Sun BI et al. (1999), derrière: a TGF-beta family member required for...

XB-ART-13413

Development 1999 Apr 01;1267:1467-82. doi: 10.1242/dev.126.7.1467.

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derrière: a TGF-beta family member required for posterior development in Xenopus.

Sun BI , Bush SM , Collins-Racie LA , LaVallie ER , DiBlasio-Smith EA , Wolfman NM , McCoy JM , Sive HL .

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TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.

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Species referenced: Xenopus
Genes referenced: a2m actl6a cdx4 egr2 en2 eomes fgf4 foxa4 gdf1 gsc hoxb9 krt12.4 lhx1 muc2 ncam1 ncoa6 nodal nodal1 nodal2 nodal3.1 nodal3.2 not odc1 otx2 sia1 sox17a tbxt tgfb1 vegt ventx1.2

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	Fig. 1. Derrière protein sequence and alignments. Amino acid residues are shown. Numbers at left indicate amino acid positions. (A) Derrière protein sequence. Arrow indicates signal sequence cleavage site predicted by von Heijne algorithm. Maturation cleavage signal (RAKR) is boxed. These four amino acids are substituted with GVDG in Cm-Derrière. The mature region of Derrière protein is underlined. The seven cysteines within the mature region are underscored with solid bars. (B) Alignment of Derrière protein sequence to Xenopus Vg1 (Vg1), zebrafish Vg1 (zVg1), chicken Vg1 (CVg1) and incomplete newt Vg1 (nVg1). Consensus amino acid residues in proteins are shaded. Sequence gaps introduced for optimal protein alignment are indicated by dashes. The percentage identities between full-length Derrière and other Vg1-related proteins are 56% (xVg1), 51% (cVg1) and 50% (zVg1), with mature region identities of 79% (xVg1), 73% (cVg1) and 76% (zVg1).
	Fig. 2. Temporal and spatial expression patterns of derrière. (A) Northern analysis of Xenopus embryos. One embryo equivalent per lane was analyzed for derrière RNA (top row) at various embryonic stages shown. Ethidium-bromide-stained 28S rRNA is a loading control (bottom row). Lanes as marked. (B) Whole-mount in situ hybridization analysis of derrière expression. Embryo orientations are indicated by vegetal (Vg), dorsal (D) and posterior (P). Bl, blastocoel. Purple staining represents derrière expression. (a) Stage 9, late blastula; (b) stage 10.5, early gastrula; (c) stage 11.5, mid-gastrula; (d) stage 12.5, late gastrula; (e) stage 14, early neurula; (f) a sagittal section of a stage 11.5 embryo. White arrow indicates superficial layer, white arrowhead indicates deep cells and black arrowhead indicates the anterior limit of archenteron.
	Fig. 3. derrière can be induced by known mesoderm inducers in animal caps. (A) Experimental scheme. Stage 8.5 animal caps of uninjected embryos were cultured with purified proteins until stage 11.5 (mid gastrula). Alternatively, embryos were injected at the animal pole of one blastomere with test RNA at the 2-cell stage. Animal caps were removed from stage 8.5 embryos and were cultured until sibling embryos reached stage 11.5. Animal caps and whole embryos were harvest for RT-PCR. (B) Induction of derrière in animal caps. ODC served as a loading control. Lanes as marked; lanes 1, 5, BSA served as a negative control; lanes 4, 10, whole embryo controls. See Materials and Methods for amount of proteins used.
	Fig. 4. derrière induces mesodermal and endodermal markers in animal caps. (A) Experimental scheme. Wild-type embryos were injected at the animal pole of one blastomere with test RNA at the 2-cell stage. Animal caps were removed from stage 8.5 embryos and were cultured until sibling embryos reached stages indicated by the triangles. Animal caps and whole embryos were harvest for RT-PCR or morphology. (B) Expression of marker genes (see Results) in animal caps after injection of 400 pg RNA. Injection of globin served as negative controls. ODC served as a loading control. VegT has a very similar expression pattern to derrière. eFGF is expressed posteriorly and in the notochord. gsc is a prechordal plate marker, siamois is expressed in the dorsal mesendoderm. eomesodermin a mesendodermal marker. Xlim-1 and Xnot are dorsal mesodermal markers. Xvent-1 is a ventral ectodermal and mesodermal marker. HoxB9 marks posterior spinal cord. Xsox17a is an endodermal marker. XK81 is a ventral ectodermal (epidermal) marker and N-CAM is a neural-specific marker. Lanes as marked. (C) Mesodermal markers respond to derrière induction in a dose-dependent manner. HoxA7 is a posterior mesodermal and ectodermal marker. At the time of harvest, Pintallavis weakly marks the dorsal mesoderm and tailbud. m-actin is a muscle-specific marker and Xbra is expressed posteriorly and in the notochord. Lanes as marked. b-globin-capped RNA was used to make up for differences in amount of test RNA injected such that all embryos received the same total amount of RNA. (D) XFD blocks derrièreinduced cap elongation. (a) 200 pg derrière and 800 pg globin; (b) 200 pg derrière and 800 pg XFD; (c) 800 pg XFD and 200 pg globin.
	Fig. 5. derrière suppresses head formation or induces a posterior secondary axis in whole embryos. (A) Experimental scheme. Wild-type or albino embryos were injected with 50 pg derrière and 80 pg lacZ RNA in the marginal zone of one blastomere at 2-cell stage, either dorsally or ventrally. Albino embryos were harvested at stage 24 (tailbud) for in situ hybridization and wild-type embryo were harvested at stage 35 (hatching) for morphological analysis. (B) Dorsal misexpression of derrière results in microcephaly. In all panels anterior (A) is to the left and posterior (P) is to the right. Light blue indicates lineage tracer b-gal. (a,b) Wild-type embryo injected with derrière RNA (a) and globin RNA (b). (c-h) Albino embryos processed for in situ hybridization (see Results). Purple staining represents probes as indicated on the left. m-actin is a muscle-specific marker, otx2 marks the forebrain, XCG marks the cement gland, en-2 marks the midbrain/hindbrain junction and Krox20 marks rhombomeres 3 and 5 in the hindbrain. (c-e) White arrow and arrowhead indicate otx2 forebrain and eye staining, respectively. Black arrowhead indicates anterior limit of HoxB9 staining. (f-h) White arrow indicates en-2 staining, white arrowhead indicates XCG staining, bracket indicates krox20 staining. (C) Ventral misexpression of derrière results in a posterior secondary axis. In all panels, anterior (A) is to the left and posterior (P) is to the right. (a,b) Wild-type embryos. White arrow indicates a secondary axis. (c-j) Albino embryos processed for in situ hybridization (see Results). Markers as for B, except for N-CAM, which is a general neural marker. Arrow indicates secondary axis. (i,j) Arrow indicates en-2 staining, bracket indicates krox20 hindbrain staining.
	Fig. 6. derrière increases dorsal character of the ventral marginal zone. (A) Experimental scheme. 25 pg derrière RNA was injected into the marginal zone of both ventral blastomeres of 4-cell stage wild-type embryos. The VMZ was dissected at stage 10.25 and cultured until harvest for RT-PCR at stage 17. (B) Dorsalization of the VMZ by derrière. Lanes as marked.
	Fig. 7. Derrière posteriorizes isolated neurectoderm indirectly. (A) Experimental scheme. Anterior dorsal ectoderm (aDE, indicated by the cut marks, see Materials and Methods) was isolated from stage 11 and 11.5 (mid-gastrula) embryos. Explants were cultured in saline alone or with added factors until harvest for RT-PCR at stage 22 (tailbud). (B) Expression of marker genes in aDE explants (see Results). BSA-treated explants served as negative controls. See Materials and Methods for amount of protein used. HoxA7 and Xcad3 are expressed in posterior mesoderm and ectoderm, m-actin is a muscle marker, XCG is a cement gland marker, en-2 marks the midbrain/hindbrain junction and Krox20 is a hindbrain marker. Lanes as marked.
	Fig. 8. Specificity of Cm-derrière. (A) Rationale for dominant negative activity. Solid bars and open bars indicate the prepro region and the mature region of Derrière protein, respectively. Gray boxes represent mutated maturation cleavage signal. The link between two open boxes represents the disulfide bond of a dimer. (B) Experimental scheme for C-E. Wild-type embryos were injected at the animal pole in one cell at the 2-cell stage with test RNA. Animal caps were removed from stage 8.5 (mid-blastula) embryos and were cultured until sibling embryos reached stage 20 (late neurula). Animal caps and control whole embryos were harvested for RT-PCR (C,D) and for morphological analysis (E). (C) Ratio of derrière: Cm-derrière at which marker expressions are inhibited in animal caps. Injection of globin alone served as a negative control. ODC was used as a loading control. Test RNA is indicated at the top. wt, wild-type derrière; Cm, Cmderrière. endodermin (Edd) is an endodermal marker. Lanes as marked; lane 7, uninjected whole embryo control. globin RNA was injected at 1 ng; In derrière and Cm-derrière injections 20 pg of derrière RNA was injected with Cm-derrière RNA to make up the indicated ratio of derrière: Cm-derrière; and globin RNA making the total mass injected equal to 1 ng. (D) Effects of Cmderrière on other TGF-b family members: molecular assay. Injection of Cm-derrière alone served as a negative control. Test RNA is indicated at the top and the presence or absence of ten-fold mass excess of Cmderrière is indicated by + or – sign. Lanes as marked; lane 16, uninjected whole embryo control. derrière, BVg1, Xnr1, Xnr2, Xnr3 and Xnr4 RNAs were injected at 50 pg and activin RNA was injected at 5 pg. (E) Effects of Cm-derrière on other TGF-b family members: animal cap elongation assay. Cm-derrière was used at a 10:1 ratio to co-injected TGF-bs, shown above the panels. derrière, BVg1, Xnr1, Xnr2, Xnr3 and Xnr4 RNAs were injected at 50 pg and activin RNA was injected at 5 pg.
	Fig. 9. Phenotype and in situ hybridization analysis of Cm-derrière-injected embryos and rescue of Cmderrière by wild-type derrière and VegT. (A) Experimental scheme. Wild-type or albino embryos were injected with test and lacZ RNA in the marginal zone. At stages indicated below, albino embryos were harvested for in situ hybridization and wild-type embryo were harvested for morphological analysis. (B) Effects of Cm-derrière expression on whole embryos. Light blue indicates lineage tracer b-gal and purple represents specific RNA expression. (a,b) Vegetal (Vg) views of stage 11-11.5 embryos injected at 4-cell stage with 250 pg Cm-derrière (a) and globin (b) into each of the two ventral blastomeres. Black arrowhead: extent of blastopore formation. (c,d) Posterior (P) views of stage 17 embryos injected at 4-cell stage with 250 pg Cmderrière (c) and globin (d) into each of the two ventral blastomeres. D, dorsal; Y, yolk cells. (e,f) Lateral views of stage 35 embryos injected at 2-cell stage with 500 pg Cm-derrière (e) and globin (f) into both blastomeres. A, anterior; P, posterior. (g-j) Albino embryos processed for in situ hybridization. (g) XCG (arrowhead) en (arrow) and krox20 (bracket) probes; dorsal view of a stage 20 embryo injected with 500 pg Cm-derrière. (h) XCG, en and krox20 probes; head-on view of a stage 20 embryo injected with 500 pg globin. (i,j) m-actin probe; lateral views of stage 26 embryos injected with 500 pg Cmderrière (i) and globin (j). (k,l) Rescue experiments. (k) 50 pg derrière and 500 pg Cm-derrière co-injected; (l) 50 pg VegT and 500 pg Cm-derrière co-injected.
	Fig. 10. Model for derrière activity. At the mid-blastula transition, we speculate that maternal transcription factors (such as VegT) and possibly secreted factors activate derrière expression in the presumptive mesoderm and endoderm (wide hatching). During early gastrula, derrière expression is maintained in the mesoderm by a positive feed-back loop that includes FGFs (such as eFGF), Xbra and zygotic VegT (close hatching). derrière expression in the endoderm is not maintained presumably because such a feed-back loop cannot be established there. During early gastrula stages, derrière may activate mesendodermal fates in both the future head region and more posteriorly. In particular, derrière may play a role in activating posterolateral (paraxial) mesodermal fates at this time, before somite formation. By mid to late gastrula, derrière is excluded from anterior mesoderm and from the dorsal midline (close hatching), and continues to promote posterolateral fates. An, animal pole; Vg, vegetal pole; A, anterior; P, posterior; meso, mesodermal fates; endo, endodermal fates.