
,
Hwang YS
,
Chae JP
,
Lee SY
,
Lee HS
,
Daar I
,
Park MJ
,
Kim J
.
???displayArticle.abstract??? Fibroblast growth factor (FGF) is established as an initiator of signaling events critical for neurogenesis and mesoderm formation during early Xenopus embryogenesis. However, less is known about the role FGF signaling plays in endoderm specification. Here, we show for the first time that endoderm-specific genes are induced when FGF signaling is blocked in animal cap explants. This block of FGF signaling is also responsible for a significant enhancement of endodermal gene expression in animal cap explants that are injected with a dominant-negative BMP-4 receptor (DNBR) RNA or treated with activin, however, neural and mesoderm gene expression is diminished. Consistent with these results, the injection of dominant-negative FGF receptor (DNFR) RNA expands endodermal cell fate boundaries while FGF treatment dramatically reduces endoderm in whole embryos. Taken together, these results indicate that inhibition of FGF signaling promotes endoderm formation, whereas the presence of active FGF signaling is necessary for neurogenesis/mesoderm formation.
Fig. 1.
(A) Truncated FGF receptor induces endoderm markers in animal cap explants. RT-PCR analysis of animal caps dissected at stage 8.5 and cultured until stages 11 and 24 to measure fate markers. EF1-α was used to normalize cDNA samples. The RT(−) lane contains all reagents except reverse transcriptase and is used as negative control. (A) Mixer, an early endodermal marker, and edd (endodermin), a late pan-endoderm marker, were induced by DNFR RNA in a dose-dependent manner. Actin, a mesoderm marker, was not induced. N-CAM, a late neural marker, was detected in dominant-negative BMP-4 receptor (DNBR)-injected animal cap. DNFR RNA (1–5 ng/embryo), wild-type FGF receptor (WTFR) (1 ng/embryo) RNA, and DNBR RNA (1 ng/embryo) were injected radially into each blastomere at two-cell stage. (B) Animal caps treated with SU5402, a chemical inhibitor of FGFR1a, also displayed endoderm marker induction in a dose-dependent fashion. (B) Induction of endoderm by DNFR expression or SU5402 treatment in activin-treated or DNBR RNA-injected animal caps. FGF signal inhibition (DNFR or SU5402) can enhance edd gene expression in the activin or DNBR-treated animal cap explants. DNFR or DNBR RNA-injected animal caps were dissected from embryos injected with 3 ng DNFR RNA or 1 ng DNBR RNA, respectively. Activin was added to a final concentration of 0.15 ng/ml (general mesoderm marker genes were induced at this concentration) and SU5402 was added to a final concentration of 10 μM. Animal caps were cultured until stage 24. (C) In situ hybridization using an edd probe. Left, uninjected explants hybridized with antisense edd probe. Center, an embryo injected with DNFR RNA. Strong expression of edd was detected when compared with sense probe treated explants (right). The explants in the center and right panels were derived from embryos injected with 3 ng DNFR RNA.
Fig. 2.
(A) Dose-dependent phenotype from injection of DNFR RNA in animal hemisphere (a) or vegetal hemisphere (b). The injection of DNFR RNA injected at varying concentrations into two-cell stage embryos and cultured until stage 34. (a) Animal hemisphere injected groups show a reduction of posterior structures and open neural tube defects in a dose-dependent fashion consistent with a previous report [22]. (b) In the vegetally injected groups, the abdominal structure was expanded with increasing doses of DNFR RNA. In contrast, mesoderm and dorsal structures were not significantly affected in this group. (B) β-Gal lineage trace of vegetal or animal pole-injected embryos. As expected, X-Gal staining was observed in the notochord region of embryos when β-Gal mRNA was injected into the animal pole, while X-Gal staining was localized within the abdominal region upon vegetal pole injection. (C) DNFR or XeFGF RNAs were injected into the animal hemisphere at the two-cell stage. An extended tail was observed in the XeFGF-injected embryos. DNFR-expressing embryos lacked most posterior structures. (D) DNFR or XeFGF RNAs were injected into the vegetal hemisphere at the two-cell stage. General ventral structures disappeared in the XeFGF RNA-injected embryos, while the DNFR RNA-injected embryos had expanded ventral structures. (E) RT-PCR analysis. In the XeFGF RNA-injected embryos, mesoderm markers were up-regulated and endoderm markers were down-regulated, and the reverse was true of DNFR-expressing embryos. The results suggest that FGF signaling may play a role in the formation and maintenance of mesoderm and a barrier for excessive expansion of the endoderm. RT(−) lane was omitted. (F) Sections of embryos that underwent DNFRor XeFGF RNA injection into the vegetal hemisphere. As shown, endodermtissue was greatly expanded when compared to control siblings. Paraffin-embedded sectioning was performed at a thickness of 10 μm at comparable depths. Scale bar: 100 μm (G). The expansion of endoderm in DNFR RNA-injected embryos. Whole-mount in situ hybridization of early gastrula (a and b) was performed using antisense Mixer probe as an endoderm marker. (a) Uninjected embryo. (b) DNFR RNA (3 ng/embryo)-injected embryos showed enlargement of endodermtissue. Whole-mount in situ hybridization of early gastrula (c and d) was performed using antisense XmyoD probe as an early mesoderm marker. (c) Uninjected embryo. (d) The embryos injected with DNFR RNA (3 ng/embryo) exhibited diminished mesodermtissue. The dashed circle indicates the boundary between sub-blastoporal endoderm and supra-blastoporal endoderm. (a–d; vegetal pole view).
Fig. 3.
Blocking FGF signaling in animal cap explants induced endoderm that lacked mesoderm inducing activity. (A) Tissue recombination technique. (B) DNFR-expressing recombinants (DNFR animal cap/control animal cap) also expressed early and later endoderm genes, Mixer and edd, as expected. However, the recombinants did not induce the early mesoderm marker, Xbra, nor the late mesoderm markers, actin and globin. An early ventralmesoderm marker (GATA2) was detected in the DNFR animal cap/control animal cap recombinants. This result indicates that the absence of FGF signaling in animal cells generates endoderm, but it is not competent to induce mesoderm in adjacent tissue. RT(−) lane was omitted.
Fig. 4.
The proposed model: the role of FGF signaling as a regulatory factor in mesendoderm formation. In the two-step model of mesendoderm formation [5] and [11], maternal factors like VegT induce TGF-β-related signaling molecules and these molecules subsequently induce mesendoderm. A modified model is proposed to explain how similar signals can induce two different germ layers and factor(s) that differentiate these two germ layers. In the proposed model, the spatially restricted existence of FGF signaling in the equatorial region acts as a natural barrier to prevent this area from succumbing to a endoderm fate in early embryogenesis. The absence of FGF signaling in the vegetal area is proposed to allow the proper microenvironment for endoderm formation driven by signals within the vegetal cells. In contrast, FGF signaling in the equatorial region is a competence factor for adjacent mesoderm formation in response to vegetal cell signals.
