Research ArticleDEVELOPMENTAL BIOLOGY

The vertebrate-specific VENTX/NANOG gene empowers neural crest with ectomesenchyme potential

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaaz1469
DOI: 10.1126/sciadv.aaz1469
  • Fig. 1 Vertebrate-specific VENTX/NANOG is required for NC specification and ectomesenchyme development.

    (A) Schematic representation of VENTX/NANOG and POU5/OCT4 evolutionary history. (B) Expression profile of pou5f3.1, ventx2, pax3, and snai2 in early-neurula-stage embryos. (C to E) Eight-cell stage embryos injected in one dorsal-animal blastomere with 10 ng of ventx2-MO and 50 pg of GFP mRNA were processed for whole-mount in situ hybridization (WISH) at the late gastrula (st.13), early neurula (st.15), and late neurula (st.18) stages with the indicated probes. (F) GFP-labeled wild-type (wt) NC efficiently migrates and populates the branchial arches. In contrast, Ventx2 morphant NC exhibited defective migration, resulting in a reduced GFP-positive cranial area. Embryos were processed for WISH with sox9 and twist1 probes. (G) Embryos injected as in (C) were processed for WISH at the tailbud stage (st.25), with foxd3 and pou4f1 probes revealing normal sensory neuron development. (H) Craniofacial morphology in tadpoles (st.45): Morphant craniofacial morphology was strongly affected compared with the control side; cartilage dissection highlighted severe craniofacial defects, with reduced pharyngeal arch area in the Ventx2 morphant side. In contrast, morphant neural crest–derived (GFP-labeled) melanocytes were detected in the dorsal cranial area above altered cartilages (live imaging). Moreover, morphant neural crest–derived cells (GFP-labeled) were detected in the mesenchyme above the neural tube and in the fin (a), in sensory Rohon-Beard neurons in the dorsal neural tube (labeled by kcna1; b), and in the dorsal root ganglia (labeled by tlx3; c). Asterisks indicate injected side. Dotted lines indicate area of interest. A.U., arbitrary units. Asterisks indicate the injected side in control (red) or morphant (blue) embryos.

  • Fig. 2 Ventx2 is required for ectoderm direct programming into NC.

    (A and B) Two-cell-stage embryos were coinjected with 50 pg of inducible Pax3-GR and 50 pg of inducible Zic1-GR mRNA, with or without 10 ng of Ventx2-MO in each blastomere. Animal caps were explanted at blastula stage 9, cultured in the presence of ethanol (control, not induced) or dexamethasone (+DEX) starting at the gastrula stage (st.10.5), collected at the late neurula stage (st.18), and processed for reverse transcription quantitative polymerase chain reaction (RT-qPCR) to detect the indicated gene expressions. (C and D) Eight-cell-stage embryos injected in one dorsal-animal blastomere with 10 ng of Ventx2-MO and 50 pg of GFP mRNA were processed for WISH at the early neurula (st.15) and tailbud (st.25) stages with the indicated probes (see fig. S4 for late neurula stage). (E) The functional redundancy between frog Ventx2 and mouse Nanog was assessed by a rescue experiment: Ventx2 depletion was efficiently rescued by mNanog mRNA overexpression, restoring twist1 expression at the late neurula stage as well as global craniofacial morphology (see full fig. S7). Red asterisks indicate injected side. For qPCR graphs, error bars represent SEM values of five independent experiments with two technical duplicates. Student’s t test was used to determine statistical significance by unpaired Student’s t test. *P < 0.05, **P < 0.005, ***P < 0.0001.

  • Fig. 3 Ventx2 promotes NC stem cell and multipotent-like state.

    (A and B) Eight-cell-stage embryos injected in one dorsal-animal blastomere with 800 pg of Ventx2-GR mRNA and 50 pg of GFP mRNA were cultured in the presence of dexamethasone (+DEX) from the early gastrula stage (st.10.5) or mid-gastrula stage (st.11.5) up to the mid-neurula stage (st.18) and processed for WISH analysis at mid-neurula (st.18) with snai2 and twist1 probes. Graphs indicate phenotype quantification. (C) RT-qPCR on whole embryos indicate that late-gastrula-stage Ventx2 gain of function promotes snai2 and twist1 expression levels and reduces the blastula stage–specific pluripotency marker pou5f3.2. (D) Embryos injected as in (A) were cultured in the presence of dexamethasone (+DEX) from the mid-gastrula stage (st.11.5) and processed for WISH analysis in late neurulas (st.18), with the indicated probes. (E) RT-qPCR on whole embryos indicate that late-gastrula-stage Ventx2 gain of function promotes the expression of several genes related with NC multipotency. For qPCR graphs, error bars represent SEM values of five independent experiments with two technical duplicates. Student’s t test was used to determine statistical significance by unpaired Student’s t test. *P < 0.05, **P < 0.005, ***P < 0.0001.

  • Fig. 4 Ventx2 confers to neural plate border specifiers Pax3 and Zic1 the capacity to reprogram non-neural ectoderm to NC identity.

    (A) Two-cell-stage embryos coinjected in one blastomere with 50 pg of Pax3-GR, 50 pg of Zic1-GR, and 50 pg of GFP mRNAs were cultured in the presence of dexamethasone (+DEX) from the late gastrula stage (st.12) and processed for WISH analysis at the mid-neurula (st.18) with the indicated probes. At this late gastrula stage, the ectopic activation of Pax3 and Zic1 repressed early NC genes (snai2, sox9, sox10, twist1, and foxd3) and enhanced the sensory neural progenitor marker tlx3 (tfap2e remaining unchanged). (B) Embryos injected as in (A) together with 800 pg of Ventx2-GR mRNA were cultured in the presence of dexamethasone (+DEX) from the late gastrula stage (st.12) and processed for WISH at the late neurula stage (st.18), with the indicated probes. When coinjected with Ventx2, Pax3 and Zic1 expanded early NC genes (snai2, sox9, sox10, twist1, and foxd3) and repressed sensory neural progenitor genes (tfap2e and tlx3) and epidermal genes (xk81a1). (C) Embryos injected as in (A) and (B) were cultured in the presence of dexamethasone (+DEX) from the late gastrula stage (st.12) and processed for WISH at tailbud (st.25) with sox10 and snai2 probes. Only the Pax3/Zic1/Ventx2 combination induced ectopic expression of sox10 in the non-neural ectoderm (a strong phenotype is shown) and weaker ectopic snai2 expression. (D) Model of Ventx2 function in vertebrate NC development. Schematic illustration of the NB in in vertebrates (left) and in vertebrates (right). We propose that the vertebrate NC (violet and red dots) evolved from an ancestral condition (violet dots) shared with invertebrates, by the introduction of Ventx/Nanog activity, which conferred multipotency and acquisition of ectomesenchyme fate.

Supplementary Materials

  • Supplementary Materials

    The vertebrate-specific VENTX/NANOG gene empowers neural crest with ectomesenchyme potential

    Pierluigi Scerbo and Anne H. Monsoro-Burq

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