New HV et al. (1997), Differential effects on Xenopus development of ...

XB-ART-17171

Mech Dev 1997 Jan 01;611-2:175-86.

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Differential effects on Xenopus development of interference with type IIA and type IIB activin receptors.

New HV , Kavka AI , Smith JC , Green JB .

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One candidate for a mesoderm-inducing factor in early amphibian development is activin, a member of the TGF beta family. Overexpression of a truncated form of an activin receptor Type IIB abolishes activin responsiveness and mesoderm formation in vivo. The Xenopus Type IIA activin receptor XSTK9 differs from the Type IIB receptor by 43 and 25% in extracellular and intracellular domains respectively, suggesting the possibility of different functions in vivo. In this paper, we compare the Type IIA receptor with the Type IIB to test such a possibility. Simple overexpression of the wild-type receptors reveals minimal differences, but experiments with dominant negative mutants of each receptor show qualitatively distinct effects. We show that while truncated (kinase domain-deleted) Type IIB receptors cause axial defects as previously described, truncated type IIA receptors cause formation of secondary axes, similar to those seen by overexpression of truncated receptors for BMP-4, another TGF beta family member. Furthermore, in animal cap assays, truncated type IIB receptors inhibit induction of all mesodermal markers tested, while truncated type IIA receptors suppress induction only of ventral markers; the anterior/dorsal marker goosecoid is virtually unaffected. The suppression of ventral development by the type IIA truncated receptor suggests either that the truncated Type IIA receptor interferes with ventral BMP pathways, or that activin signaling through the Type IIA receptor is necessary for ventral patterning.

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Species referenced: Xenopus laevis
Genes referenced: actl6a acvr2a acvr2b cdkl5 gsc mt-tr nog tbxt trna

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	Fig. 2. RNAase protection analysis of XSTK9 expression. (A) Temporal expression pattern. Total RNA from two embryos was extracted at the indicated stages. (B) Spatial expression pattern at stage 8. RNA was extracted from animal caps (An), marginal zone regions (Mz). and vegetal poles (Vg). Yeast tRNA (tRNA) was used as a negative control for protection. Pigment differences were used to define dorsal (DMz), lateral (LMz) and ventral (VMz) marginal zones. To confirm these assignments, additional explants were allowed to develop to stage 10, when they were analyzed for expression of goosecoid. These results were observed in three separate experiments. Samples were co-protected with EFlor to control for amount of RNA loaded. The difference in appearance of EFlor between the XSTK9 and goosecoid protections results from the use of RNAase A with XSTK9.
	Fig. 3. Effects of over-expression of XSTK9 and XARl. (A) Graph comparing phenotypes of tadpoles (stage 40-41) after injection of 2 ng XSTKO or XAR1 RNA into a ventral-animal blastomere at the 16-cell stage. It shows the means f SEM for four experiments, with a total of 98-l 10 embryos injected for each condition. As controls, 1 ng of the XIDM or ASTK-7 was injected, to produce similar molar amounts of RNA. The graph shows results obtained after injection of 2 ng of ASTK- 7, which gave similar results to the other controls. Scoring criteria: ormal/slightly smallincludes some embryos which were slightly short or had slightly small eyes. This small subjective variation was also present in uninjected embryos. on-specific abnormalities includes major abnormalities found in both control and receptor-injected embryos, such as small eyes, truncated heads or tails, and spina bifida. ailsdescribes embryos with small tail-like protrusions. ther specific abnormalities only seen in receptor injected embryos, include brown pigment patches, usually at the base of the tail, small tail-like blebs, and fine xiallines of pigment running anteriorly from the region of the tail. (B,C) Examples of ailphenotype with XSTK9 (B) and XARl (C). The tails observed after injection of XARl RNA are often smaller than this. (D-G) Sections of ailphenotypes. (D,E) Ectopic structure obtained after injection of XSTK9 RNA. (El shows muscle (mus) and pigmented endoderm. (F) Endodermal-like tissue obtained after injection of XARl RNA, at mid-trunk level, associated with (G) small tail-like structure. Scale bar. (B,C) 1 mm; (D,F,G) 150 Frn: (El 85 um.
	Fig. 4. Effects of wild type XSTK9 and XAR1 overexpression in animal caps. Embryos at the one cell stage received injections of 10 ng XSTKB, XARl or delta-STK-7, or 5 ng of delta-STK-7 or XIDM RNA (to give equimolar amounts of nucleic acid), and caps were dissected at stage 8. Injections of 2 or 10 ng XSTK9 RNA gave identical results. (A-D) Intact stage 16 animal caps, fused as pairs injected with (A) delta-STK-7; (B) delta-STK-7 treated with activin; (C) XSTK9; (D) XAR 1. These sections are representative of two experiments. Note formation of pigmented protrusions in (C) and (D). (E-H) Sections of animal caps at control stage 40. (E) delta-STK-7; (F) delta-STK-7 treated with activin; (G) XSTK9; (H) XARl. Note formation of notochord (not) in response to activin (F) and mesenchyme (mes) and muscle (mus) in response to XSTK9 and XARl respectively (G,H). (1.J) RNAase protections on stage 16 animal caps. Whole embryo RNA is from one uninjected control in each case. (I) RNAase protection with Xwnt-8, noggin, Xbra and N-CAM probes, using EFlor loading control. (J) RNAase protection with actin probe, which hybridizes with both muscle actin and cytoskeletal actin. The same result was obtained in three experiments. Scale bar, (A.D) 1 mm; (E-H) 200 um.
	Fig. 5. Effects of truncated activin receptors in whole embryos. Embryos were injected at 1-2 cell stage with truncated activin receptor constructs. (A) Control embryos injected with 10 nl water. (B) Embryos injected with 2.5 ng delta-IXARl. Note short, deformed axes and reduced or absent eyes and cement glands. (C) Embryos injected with 2.5 ng delta-STK + 10. Embryos have varying degrees of axis duplication. Arrows indicate secondary axes. (D) Embryo injected with 2.5 ng delta-STK + 10 which has developed, along with secondary axis, anterior structures such as a cement gland (white arrow) and an eye (black arrow). Scale bar, (D) 1 mm.
	Fig 6. Effects of truncated activin receptors on animal cap morphology and differentiation. Embryos at the one cell stage received injections of 5 ng delta-STK + 10, delta-lXARI, or control RNAs delta-STK-7 and XIDM. Animal caps were dissected at stage 8. (A-F) Appearances of intact animal caps, fused as pairs, at control stage 16. (A.B) XIDM; (C,D) delta-STK + 10; (E,F) delta-IXARI. Caps in (A,C,E) received no added factor. (B,D,F) were incubated in activin A. Note that delta-IXARI (F) inhibits activin-induced elongation more effectively than delta-STK + 10 (D). (G,H) Appearances at stage 37 of caps injected with delta-STK-7 control (G) and ASTK + 10 (H). (I-N) Sections of animal caps at control stage 40. (1,J) XIDM injected caps; (K,L) delta-STK + IO; (M,N) delta-lXARl. Caps in (I.K,M) received no added factor. (J,L,N) were incubated in activin A. Note muscle formation (mus) in control injected cap (J), neural tissue (tit) in caps injected with dominant negative constructs (K) to (N), cement gland (cg) in dominant negative injected caps without addition of activin (K) and (M), and notochord (not) in activin treated caps injected with ASTK + IO (L) but not in caps injected with delta-lXARI (N). These results are representative of two experiments. Scale bar, (A-H) 1 mm; (I,J-N) 200 um.