XB-ART-3015Development 2004 Oct 01;13120:4977-86. doi: 10.1242/dev.01323.
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Activin redux: specification of mesodermal pattern in Xenopus by graded concentrations of endogenous activin B.
Mesoderm formation in the amphibian embryo occurs through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial cells. The first candidate mesoderm-inducing factor to be identified was activin, a member of the transforming growth factor type beta family, and it is now clear that members of this family are indeed involved in mesoderm and endoderm formation. In particular, Derrière and five nodal-related genes are all considered to be strong candidates for endogenous mesoderm-inducing agents. Here, we show that activin, the function of which in mesoderm induction has hitherto been unclear, also plays a role in mesoderm formation. Inhibition of activin function using antisense morpholino oligonucleotides interferes with mesoderm formation in a concentration-dependent manner and also changes the expression levels of other inducing agents such as Xnr2 and Derrière. This work reinstates activin as a key player in mesodermal patterning. It also emphasises the importance of checking for polymorphisms in the 5' untranslated region of the gene of interest when carrying out antisense morpholino experiments in Xenopus laevis.
PubMed ID: 15371302
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: cer1 chrd.1 fst gdf3 gsc hhex inhbb nodal nodal1 nodal2 nodal5 nodal6 not tbxt ventx1.2 wnt8a zfyve9
Antibodies: Notochord Ab2 Somite Ab1
Morpholinos: inhbb MO3 inhbb MO4 inhbb MO5
Article Images: [+] show captions
|Fig. 3. MO1 disrupts axial development in Xenopus and exerts dose-dependent effects on gene expression in the early gastrula. (A-C) Xenopus embryos were injected at the one-cell stage with mMO1 (A; 40 ng) or MO1 (B,C; 40 ng) and allowed to develop to tadpole stage 33. MO1 causes axial defects and a disruption of anterior development. (D-G) The phenotype illustrated in B,C is presaged by disruption of expression of Xnot (D,E) and Chordin (F,G). Xnot expression persists around the blastopore of embryos injected with MO1. (E) Expression of Chordin in embryos injected with MO1 (F) is more diffuse than that in embryos injected with mMO1 (G). (H-K) Although axial morphogenesis is disrupted in embryos injected with antisense morpholino oligonucleotide MO1, notochord and muscle do nevertheless form in such embryos. (H,J) Embryos injected with mMO1 stained with monoclonal antibody MZ15 (H) or 12/101 (J). (I,K) Embryos injected with MO1 stained with MZ15 (I) or 12/101 (K). (L) Embryos were injected with the indicated amounts of MO1 and allowed to develop to early gastrula stage 10.5, when gene expression was assessed by real-time RT-PCR. Levels of gene expression are normalised to those of ornithine decarboxylase. Increasing concentrations of MO1 cause the downregulation first of dorsally expressed genes such as Goosecoid and chordin and then the downregulation of Xbra, which is expressed throughout the marginal zone. Expression of Xwnt8, which occurs in lateral and ventral tissue, is little affected.|
|Fig. 4. Gene expression patterns in embryos injected with MO1. (A,B) Expression of Goosecoid is downregulated in response to MO1 (B), but its expression domain is unaltered. (C,D) Downregulation of Xbra in response to MO1 (D). See text for details. Embryos in A-D were fixed at similar morphological stages rather than at different times after fertilisation to compensate for slight delays in development. (E-H) The expression domains of Xvent-1 (E,F) and Xwnt-8 (G,H) are unaffected by MO1, even though their expression levels are very slightly (Xwnt-8; Fig. 3) or significantly (Xvent-1; Fig. 6) elevated. Embryos were injected at the two-cell stage, with 20 ng MO1 into each blastomere.|
|Fig. 7. Inhibition of activin function causes an upregulation of Xnr2 expression and a downregulation of Derrière. (A) Embryos were injected with the indicated antisense morpholino oligonucleotides and allowed to develop to early gastrula stage 10.5, when they were assayed for expression of nodal-related genes and Derrière by real-time RT PCR. (B-G) Upregulation of Xnr2 expression is not accompanied by an expansion of its expression domain. Embryos were subjected to in situ hybridisation to detect Xnr2 RNA and viewed from the vegetal pole (B-D) or from the side (E-G). Transcription of Xnr2 in embryos injected with MO1 (C,D) or MO3 (F,G) is not observed beyond its normal expression domain. (H-J) Downregulation of Derrière is not accompanied by a restriction in its normal expression domain. All embryos were injected at the two-cell stage, with 20 ng MO1 or 15 ng MO3 into each blastomere.|
|Fig. 2. Design and verification of activin B antisense morpholino oligonucleotides. (A) Sequence of the 5′ untranslated region of activin B derived from GenBank (i) and derived from the Xenopus colony at the Wellcome Trust/Cancer Research UK Gurdon Institute (ii). Differences between the two sequences are highlighted and the sequences targeted by the antisense morpholino oligonucleotides used in this paper are boxed. (B) MO1 inhibits in vitro translation of activin B. Arrow indicates activin B. mMO1 was included in one reaction at a final concentration of 5 μM and MO1 was included at 0.5, 2.0 and 5.0 μM. – indicates no addition of morpholino oligonucleotide. (C) MO1 inhibits, in a dose-dependent fashion, translation of RNA encoding HA-tagged activin B following injection into Xenopus embryos. RNA encoding activin B-HA, together with RNA encoding an HA-tagged version of the FYVE and SBD domains of SARA (see Materials and methods), was injected into Xenopus embryos at the one-cell stage in the absence of MO1 or at the indicated concentrations of morpholino. Embryos were cultured to early gastrula stage 10 and subjected to western blotting using an anti-HA antibody. Activin B-HA and FYVE/SBD-HA are indicated by arrows. Inhibition of activin B translation is not observed with mMO1. (D) MO1 prevents activin-induced expression of Xbra in Xenopus animal caps. Xenopus embryos were injected with the indicated combinations of activin and MO1. They were cultured to early gastrula stage 10.5 and assayed for expression of Xbra by real-time RT-PCR. (E-G) MO1 inhibits activin-induced elongation of animal caps. Animal pole regions were derived from uninjected embryos (E) or embryos injected with RNA (5 pg) encoding activin B in the absence (F) or the presence (G) of 40 ng MO1. MO1 inhibits the elongation of animal pole regions (G). (H-M) MO1, but not mMO1, inhibits the function of exogenous activin in intact Xenopus embryos; mMO1 but not MO1 inhibits the function of a mutated form of activin in which the sequence has been mutated to match that of mMO1. Embryos were injected with wild-type activin (H-J; Activin B-HA; 10 pg) or mutated activin (K-M; mActivin B-HA; 10 pg) in the absence of morpholino oligonucleotides or in the presence of MO1 (40 ng) or mMO1 (40 ng).|
|Fig. 5. Antisense morpholino oligonucleotide MO1 does not affect the activin signal transduction pathway (A-F). Exogenous activin B can `rescue' the effects of MO1 (G-J). (A-F) Animal pole regions derived from uninjected Xenopus embryos or from those injected with MO1 (40 ng) form spheres (A,D), while those treated with activin (16 U ml–1) elongate (B). Elongation is substantially inhibited in animal caps derived from embryos injected with RNA (500 pg) encoding Xenopus follistatin (C) but not by 40 ng mMO1 (E) or MO1 (F). (G-H) Thirty-six percent of embryos injected into one cell at the four-cell stage with 2 pg RNA encoding mutated activin B-HA suffer defects in early development (H; compare with normal embryos in G), while 64% of embryos injected with 20 ng MO1 display a `knockdown' phenotype (I). Co-injection of mutated activin B-HA and MO1 `rescues' development, such that only 24% are abnormal (J).|
|MO3 causes a phenotype similar to that caused by MO1. (A-D) Embryos injected with increasing concentrations of an alternative activin B antisense morpholino oligonucleotide termed MO3 (B-D) show a similar phenotype to that observed with MO1, although MO3 is effective at lower concentrations (compare C with Fig. 3B). (E-J) Expression of Goosecoid and Chordin is reduced by both MO1 and MO3, and expression of Xvent1 is elevated. For MO1, mMO1 was used as a control, and for MO3, MO2 was used as a control. Embryos were analysed at the indicated stages. In this experiment, inhibition of Goosecoid expression by MO3 was most marked at stage 11 (H).|
|MZ15 staining in notochord|