Casellas R and Brivanlou AH (1998), Xenopus Smad7 inhibits both the activin and BMP...

XB-ART-14706

Dev Biol 1998 Jun 01;1981:1-12. doi: 10.1006/dbio.1998.8893.

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Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer.

Casellas R , Brivanlou AH .

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Smads are proteins that transduce signals on behalf of members of the TGF beta superfamily of growth factors. Recently, inhibitory Smads, Smad6, Smad7, and Dad, were isolated from human, mouse, and fly. These anti-Smads were shown to inhibit TGF beta signaling by stably associating to TGF beta type I receptors or, as it was shown for Smad6, by binding to receptor-activated Smad1. We report the cloning, distribution, and embryological activity of the Xenopus Smad7 (XSmad7). We report that XSmad7 inhibits signaling from the activin and BMP pathways in animal explants although at different thresholds. When expressed in the embryo, low concentrations of XSmad7 dorsalize the ventral mesoderm, thus inducing a secondary axis. At higher concentrations however, XSmad7 inhibits both mesoderm induction and primary axis specification. In addition, we show that XSmad7 acts as a direct neural inducer both in the context of ectodermal explants and in vivo. We discuss the implications of these findings in the biochemical context of the activin and BMP pathways as well as their implications in mesodermal, neural, and axis specification.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a acvr1b bmp4 bmpr1b chrd eef1a2 egr2 en2 gdf1 hoxb9 ncam1 odc1 otx2 smad1 smad2 smad6 smad6.2 smad7 snai2 tbx2 tbxt twist1 wnt8a

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	FIG. 1. Structure of XSmad7. (A) Nucleotide and deduced amino acid sequence of XSmad7. The underlined nucleotides are identical to sequences found in the 39 UTR of human Smad7. (B) Comparison of domain structures between Smad1, Smad6, Smad7, and Xsmad7, drawn to scale. The amino- (N) and carboxy- (C) terminal homologous regions are shown as dark gray boxes. A region conserved only between Smad6, Smad7, and XSmad7 is shown in black.
	FIG. 2. Developmental expression of XSmad7. (A) RT-PCR analysis of Xsmad7 expression during different stages of Xenopus development: (2, 4) maternal, (8 –9) blastula, (10 –12.5) gastrula, and (14 –19) neurula. Ornithine decarboxylase (ODC) is used as a loading control. (B) Expression of XSmad7 in early gastrulae. Stage 10.25 embryos were dissected into animal–vegetal and dorsal–ventral halves, and total RNA was harvested. The RNA was analyzed by RT-PCR for the distribution of Xsmad7 in different regions of the embryo. Vg1, chordin, and Xwnt8 are dissection controls. Vg1 is localized in the vegetal pole, chordin is a marker of the organizer, Xwnt 8 is a marker of the ventral mesoderm, and ODC is used as a loading control.
	FIG. 3. Localization of XSmad7 transcripts in Xenopus embryos. Whole-mount in situ hybridization showing the localization of XSmad7 at different stages of Xenopus development. At neural plate stages (A), XSmad7 transcripts can be detected in the lateral edge of the anterior neural plate (white arrowheads), as well as in the heart primordium (black arrowhead). Anterior is to the right and dorsal is facing the reader. When the neural tube is closed (B and C), XSmad7 is strongly expressed in the ventral mesoderm, including the blood islands (C, red arrowheads), heart (B and C, blue arrows), eyes (B and C), and hindbrain (black arrowheads in B and white arrowhead in C). B is a head-on view and C is a lateral view. At tailbud stages (D and E) the ventral expression of XSmad7 is detected in the blood islands (red arrows, D and E), in a pattern reminiscent of the expression of globin transcripts (Hemmati-Brivanlou et al., 1990). Strong expression in the heart is maintained (blue arrow in E). D is a side view and E is a ventral view. At tadpole stages (F), the strongest levels of expression remain in the heart (blue arrow), the eyes, and the otic vesicle (black arrowhead).
	FIG. 4. Inhibition of the activin pathway by XSmad7. (A) XSmad7 RNA-injected or uninjected animal caps were cultured until stage 23 in the presence (lanes 1 to 6) or absence (lane 7, control) of activin protein (1/1000 dilution of activin-injected oocyte supernatant). EF1a, which is ubiquitously expressed, was used as a loading control. RNA from embryos provides a positive control. The RT- lane is identical to that of the embryo with the omission of reverse transcriptase, thus serving as a negative control. Uninjected animal caps cultured in the presence of activin protein express the dorsal mesoderm-specific marker cardiac actin (C. Actin), which is gradually downregulated by injection of XSmad7 RNA (0.25 to 2 ng). Similarly, animal caps injected with 1 ng of constitutively active activin receptor ALK4 (CA-ALK4) or its signal transducer Smad2 induce the general mesoderm marker Xbra at gastrula stages (lanes 1 and 2, B) and the dorsal mesoderm marker cardiac actin at neurula stages (lane 1, C). These inductions are efficiently inhibited by coinjections of 2 ng of XSmad7 RNA (lanes 4 and 5, B; and lane 3, C).
	FIG. 5. XSmad7 induces anterior neural tissue. (A) Animal caps were injected with 0.1, 0.4, and 2 ng of XSmad7 RNA. Total RNA was harvested at stage 16 (neurula) and analyzed by RT-PCR for the presence of the indicated transcripts. XSmad7 alone induces the expression of the neural marker NCAM in a concentration-dependent manner. (B) Animal caps injected with 1 ng of XSmad7 were cultured until sibling embryos reached stage 16. By using specific primers in RT-PCR assays we show that XSmad7 induces anterior neural markers OtxA (a marker of forebrain, midbrain, and eyes; Lamb et al., 1993) but not Engrailed-2 (En2, a marker of midbrain–hindbrain), Krox20 (hindbrain marker), HoxB9 (spinal cord and posterior lateral mesoderm marker), or Xslug or Twist (markers of neural crest).
	FIG. 6. XSmad7 blocks the BMP pathway in animal cap explants. (A) BMP4 RNA (1 ng) was injected alone or coinjected with increasing concentrations of XSmad7 RNA in the animal poles of two-cell-stage embryos. Animal cap explants from these embryos were cultured until midgastrula stages, when RNA was extracted, reverse transcribed, and amplified by PCR in the presence of Xbra and Xwnt8 specific primers. Although BMP4 alone is able to induce these markers (lane 1), in the presence of as low as 0.25 ng of XSmad7, this induction is completely abolished. (B) As previously reported, constitutively active BMP receptor ALK6 [CA-ALK6 (1 ng), Kretzschmar et al., 1997] as well as its signal transducer Smad1 [(1 ng), Graff et al., 1996] induces early (Xbra and Xwnt8, at gastrula stages) and late (globin, late neurula stages) ventral mesoderm markers in animal caps (lanes 1 and 2). As shown with BMP4 in A, these inductive events are blocked in the presence of XSmad7 (1 ng, lanes 4 and 5).
	FIG. 7. XSmad7 induces secondary axes and ectopic neural tissue in vivo. XSmad7 RNA (0.1– 0.3 ng) was injected alone (A) or coinjected with nuclear b-Gal RNA (100 pg) into the dorsal (C) or ventral marginal zone (D) of four-cell-stage embryos. Unlike control b-Gal (B) or XSmad7/b-Gal dorsal injections (C), ventral expression of XSmad7 or XSmad7/b-Gal RNA induced ectopic secondary axes in embryos (D, F, and H). b-Galactosidase staining of injected embryos revealed that the progeny of the injected blastomere contributed directly to the secondary axis (D). N-CAM staining of XSmad7 ventrally injected embryos (F) shows that the secondary axis contains neural tissue; E corresponds to XSmad7 dorsally injected controls. Muscle actin staining shows that although muscle is present in the primary axis [arrowheads, G (control) and H (ventral injection)], this expression is absent in the secondary axis (H and data not shown). Animal pole injections of XSmad7 RNA in two-cell-stage embryos induce exaggerated neural tissue at tadpole stages, including the formation of ectopic eyes (J, arrowhead). (I) Uninjected control.
	FIG. 8. Mesoderm inhibition by XSmad7. XSmad7 RNA (50–400 pg) was either injected alone or coinjected with the lineage tracer b-Gal (same concentrations) dorsally or radially in the marginal zone of four-cell-stage embryos. Embryos were cultured until gastrula stage 10.5 (A, C, E, and G, vegetal pole view) or stage 28 (B, D, F, and H). Stage 10.5 embryos were stained for b-Gal (red) and assayed for Xbra expression by whole-mount in situ hybridization (blue). Although embryos injected dorsally with b-Gal alone displayed the typical Xbra ring (A), coinjections in the dorsal marginal zone with 50 or 400 pg (C and E, respectively) of XSmad7 inhibited Xbra at the site of injection (shown with arrowheads). Radial coinjections (300 pg) completely abolished Xbra expression (G). At tadpole stages, dorsal injections of XSmad7/b-Gal (50 pg) had no visible effects (D). At higher concentrations of XSmad7, embryos showed a reduction in the primary axis (350 pg, F). Radial injections of XSmad7 (300 pg) completely inhibited mesoderm induction leading to the formation of ‘‘bubble’’ embryos (H, animal pole view).