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Ventx factors function as Nanog-like guardians of developmental potential in Xenopus.

Scerbo P, Girardot F, Vivien C, Markov GV, Luxardi G, Demeneix B, Kodjabachian L, Coen L - PLoS ONE (2012)

Bottom Line: Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization.Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells.We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

View Article: PubMed Central - PubMed

Affiliation: Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris, France.

ABSTRACT
Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

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mNanog and ventx1/2 overexpression cause similar effects in Xenopus embryos.(A) Four-cell stage embryos (NF3) were injected in both dorsal blastomeres, with a 1∶3 mix of ventx1.2 and ventx2.1-b mRNAs (ventx1/2; 0.5 ng per blastomere), with mouse Nanog mRNA (mNanog; 0.15 ng/blastomere), or with water for control. Representative phenotypes observed at tailbud stage (NF28) are shown (lateral views, anterior to the left, dorsal to the top). (B) Percentages of observed phenotypes in three independent experiments for mock (n = 14), ventx1/2 (n = 31) and mNanog (n = 36) mRNAs injections. (C) Embryos injected as in (A) were collected at early gastrulae (NF10.5; whole embryos: ventral view, dorsal side to the top; hemisected embryos: lateral view, dorsal to the left, animal side to the top) and tailbud (NF28; ventral view, anterior to the left) stages and processed for whole-mount in situ hybridization (WISH) with a gsc or hhex probe, or with hba4 (black arrowheads) and egr2 (white arrowheads), respectively. The number of embryos showing staining similar to the one photographed over the total number of embryos assayed is indicated.
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pone-0036855-g001: mNanog and ventx1/2 overexpression cause similar effects in Xenopus embryos.(A) Four-cell stage embryos (NF3) were injected in both dorsal blastomeres, with a 1∶3 mix of ventx1.2 and ventx2.1-b mRNAs (ventx1/2; 0.5 ng per blastomere), with mouse Nanog mRNA (mNanog; 0.15 ng/blastomere), or with water for control. Representative phenotypes observed at tailbud stage (NF28) are shown (lateral views, anterior to the left, dorsal to the top). (B) Percentages of observed phenotypes in three independent experiments for mock (n = 14), ventx1/2 (n = 31) and mNanog (n = 36) mRNAs injections. (C) Embryos injected as in (A) were collected at early gastrulae (NF10.5; whole embryos: ventral view, dorsal side to the top; hemisected embryos: lateral view, dorsal to the left, animal side to the top) and tailbud (NF28; ventral view, anterior to the left) stages and processed for whole-mount in situ hybridization (WISH) with a gsc or hhex probe, or with hba4 (black arrowheads) and egr2 (white arrowheads), respectively. The number of embryos showing staining similar to the one photographed over the total number of embryos assayed is indicated.

Mentions: These extensive similarities prompted us to compare the effects of overexpression of mouse Nanog (mNanog) to combined Xenopus ventx1.2[32] and ventx2.1-b[47] (referred to as ventx1/2 from now on) overexpression on Xenopus embryonic development. The relevant mRNAs were dorsally injected at the 4-cell stage (NF3 [48]), using previously described doses for ventx1/2 (0.5 ng per blastomere [34]) and half the lethal dose for mNanog (0.6 ng per blastomere, see Fig. S2). As expected [34], ventx1/2 overexpression led at tailbud stage (NF28) to severely ventralized phenotypes with truncated anterior structures (Fig. 1A). Remarkably, mNanog overexpression produced similar defects with comparable penetrance (Fig. 1, A and B, and Fig. S3C). In contrast, overexpression of the medaka ortholog OlNanog led to phenotypes clearly distinct from those obtained with mNanog, no ventralization being observed at any of the doses assayed, with all embryos displaying clear head features (Fig. S3A).


Ventx factors function as Nanog-like guardians of developmental potential in Xenopus.

Scerbo P, Girardot F, Vivien C, Markov GV, Luxardi G, Demeneix B, Kodjabachian L, Coen L - PLoS ONE (2012)

mNanog and ventx1/2 overexpression cause similar effects in Xenopus embryos.(A) Four-cell stage embryos (NF3) were injected in both dorsal blastomeres, with a 1∶3 mix of ventx1.2 and ventx2.1-b mRNAs (ventx1/2; 0.5 ng per blastomere), with mouse Nanog mRNA (mNanog; 0.15 ng/blastomere), or with water for control. Representative phenotypes observed at tailbud stage (NF28) are shown (lateral views, anterior to the left, dorsal to the top). (B) Percentages of observed phenotypes in three independent experiments for mock (n = 14), ventx1/2 (n = 31) and mNanog (n = 36) mRNAs injections. (C) Embryos injected as in (A) were collected at early gastrulae (NF10.5; whole embryos: ventral view, dorsal side to the top; hemisected embryos: lateral view, dorsal to the left, animal side to the top) and tailbud (NF28; ventral view, anterior to the left) stages and processed for whole-mount in situ hybridization (WISH) with a gsc or hhex probe, or with hba4 (black arrowheads) and egr2 (white arrowheads), respectively. The number of embryos showing staining similar to the one photographed over the total number of embryos assayed is indicated.
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Related In: Results  -  Collection

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pone-0036855-g001: mNanog and ventx1/2 overexpression cause similar effects in Xenopus embryos.(A) Four-cell stage embryos (NF3) were injected in both dorsal blastomeres, with a 1∶3 mix of ventx1.2 and ventx2.1-b mRNAs (ventx1/2; 0.5 ng per blastomere), with mouse Nanog mRNA (mNanog; 0.15 ng/blastomere), or with water for control. Representative phenotypes observed at tailbud stage (NF28) are shown (lateral views, anterior to the left, dorsal to the top). (B) Percentages of observed phenotypes in three independent experiments for mock (n = 14), ventx1/2 (n = 31) and mNanog (n = 36) mRNAs injections. (C) Embryos injected as in (A) were collected at early gastrulae (NF10.5; whole embryos: ventral view, dorsal side to the top; hemisected embryos: lateral view, dorsal to the left, animal side to the top) and tailbud (NF28; ventral view, anterior to the left) stages and processed for whole-mount in situ hybridization (WISH) with a gsc or hhex probe, or with hba4 (black arrowheads) and egr2 (white arrowheads), respectively. The number of embryos showing staining similar to the one photographed over the total number of embryos assayed is indicated.
Mentions: These extensive similarities prompted us to compare the effects of overexpression of mouse Nanog (mNanog) to combined Xenopus ventx1.2[32] and ventx2.1-b[47] (referred to as ventx1/2 from now on) overexpression on Xenopus embryonic development. The relevant mRNAs were dorsally injected at the 4-cell stage (NF3 [48]), using previously described doses for ventx1/2 (0.5 ng per blastomere [34]) and half the lethal dose for mNanog (0.6 ng per blastomere, see Fig. S2). As expected [34], ventx1/2 overexpression led at tailbud stage (NF28) to severely ventralized phenotypes with truncated anterior structures (Fig. 1A). Remarkably, mNanog overexpression produced similar defects with comparable penetrance (Fig. 1, A and B, and Fig. S3C). In contrast, overexpression of the medaka ortholog OlNanog led to phenotypes clearly distinct from those obtained with mNanog, no ventralization being observed at any of the doses assayed, with all embryos displaying clear head features (Fig. S3A).

Bottom Line: Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization.Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells.We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

View Article: PubMed Central - PubMed

Affiliation: Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris, France.

ABSTRACT
Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

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