Limits...
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.

Show MeSH
mNanog expression rescues specifically ventx1/2 morphant embryos.(A) Two-cell stage embryos (NF2) were first injected radially twice with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1 and ventx2 MOs (ventx1/2 MOs; 30 ng/blastomere), and subsequently injected radially at NF3 in all blastomeres with mNanog mRNA (0.15 ng/blastomere), msx1 mRNA (0.15 ng/blastomere), or with water. (B) Range of phenotypes observed in rescue of ventx1/2 knockdown experiment. (C) Percentages observed for each phenotypic category in three independent replicates of the rescue experiment. The combined injections performed are indicated at the bottom of the graph, and the number of injected embryos for each condition is indicated on the top of each bar. NF28 embryos were processed for WISH with six6, egr2 and hoxb9 (D), or six6, shh and hba4 (E) probes (anterior to the left, dorsal to the top).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351468&req=5

pone-0036855-g005: mNanog expression rescues specifically ventx1/2 morphant embryos.(A) Two-cell stage embryos (NF2) were first injected radially twice with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1 and ventx2 MOs (ventx1/2 MOs; 30 ng/blastomere), and subsequently injected radially at NF3 in all blastomeres with mNanog mRNA (0.15 ng/blastomere), msx1 mRNA (0.15 ng/blastomere), or with water. (B) Range of phenotypes observed in rescue of ventx1/2 knockdown experiment. (C) Percentages observed for each phenotypic category in three independent replicates of the rescue experiment. The combined injections performed are indicated at the bottom of the graph, and the number of injected embryos for each condition is indicated on the top of each bar. NF28 embryos were processed for WISH with six6, egr2 and hoxb9 (D), or six6, shh and hba4 (E) probes (anterior to the left, dorsal to the top).

Mentions: Based on the above data, we tested the ability of mNanog to rescue development of ventx1/2 knock-downed embryos. MOs directed against ventx1/2[37] or control MO were first injected radially in each blastomere at the 2-cell stage (NF2), followed by radial injections in all blastomeres at stage NF3 of either mNanog or msx1 mRNAs (Fig. 5A). Injections of control MO+mNanog and control MO+msx1 led to a high proportion of ventralized embryos (Fig. 5, B and C, about 80% and 60% respectively). As described [37], ventx1/2 MOs injection caused dorsalization defects in 80% of embryos, whereas control injections (control MO+water) yielded 90% of normal embryos. Quite remarkably, the addition of mNanog mRNA to ventx1/2 MOs produced 50% of embryos with normal morphology. Antero-posterior (Fig. 5D) and dorso-ventral axes (Fig. 5E) were correctly restored in such embryos, as revealed by whole mount in situ hybridization for markers of notochord (shh), ventral blood islands (hba4), spinal cord (hoxb9) and brain (six6 and egr2, also known as optx2 and krox-20 respectively). In contrast, msx1 overexpression failed to restore a normal morphology in ventx1/2 morphant embryos. In this condition, about 60% of embryos remained dorsalized and about 40% became ventralized, confirming that msx1 ventralizes embryos through mechanisms distinct from the ones induced by ventx1/2 and mNanog (Fig. 5E). Altogether, these results demonstrate that mNanog is able to substitute for ventx1/2 in Xenopus development and that this effect is specific.


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 expression rescues specifically ventx1/2 morphant embryos.(A) Two-cell stage embryos (NF2) were first injected radially twice with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1 and ventx2 MOs (ventx1/2 MOs; 30 ng/blastomere), and subsequently injected radially at NF3 in all blastomeres with mNanog mRNA (0.15 ng/blastomere), msx1 mRNA (0.15 ng/blastomere), or with water. (B) Range of phenotypes observed in rescue of ventx1/2 knockdown experiment. (C) Percentages observed for each phenotypic category in three independent replicates of the rescue experiment. The combined injections performed are indicated at the bottom of the graph, and the number of injected embryos for each condition is indicated on the top of each bar. NF28 embryos were processed for WISH with six6, egr2 and hoxb9 (D), or six6, shh and hba4 (E) probes (anterior to the left, dorsal to the top).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351468&req=5

pone-0036855-g005: mNanog expression rescues specifically ventx1/2 morphant embryos.(A) Two-cell stage embryos (NF2) were first injected radially twice with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1 and ventx2 MOs (ventx1/2 MOs; 30 ng/blastomere), and subsequently injected radially at NF3 in all blastomeres with mNanog mRNA (0.15 ng/blastomere), msx1 mRNA (0.15 ng/blastomere), or with water. (B) Range of phenotypes observed in rescue of ventx1/2 knockdown experiment. (C) Percentages observed for each phenotypic category in three independent replicates of the rescue experiment. The combined injections performed are indicated at the bottom of the graph, and the number of injected embryos for each condition is indicated on the top of each bar. NF28 embryos were processed for WISH with six6, egr2 and hoxb9 (D), or six6, shh and hba4 (E) probes (anterior to the left, dorsal to the top).
Mentions: Based on the above data, we tested the ability of mNanog to rescue development of ventx1/2 knock-downed embryos. MOs directed against ventx1/2[37] or control MO were first injected radially in each blastomere at the 2-cell stage (NF2), followed by radial injections in all blastomeres at stage NF3 of either mNanog or msx1 mRNAs (Fig. 5A). Injections of control MO+mNanog and control MO+msx1 led to a high proportion of ventralized embryos (Fig. 5, B and C, about 80% and 60% respectively). As described [37], ventx1/2 MOs injection caused dorsalization defects in 80% of embryos, whereas control injections (control MO+water) yielded 90% of normal embryos. Quite remarkably, the addition of mNanog mRNA to ventx1/2 MOs produced 50% of embryos with normal morphology. Antero-posterior (Fig. 5D) and dorso-ventral axes (Fig. 5E) were correctly restored in such embryos, as revealed by whole mount in situ hybridization for markers of notochord (shh), ventral blood islands (hba4), spinal cord (hoxb9) and brain (six6 and egr2, also known as optx2 and krox-20 respectively). In contrast, msx1 overexpression failed to restore a normal morphology in ventx1/2 morphant embryos. In this condition, about 60% of embryos remained dorsalized and about 40% became ventralized, confirming that msx1 ventralizes embryos through mechanisms distinct from the ones induced by ventx1/2 and mNanog (Fig. 5E). Altogether, these results demonstrate that mNanog is able to substitute for ventx1/2 in Xenopus development and that this effect is specific.

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.

Show MeSH