Ishimura A et al. (2000), Involvement of BMP-4/msx-1 and FGF pathways in ...

XB-ART-10395

Dev Growth Differ 2000 Aug 01;424:307-16. doi: 10.1046/j.1440-169x.2000.00514.x.

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Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo.

Ishimura A , Maeda R , Takeda M , Kikkawa M , Daar IO , Maéno M .

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The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription-polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4-injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 cer1 egr2 en2 hoxb9 msx1 ncam1 nog otx2

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	Fig. 1. Xmsx-1 and neural-inducing genes antagonized mutual expression. (A,B) The expression of noggin in a control uninjected stage 11 embryo (A) and an embryo injected with Xmsx-1 ribonucleic acid (RNA; 5 ng/embryo) in two dorsal blastomeres at the 4-cell stage (B). Fixed embryos were processed for whole-mount in situ hybridization analysis. Note that the expression of endogenous noggin at the gastrula stage was completely suppressed by the misexpression of Xmsx-1. (C,D) The expression of Xmsx-1 in animal cap explants. Two-cell stage embryos were injected with noggin (C) or cerberus (Xcer, (D)) RNA, and the animal caps were excised at stage 10 for reverse transcription–polymerase chain reaction (RT-PCR) analysis to detect Xmsx-1 expression. In animal caps, the expression of Xmsx-1 was downregulated by the introduction of neural-inducing agents.
	Fig. 2. Bone morphogenetic proteins (BMP)-4/Xmsx-1 inhibited neural tissue differentiation induced by noggin (A) and cerberus. (Xcer, (B)). After the injection of ribonucleic acid (RNA) as indicated (5 ng/embryo for BMP-4 and Xmsx-1, 0.1 ng for noggin and 1 ng for Xcer), animal caps were excised at stage 10 and cultured for 2 days (stage 35/36 in sibling control embryos). The lysates of explants were subjected to western blot analysis to detect a neural marker, N-CAM. S1 and S2 are the standard brain lysates from stage 56 tadpoles (the loading protein in S2 is five times more than that in S1). A major protein (34 kDa) in the lysates of explants is shown as a protein loading control.
	Fig. 3. A dominant-negative Xmsx-1 induced the expression of N-CAM in the animal cap. Animal caps that had been injected with Xmsx-1/VP-16 ribonucleic acid RNA (2 ng/embryo) were excised at stage 9 and cultured until the sibling embryos reached stage 27. The expression of N-CAM and elongation factor (EF)1a was determined by reverse transcription–polymerase chain reaction (RT-PCR) analysis. The effect of Xmsx-1/VP-16 can be reverted by the simultaneous injection of wild-type Xmsx-1 (5 ng/embryo) or bone morphogenetic protein (BMP)-4 (5 ng/embryo).
	Fig. 4. Bone morphogenetic protein (BMP)-4 and Xmsx-1 failed to inhibit the neurulation induced by the prospective dorsal mesoderm in the combination explant assay. (A,B) The animal pole region of 2-cell stage embryos or dorsal blastomeres of 4-cell-stage embryos were injected with Xmsx-1 (A, 2 ng/embryo) or BMP-4 ribonucleic acid (RNA; B, 5 ng/embryo), and the resultant animal pole tissue (AP) or dorsal mesoderm (DM) excised from early gastrula embryos was combined with the uninjected counterpart. As a control experiment, an AP or DM explant was cultured alone (A). All the explants were cultured until stage 22 and processed for reverse transcription–polymerase chain reaction (RT-PCR) analysis to detect the N-CAM and elongation factor (EF)-1a RNA. (C) Western blot analysis was also performed to show the expression of N-CAM protein in the combination explants. A major protein (34 kDa) in the lysates of explants is shown as a protein loading control. Note that injection of BMP-4 in the dorsal mesoderm caused suppression of N-CAM expression while injection of RNA in the animal pole cells did not affect the expression of N-CAM.
	Fig. 5. Cell lineage tracing in explants using the Xenopus borealis cell as a marker. The animal pole region of 2-cell-stage X. laevis embryos was injected with bone morphogenetic protein (BMP)-4 ribonucleic acid (RNA; 5 ng/embryo), and animal pole tissue (AP) excised at the early gastrula stage was combined with the uninjected dorsal mesoderm (DM) of X. borealis embryos. The combination explants were cultured for 2 days. Sections of these explants were immunostained with an anti-N-CAM antibody (A,B), and counterstained with quinacrine (C). (B) shows a higher magnification of (A). The N-CAMpositive cells were located close to the notochord and were derived from the AP as indicated by dark nuclei (X. laevis cell). The notochord was derived from the dorsal mesoderm as indicated by bright nuclei (X. borealis cell). Darker staining at the peripheral region of the explant represents accumulation of melanin granules. The area of specific staining by the antibody is indicated by arrowheads in (A). Bars, 100 μm (A–C).
	Fig. 6. The expression of anterior/posterior neural markers in the combination explants injected with bone morphogenetic proteins (BMP)-4. The animal pole region of 2-cell-stage embryos was injected with BMP-4 ribonucleic acid (RNA; 5 ng/embryo), and animal pole tissue (AP) excised at the early gastrula stage was combined with the uninjected dorsal mesoderm (DM). The combination explants were cultured until the sibling embryos reached stage 27. The reverse transcription–polymerase chain reaction (RT-PCR) analysis was performed to detect otx-2 (fore- and midbrain marker), en2 (mid- and hindbrain boundary marker), krox20 (hindbrain marker), hoxB9 (spinal cord marker), N-CAM (pan-neural marker), and elongation factor (EF)1a. Note that the neural tissue induced in the BMP-4-loaded ectoderm exhibited a posterior phenotype in comparison with the uninjected control ectoderm.
	Fig. 7. Possible involvement of the fibroblast growth factor (FGF) signal in neural tissue differentiation induced by prospective dorsal mesoderm in combination explants. The animal pole region of 2-cell-stage embryos was injected with XFD (a dominant-negative form of the FGF receptor). Ribonucleic acid (RNA) alone (A), or XFD and BMP-4 RNA (B), and animal pole tissue (AP) excised at the early gastrula stage was combined with the uninjected dorsal mesoderm (DM). Simultaneous injection of XFD and BMP-4 inhibited the expression of N-CAM and otx2 in AP/DM combination explants, while XFD alone did not suppress the expression of N-CAM and otx2.