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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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ventx1/2 activity is necessary to maintain an uncommitted cell population in early gastrulae.(A) NF2-embryos were injected radially twice in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1/2 MOs (30 ng/blastomere). Variations of gene expression at 516-, 1000-, 2000-, 4000-cell and NF10.5 stages were assessed by RT-QPCR as in Fig. 2. Dorsal (siamois, gsc, hhex), and ventral (wnt8) mesendoderm, endoderm (xnr5, mixer) and ectoderm (tfap2a, k81a1) markers were monitored. Kinetic graphs represent means of fold-change relative to NF10.5 controls +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005), and undetectable levels of transcript noted as Φ. (B) Animal injections were performed twice in a single blastomere NF2-embryos, using MO conditions described in (A); fldx was used as a lineage tracer. WISH with an oct91 probe (left panel) were performed at stage NF10.5 and the progeny of the injected blastomere was revealed by fluorescence (right panel). Embryos are positioned with the animal side upwards; white arrows indicate the injected side. (C) Injections were performed using mRNA and MO conditions described in Fig. 2. All embryos were collected at stage NF10.5 and processed for RT-QPCRs using the pluripotency marker oct91. Data and graphs are presented as in Fig. 2.
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pone-0036855-g004: ventx1/2 activity is necessary to maintain an uncommitted cell population in early gastrulae.(A) NF2-embryos were injected radially twice in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1/2 MOs (30 ng/blastomere). Variations of gene expression at 516-, 1000-, 2000-, 4000-cell and NF10.5 stages were assessed by RT-QPCR as in Fig. 2. Dorsal (siamois, gsc, hhex), and ventral (wnt8) mesendoderm, endoderm (xnr5, mixer) and ectoderm (tfap2a, k81a1) markers were monitored. Kinetic graphs represent means of fold-change relative to NF10.5 controls +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005), and undetectable levels of transcript noted as Φ. (B) Animal injections were performed twice in a single blastomere NF2-embryos, using MO conditions described in (A); fldx was used as a lineage tracer. WISH with an oct91 probe (left panel) were performed at stage NF10.5 and the progeny of the injected blastomere was revealed by fluorescence (right panel). Embryos are positioned with the animal side upwards; white arrows indicate the injected side. (C) Injections were performed using mRNA and MO conditions described in Fig. 2. All embryos were collected at stage NF10.5 and processed for RT-QPCRs using the pluripotency marker oct91. Data and graphs are presented as in Fig. 2.

Mentions: To test whether ventx1/2 are functionally required to restrict cellular commitment during early Xenopus development, we next performed RT-QPCR to monitor kinetics of expression following ventx1/2 knockdown (Fig. 4A). Time-course experiments revealed that in ventx1/2 morphant embryos, expression levels of early dorsal mesendoderm (siamois, gsc, hhex), ventral mesendoderm (wnt8), pan-endodermal (mixer) and ventral ectoderm (tfap2a, k81a1) markers are higher at the 4000-cell stage when activation of zygotic transcription or “mid-blastula transition” (MBT) occurs. Remarkably, stronger expression was also observed at pre-MBT for xnr5, a nodal-related factor that participates in primary germ layer induction at these stages and pre-patterns the dorsal side of the embryo [56]. Overall, the expression profiles seem to be shifted to earlier time-points and to reach higher levels in ventx1/2 morphant embryos. These results suggest that in morphant embryos, cells are no longer protected against premature commitment. Interestingly, embryos in which the POU5F1 family member oct91 is knocked-down also fail to maintain a multipotent uncommitted cell population [25], [29]. We thus tested whether up-regulation of pro-differentiation markers in ventx1/2 LOF is accompanied by down-regulation of this marker of the uncommitted state. The expression of oct91 was significantly down-regulated after ventx1/2 knockdown, as assessed by RT-Q-PCR and WISH (Fig. 4, B and C). However, no significant up-regulation of oct91 was seen following ventx1/2 overexpression.


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)

ventx1/2 activity is necessary to maintain an uncommitted cell population in early gastrulae.(A) NF2-embryos were injected radially twice in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1/2 MOs (30 ng/blastomere). Variations of gene expression at 516-, 1000-, 2000-, 4000-cell and NF10.5 stages were assessed by RT-QPCR as in Fig. 2. Dorsal (siamois, gsc, hhex), and ventral (wnt8) mesendoderm, endoderm (xnr5, mixer) and ectoderm (tfap2a, k81a1) markers were monitored. Kinetic graphs represent means of fold-change relative to NF10.5 controls +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005), and undetectable levels of transcript noted as Φ. (B) Animal injections were performed twice in a single blastomere NF2-embryos, using MO conditions described in (A); fldx was used as a lineage tracer. WISH with an oct91 probe (left panel) were performed at stage NF10.5 and the progeny of the injected blastomere was revealed by fluorescence (right panel). Embryos are positioned with the animal side upwards; white arrows indicate the injected side. (C) Injections were performed using mRNA and MO conditions described in Fig. 2. All embryos were collected at stage NF10.5 and processed for RT-QPCRs using the pluripotency marker oct91. Data and graphs are presented as in Fig. 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351468&req=5

pone-0036855-g004: ventx1/2 activity is necessary to maintain an uncommitted cell population in early gastrulae.(A) NF2-embryos were injected radially twice in both blastomeres with control MO (30 ng/blastomere), or a 1∶1 mix of ventx1/2 MOs (30 ng/blastomere). Variations of gene expression at 516-, 1000-, 2000-, 4000-cell and NF10.5 stages were assessed by RT-QPCR as in Fig. 2. Dorsal (siamois, gsc, hhex), and ventral (wnt8) mesendoderm, endoderm (xnr5, mixer) and ectoderm (tfap2a, k81a1) markers were monitored. Kinetic graphs represent means of fold-change relative to NF10.5 controls +/− s.e.m, and significance was assessed using paired t-test (*p≤0.05, **p≤0.005, ***p≤0.0005), and undetectable levels of transcript noted as Φ. (B) Animal injections were performed twice in a single blastomere NF2-embryos, using MO conditions described in (A); fldx was used as a lineage tracer. WISH with an oct91 probe (left panel) were performed at stage NF10.5 and the progeny of the injected blastomere was revealed by fluorescence (right panel). Embryos are positioned with the animal side upwards; white arrows indicate the injected side. (C) Injections were performed using mRNA and MO conditions described in Fig. 2. All embryos were collected at stage NF10.5 and processed for RT-QPCRs using the pluripotency marker oct91. Data and graphs are presented as in Fig. 2.
Mentions: To test whether ventx1/2 are functionally required to restrict cellular commitment during early Xenopus development, we next performed RT-QPCR to monitor kinetics of expression following ventx1/2 knockdown (Fig. 4A). Time-course experiments revealed that in ventx1/2 morphant embryos, expression levels of early dorsal mesendoderm (siamois, gsc, hhex), ventral mesendoderm (wnt8), pan-endodermal (mixer) and ventral ectoderm (tfap2a, k81a1) markers are higher at the 4000-cell stage when activation of zygotic transcription or “mid-blastula transition” (MBT) occurs. Remarkably, stronger expression was also observed at pre-MBT for xnr5, a nodal-related factor that participates in primary germ layer induction at these stages and pre-patterns the dorsal side of the embryo [56]. Overall, the expression profiles seem to be shifted to earlier time-points and to reach higher levels in ventx1/2 morphant embryos. These results suggest that in morphant embryos, cells are no longer protected against premature commitment. Interestingly, embryos in which the POU5F1 family member oct91 is knocked-down also fail to maintain a multipotent uncommitted cell population [25], [29]. We thus tested whether up-regulation of pro-differentiation markers in ventx1/2 LOF is accompanied by down-regulation of this marker of the uncommitted state. The expression of oct91 was significantly down-regulated after ventx1/2 knockdown, as assessed by RT-Q-PCR and WISH (Fig. 4, B and C). However, no significant up-regulation of oct91 was seen following ventx1/2 overexpression.

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