Swiers G et al. (2010), A conserved mechanism for vertebrate mesoderm s...

XB-ART-41453

Dev Biol 2010 Jul 01;3431-2:138-52. doi: 10.1016/j.ydbio.2010.04.002.

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A conserved mechanism for vertebrate mesoderm specification in urodele amphibians and mammals.

Swiers G , Chen YH , Johnson AD , Loose M .

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Understanding how mesoderm is specified during development is a fundamental issue in biology, and it has been studied intensively in embryos from Xenopus. The gene regulatory network (GRN) for Xenopus is surprisingly complex and is not conserved in vertebrates, including mammals, which have single copies of the key genes Nodal and Mix. Why the Xenopus GRN should express multiple copies of Nodal and Mix genes is not known. To understand how these expanded gene families evolved, we investigated mesoderm specification in embryos from axolotls, representing urodele amphibians, since urodele embryology is basal to amphibians and was conserved during the evolution of amniotes, including mammals. We show that single copies of Nodal and Mix are required for mesoderm specification in axolotl embryos, suggesting the ancestral vertebrate state. Furthermore, we uncovered a novel genetic interaction in which Mix induces Brachyury expression, standing in contrast to the relationship of these molecules in Xenopus. However, we demonstrate that this functional relationship is conserved in mammals by showing that it is involved in the production of mesoderm from mouse embryonic stem cells. From our results, we produced an ancestral mesoderm (m)GRN, which we suggest is conserved in vertebrates. The results are discussed within the context of a theory in which the evolution of mechanisms governing early somatic development is constrained by the ancestral germ line-soma relationship, in which germ cells are produced by epigenesis.

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Species referenced: Xenopus
Genes referenced: grn nodal nodal1 odc1 sox17a sox17b.1 tbxt

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	Fig. 3. Analysis of the effect of SB 431542 Nodal antagonist during early axolotl development. (A) Representative embryos showing the complete block to gastrulation caused by SB 431542 treatment, as seen in Xenopus embryos. Panels i, iv, v, vi, vii, ix, and x are vegetal views. Panels vii and xi show the animal view of the embryos in vi and x, respectively. Embryos were treated from the two-cell stage. (B) qRT–PCR analysis of inhibitor treated embryos (five embryos per sample).
	Fig. 4. AxNodal-1 and AxNodal-2 gene knockdown. (A) Schematic illustrating the action of the two splice morpholinos targeted to AxNodal-1 and AxNodal-2 (shown as M:A and M:B). Approximate location of PCR primers indicated by arrows. (B) PCR demonstrates effectiveness of AxNodal-1 and AxNodal-2 morpholinos (MO:AxNodal-1 and MO:AxNodal-2). MO:Control = Control. About 80 ng of each of M:A and M:B, 160 ng in total; 160 ng of MO:Contol. (C) AxNodal-1 and AxNodal-2 morphant embryos. Vegetal views, except uninjected (iii and iv) and MO:AxNodal-2, stage 28 (xii), lateral view. AxNodal-2 morphants gastrulate, subsequent axial patterning is disrupted. AxNodal-1 morphants fail to gastrulate, remaining phenotypicaly at stage 9. Each morpholino combination is 80 ng of two splice morpholinos, 160 ng in total. Dorsal lips indicated by arrows. (D) qPCR analysis of MO:AxNodal embryos at stages 12 and 15.
	Fig. 5. AxMix gene knockdown. (A) Schematic illustrating the action of two splice morpholinos targeted to AxMix. Approximate location of PCR primers is indicated by arrows. (B) PCR demonstrates the effectiveness of MO:AxMix (80 ng of each M:A and M:B, 160 ng in total). MO:Control = Control, 160 ng. Note that primers designed to amplify both the normal and mis-spliced transcripts have been used. (C) AxMix morphants are unable to gastrulate. MO:Control = 160 ng control morpholino, MO:AxMix = 80 ng of Mix splice morpholinos A and B, 160 ng total. (i, iii, v, and vi) Vegetal view. (ii and iv) Dorsal view. (D and E) qPCR analysis of MO:AxMix embryos, normalised to uninjected controls at each time point.
	Fig. 6. AxBrachyury gene knockdown. (A) Schematic illustrating the action of two splice morpholinos targeted to AxBra. Approximate location of PCR primers indicated by arrows. Note that AxBra is predicted to have eight exons. Exon 4, likely to be required for DNA binding was targeted for disruption and is only 62 bp in length. (B) PCR demonstrates effectiveness of MO:AxBra (80 ng of each M:A and M:B, 160 ng in total). MO:Control = Control, 160 ng. Orange shading indicates predicted DNA binding domain. (C) AxBra morphants are unable to gastrulate. MO:AxBra = 80 ng of both Brachyury splice morpholino A and B, 160 ng in total. (D and E)qPCR analysis of MO:AxBra embryos, normalised to uninjected controls at each time point.
	Fig. 7. The relationship between AxMix and AxBra. Overexpression of 200 pg of either AxMix or AxBra in whole embryos. AxMix injected dorsally leads to an up-regulation of Brachyury expression. Conversely, AxBra injected ventrally leads to a down-regulation of Mix expression.
	Fig. 9. Rescuing mesoderm induction with AxBra overexpression. (A) Axolotl animal caps injected with 1 pg of Activin mRNA induce mesoderm in the presence and absence of Mo:AxMix. The elongation phenotype, characteristic of mesoderm, can be rescued by overexpression of 200 pg of AxBra mRNA. (B) qPCR analysis of AxBra, AxSox17, and AxFGF-8 expression in animal caps. The AxBra primers detect endogenous, not exogenous Brachyury. The up-regulation of AxSox17 is rescued by the overexpression of AxBra. However, AxFGF-8 levels are not significantly reduced, supporting the existence of a Brachyury-independent FGF-8 pathway.
	Fig. 1. Analysis of AxNodal-1 and AxNodal-2 expression during axolotl early development. (A) qPCR of AxNodal-1 and AxNodal-2 expression, normalised to ODC, and then AxNodal-1 at stage 12 to compare levels between AxNodal-1 and AxNodal-2. (B) In situ hybridization for AxNodal-1 and AxNodal-2 on hemisectioned embryos (dorsal to the left). (C) Characteristic asymmetrical expression of AxNodal-1, but not AxNodal-2, at stage 20.
	Fig. 2. Analysis of AxMix and AxBrachyury expression during axolotl early development. (A) qPCR of AxMix and AxBra, normalised to ODC and then stage 12. (B) In situ hybridization on hemisectioned embryos. Stage 10.5, 10.75, and 12 images are the same embryo: dorsal = top, vegetal = left. The cartoons below represent the combined expression of AxMix (blue) and AxBra (yellow).
	Fig. 3. Analysis of the effect of SB 431542 Nodal antagonist during early axolotl development. (A) Representative embryos showing the complete block to gastrulation caused by SB 431542 treatment, as seen in Xenopus embryos. Panels i, iv, v, vi, vii, ix, and x are vegetal views. Panels vii and xi show the animal view of the embryos in vi and x, respectively. Embryos were treated from the two-cell stage. (B) qRT–PCR analysis of inhibitor treated embryos (five embryos per sample).
	Fig. 4. AxNodal-1 and AxNodal-2 gene knockdown. (A) Schematic illustrating the action of the two splice morpholinos targeted to AxNodal-1 and AxNodal-2 (shown as M:A and M:B). Approximate location of PCR primers indicated by arrows. (B) PCR demonstrates effectiveness of AxNodal-1 and AxNodal-2 morpholinos (MO:AxNodal-1 and MO:AxNodal-2). MO:Control = Control. About 80 ng of each of M:A and M:B, 160 ng in total; 160 ng of MO:Contol. (C) AxNodal-1 and AxNodal-2 morphant embryos. Vegetal views, except uninjected (iii and iv) and MO:AxNodal-2, stage 28 (xii), lateral view. AxNodal-2 morphants gastrulate, subsequent axial patterning is disrupted. AxNodal-1 morphants fail to gastrulate, remaining phenotypicaly at stage 9. Each morpholino combination is 80 ng of two splice morpholinos, 160 ng in total. Dorsal lips indicated by arrows. (D) qPCR analysis of MO:AxNodal embryos at stages 12 and 15.
	Fig. 5. AxMix gene knockdown. (A) Schematic illustrating the action of two splice morpholinos targeted to AxMix. Approximate location of PCR primers is indicated by arrows. (B) PCR demonstrates the effectiveness of MO:AxMix (80 ng of each M:A and M:B, 160 ng in total). MO:Control = Control, 160 ng. Note that primers designed to amplify both the normal and mis-spliced transcripts have been used. (C) AxMix morphants are unable to gastrulate. MO:Control = 160 ng control morpholino, MO:AxMix = 80 ng of Mix splice morpholinos A and B, 160 ng total. (i, iii, v, and vi) Vegetal view. (ii and iv) Dorsal view. (D and E) qPCR analysis of MO:AxMix embryos, normalised to uninjected controls at each time point.
	Fig. 6. AxBrachyury gene knockdown. (A) Schematic illustrating the action of two splice morpholinos targeted to AxBra. Approximate location of PCR primers indicated by arrows. Note that AxBra is predicted to have eight exons. Exon 4, likely to be required for DNA binding was targeted for disruption and is only 62 bp in length. (B) PCR demonstrates effectiveness of MO:AxBra (80 ng of each M:A and M:B, 160 ng in total). MO:Control = Control, 160 ng. Orange shading indicates predicted DNA binding domain. (C) AxBra morphants are unable to gastrulate. MO:AxBra = 80 ng of both Brachyury splice morpholino A and B, 160 ng in total. (D and E)qPCR analysis of MO:AxBra embryos, normalised to uninjected controls at each time point.
	Fig. 7. The relationship between AxMix and AxBra. Overexpression of 200 pg of either AxMix or AxBra in whole embryos. AxMix injected dorsally leads to an up-regulation of Brachyury expression. Conversely, AxBra injected ventrally leads to a down-regulation of Mix expression.
	Fig. 8. Rescuing mesoderm induction with AxMix overexpression. (A) Schematic illustrating animal cap explants. (B) Axolotl animal caps injected with 1 pg of Activin mRNA to induce mesoderm in the presence or absence of Mo:AxMix. The Mo:AxMix can be rescued by overexpression of low levels of AxMix mRNA. High levels of AxMix mRNA fail to rescue. (C) qPCR analysis of AxBra, AxSox17, and AxFGF-8 expression in animal caps. Clear rescue of Brachyury expression is observed at low levels of AxMix mRNA, whereas high AxMix mRNA levels lead to an up-regulation of Sox17 and loss of Brachyury expression. The expression of FGF-8 in the animal cap explants mirrors that seen in whole embryos.
	Fig. 9. Rescuing mesoderm induction with AxBra overexpression. (A) Axolotl animal caps injected with 1 pg of Activin mRNA induce mesoderm in the presence and absence of Mo:AxMix. The elongation phenotype, characteristic of mesoderm, can be rescued by overexpression of 200 pg of AxBra mRNA. (B) qPCR analysis of AxBra, AxSox17, and AxFGF-8 expression in animal caps. The AxBra primers detect endogenous, not exogenous Brachyury. The up-regulation of AxSox17 is rescued by the overexpression of AxBra. However, AxFGF-8 levels are not significantly reduced, supporting the existence of a Brachyury-independent FGF-8 pathway.