Armes NA and Smith JC (1997), The ALK-2 and ALK-4 activin receptors transduce...

XB-ART-15793

Development 1997 Oct 01;12419:3797-804. doi: 10.1242/dev.124.19.3797.

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The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds.

Armes NA , Smith JC .

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The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.

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Species referenced: Xenopus laevis
Genes referenced: a2m acta4 actc1 actl6a acvr1 acvr1b alk evx1 foxa4 gsc tbxt tgfb1

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	Fig. 1. Constitutively active ALK-4 (ALK-4) induces the early mesodermal markers Xbra and goosecoid in a concentrationdependent fashion. Constitutively active ALK-2 (ALK-2) induces only Xbra. Embryos were injected at the one cell stage with increasing quantities of capped RNA encoding either ALK-2* or ALK-4*. Animal cap explants were cut at stage 8.5 and expression of Xbra and goosecoid was assayed by RNAse protection at stage 10.5. EF-1a was used as a loading control; shorter exposures of the gel confirmed equal loading.
	Fig. 2. Comparison of the effects of ALK-2* and ALK-4* on animal cap explant morphology. Explants from uninjected embryos (A,B) or embryos injected at the one-cell stage with RNA encoding either ALK- 2* (C,D) or ALK-4* (E,F) were cultured until early gastrula stage 10.5 (A,C,E) or neurula stage 17 (B,D,F). At the early gastrula stage, explants from uninjected embryos (A), or from embryos injected with 100 pg (C) or 1 ng (not shown) ALK-2* RNA remain spherical. In contrast, explants injected with 1 ng ALK-4* RNA (E) undergo inversion movements. At the neurula stage, explants from uninjected embryos (B), or from embryos injected with 100 pg (not shown) or 1 ng (D) ALK-2* RNA remain spherical. Animal caps derived from embryos injected with 100 pg ALK-4* undergo elongation, reminiscent of activin-treated animal caps (F), while caps from embryos injected with 1 ng ALK-4* RNA remain spherical (not shown).
	Fig. 3. ALK-4* induces the late mesodermal markers muscle-specific actin and Xhox3 in a concentration-dependent fashion. ALK-2* induces only Xhox3. Embryos were injected at the one cell stage with increasing quantities of capped RNA encoding either ALK-2* or ALK-4*. Animal cap explants were dissected at stage 8.5 and assayed by RNAse protection at stage 17 for expression of musclespecific actin and Xhox3.
	Fig. 4. ALK-4* induces a range of mesodermal cell types and perhaps endodermal tissues; ALK-2* induces only ventral mesoderm. Animal caps were dissected from embryos injected with increasing quantities of capped RNA encoding either ALK-2* or ALK-4. They were cultured to the equivalent of stage 35, when they were fixed, sectioned and stained by the Feulgen/Light Green/Orange G technique (Smith, 1993). Control caps (A,E) form atypical epidermis. Explants derived from embryos injected with 10 pg ALK-4 RNA (B) form mesenchyme (Mes), explants derived from embryos injected with 100 pg ALK-4* (C) form muscle (Mus) and notochord (Not), and those derived from embryos injected with 1 ng ALK-4* RNA (D) form an unfamiliar tissue which may be endoderm (see text). Explants expressing ALK-2* form mesenchyme at all doses of RNA. (F) shows a cap derived from an embryo injected with 1 ng ALK-2* RNA. Scale bar in A, 50 mm.
	Fig. 5. ALK-4* induces the pan-endodermal marker endodermin. Explants from embryos injected with increasing quantities of RNA encoding ALK-4* were assayed at stage 13 by RNAse protection for expression of the endoderm marker endodermin.
	Fig. 6. ALK-4* but not ALK-2* can induce secondary axes when expressed in ventral blastomeres of the Xenopus embryo. 150 pg of RNA encoding either ALK-2* or ALK-4* was injected into each of the two most ventral B-tier blastomeres of 32-cell stage embryos. Embryos were allowed to develop to stage 33 before being fixed and lightly stained for muscle using the monoclonal antibody 12/101. (A) Control embryo. (B) ALK-4* induces a partial secondary axis (arrow) in 58% of embryos. Axes contained muscle and in some instances a single eye at their anterior end. (C,D) Injection of ALK- 2* RNA never produced secondary axes but often produced defects on the ventral side of the embryo. Sometimes the ventral region formed a bulge (C) and sometimes the ventral tissue mass was decreased (arrow in D).
	Fig. 7. ALK-2* abolishes the dorsalising effects of ALK-4. Animal caps were dissected from embryos injected with 1 ng of ALK-4 alone or with 1 ng ALK-4* together with increasing quantities of RNA encoding ALK-2*. Explants were analysed at stage 17 for the expression of the dorsal mesoderm marker muscle actin.
	Fig. 8. The inhibitory effects of ALK-2* on dorsal mesoderm induction caused by ALK-4* take place rapidly, suggesting that signal interference occurs prior to immediate transcriptional responses. A kinase-inactive ALK-2* is a poor inhibitor of ALK-4* dorsal mesoderm induction. (A) Animal cap explants were dissected at stage 8 from embryos injected with ALK-4* RNA alone or with the indicated combinations of ALK- 4* and ALK-2* RNA. Explants were analysed for expression of the early mesodermal markers Pintallavis, goosecoid and Xbra by RNAse protection after 1.5, 2.5, 3.5 or 6 hours of subsequent culture. (B) Animal cap explants were dissected at stage 8 from embryos injected with ALK-4* RNA alone or with the indicated combinations of ALK-4* with either ALK-2* or ALK- 2* kinase-inactive RNA. Explants were analysed for expression of the early mesodermal markers Pintallavis, goosecoid and Xbra by RNAse protection at stage 10.5.