Scerbo P et al. (2012), Ventx factors function as Nanog-like guardians ...

XB-ART-45293

PLoS One 2012 Jan 01;75:e36855. doi: 10.1371/journal.pone.0036855.

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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 .

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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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Species referenced: Xenopus laevis
Genes referenced: egr2 gsc hba4 hhex hoxb9 mixer msx1 myf5 nf2 nodal5.2 nodal5.4 pou5f3 shh sia1 six6 tfap2a ventx1 ventx1.2 ventx2 ventx2.2 wnt8a

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	Figure 1. 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.
	Figure 2. mNanog, ventx1/2, and msx1 cause distinct effects on early patterning gene expression. For gain-of-function experiments, NF3-embryos were injected radially in all blastomeres with water, msx1 mRNA (0.3 ng/blastomere, red), mNanog mRNA (0.15 ng/blastomere, blue) or ventx1/2 mRNAs (0.5 ng/blastomere, green); For loss-of-function experiments, NF-2 embryos were injected twice radially in both blastomeres with control MO (30 ng/blastomere), or a 1:1 mix of ventx1/2 MOs (30 ng/blastomere, purple). All embryos were collected at stage NF10.5 and processed for RT-QPCRs. Ectodermal, mesodermal and endodermal markers were assayed (each quantification was performed at least 3 times independently). For all RT-QPCR, graphs represent means of the fold-change calculated versus the appropriate control (fldx injected embryos in cases of overexpression and control MO for ventx1/2 knock-down) +/− s.e.m, and significance was assessed using paired t-test (p≤0.05, p≤0.005, **p≤0.0005).
	Figure 3. ventx1/2 overexpression prevents multiple lineage commitment. (A) NF2-embryos were injected twice in one blastomere, either with msx1 mRNAs (0.6 ng/blastomere), ventx1/2 mRNAs (1 ng/blastomere), mNanog mRNA (0,3 ng/blastomere), or with water for control; fldx was used as a lineage tracer. WISH with a k81a1 probe were performed at stage NF10.5 (left panels, animal views, dorsal side to the top). The progeny of the injected blastomere was revealed by fluorescence; white arrows indicate the injected side (right panels). (B) Sixteen-cell stage embryos (NF5) were injected in one AB4 blastomere with msx1 mRNA (0.15 ng), ventx1/2 mRNAs (0.5 ng), mNanog mRNA (0.15 ng), or water, collected at stage NF10.5 and processed for WISH with a k81a1 probe (animal views). Black stripped lines mark the border between injected and uninjected domains. (C) NF2-embryos were injected twice in one blastomere with msx1 mRNA (5 ng/blastomere), ventx1/2 mRNAs (5 ng/blastomere), ventx1/2+msx1 mRNAs (5 ng +5 ng/blastomere), or with water. WISH with a myf5 probe were performed at stage NF10.5; black arrowheads point to myf5-expressing territories (left panels, ventral views, dorsal side to the top). The progeny of the injected blastomere was revealed by fldx fluorescence; white arrows point to the injected side (right panels).
	Figure 4. 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.
	Figure 5. 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).
	Determination of lethal doses of mNanog and OlNanog. NF3-embryos were injected radially in all blastomeres, with water, mNanog mRNA or OlNanog mRNA at multiples doses and embryonic lethality was assessed at late blastula (NF9) and early tadpole (NF31). The doses indicated correspond to the total amount of mRNA injected per embryo. (A-C) Representative NF9 embryos observed in the indicated conditions (top panels), an arrow points to the embryos shown at greater magnification (bottom panels). The number of embryos injected is indicated. (D-E) Percentage of lethality observed at NF9 and NF31 after mNanog and OlNanog injection, respectively. The numbers of dead and living embryos observed at both time points is given under the graphs. Note that a dose of 1,2 ng of mNanog results in 100% embryonic lethality at NF9, while 0,6 ng (half the lethal dose) had no toxic effect; hence this condition was retained for further study. Conversely, OlNanog overexpression led to increased lethality beyond the 2 ng injection condition of OlNanog RNA (dotted line). Embryo death arose from 5 ng injected embryos, and about 40% or 100% lethality was observed at NF31 for the 5 ng or the 10 ng conditions respectively. Hence the condition with 1,2 ng injected embryos was retained for further study.
	OlNanog overexpression leads to phenotypes that strongly differ from those observed upon mNanog or ventx1/2 overexpression. (A) NF3 embryos were injected radially with OlNanog mRNA (0.6 ng, 1.2 ng, 2 ng and 5 ng./embryo), or with water for control. Representative phenotypes observed at early tadpole stage (NF31) are shown (lateral views, anterior to the left, dorsal to the top). (B) Percentages of observed phenotypes for the different OlNanog mRNAs doses assayed. Across the whole range of concentration used, the phenotypes obtained in OlNanog-injected embryos strongly differed from those resulting from mNanog overexpression (C and D). No cues of ventralization were observed as seen with mNanog-injected embryos (see black arrowheads in C), the embryos retaining distinguishable head structures. The main effect was a shortened axis, resulting from defects in blastopore closure (see white arrowheads in A).

References [+] :

Abu-Remaileh, Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/β-catenin signalling. 2010, Pubmed, Xenbase