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Experiment details for pax3

Simon E et al. (2017) Assay

Vestigial-like 3 is a novel Ets1 interacting partner and regulates trigeminal nerve formation and cranial neural crest migration.

Gene Clone Species Stages Anatomy
pax3.S laevis NF stage 10 neuroectoderm
pax3.S laevis NF stage 13 neuroectoderm , neural plate border

  Fig. 7. Vgll3 interacts with tead proteins and can activate pax3 independently of tead. (A) Embryos injected with vgll3 (1ng or lacZ) mRNA and T1MO/T2MO or cMO were fixed at stage 10 or 13 and analysed for pax3. Vgll3 induces ectopic pax3 expression when tead1 and tead2 have been knocked down. Lower right panel (IB myc): embryos were injected with 50 pg of myc-tead1 or myc-tead2 mRNAs with 15 ng of T1MO or T2MO and analysed by IB. T1MO and T2MO block efficiently tead1 and tead2 expression respectively. Erk2 is used as control. ni: non injected embryo. (B) Embryos injected with HA-vgll3, myc-tead1 or myc-tead2 mRNAs were processed for immunoprecipitation with HA (IP HA) or myc antibodies (IP myc) followed by IB with antibodies. Vgll3 interacts with tead1 and tead2 in the embryo. Lysate, IB of injected embryo before immunoprecipitation. Vertical white lines indicate spliced images in order to remove results not described in this paper (IP myc).

Gene Clone Species Stages Anatomy
pax3.S laevis NF stage 11 neuroectoderm

  Figure S8. The histidine repeat in vgll3 is required for pax3 activation. (A) Sequence alignment of histidine (H) tract in Vgll3 proteins from different vertebrate species. Numbers indicate the relative position of the histidine tract. Xenlae, Xenopus laevis, Xentrop, Xenopus tropicalis. (B) Embryos were injected with 1 ng of vgll3 (vgll3 wt) or vgll3Dhis, fixed at stage 11 and analysed for pax3 expression. Vgll3Dhis cannot activate pax3 (100%, n=30). (*) injected side. (C) HEK293 cells, recently tested for contamination, were transfected with 1μg of plasmid encoding HA-vgll3wt or HAvgll3Dhis, fixed and analysed by immunocytochemistry using HA antibody. The deletion of histidine tract from vgll3 does not impair its nuclear localization. White arrow indicates nuclear staining. Scale bars, 10 μm.

Gene Clone Species Stages Anatomy
pax3.L laevis NF stage 14 to NF stage 20 neuroectoderm , neural plate , cranial neural crest , preplacodal ectoderm , neural plate border

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  Figure S1. Vgll3 does not co-localise with trigeminal placode markers. (A) Wholemount ISH for vgll3 and trigeminal placode genes islet1, neuroD, pax3 and foxi1c. (B) Whole-mount ISH for trigeminal placodal genes islet1, neuroD, pax3 and foxi1c. Blue arrow indicates the position of vgll3 expression domain. All staining are visualized in blue. Analysed embryos are from stage 14 to stage 20 depending on the trigeminal markers used. All views are dorsal-anterior.

Gene Clone Species Stages Anatomy
pax3.L laevis NF stage 15 neuroectoderm , neural crest , neural plate , preplacodal ectoderm , presumptive rhombomere
pax3.L laevis NF stage 18 neuroectoderm , neural crest , neural plate , preplacodal ectoderm , presumptive rhombomere
pax3.L laevis NF stage 19 neuroectoderm , neural plate , cranial neural crest , preplacodal ectoderm
pax3.L laevis NF stage 21 neuroectoderm , neural crest , neural plate , preplacodal ectoderm

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  Fig. 3. Vgll3 stimulates pax3 and zic1 expression. (A) Embryos were injected with v3MO or vgll3 mRNA and analysed at stage 19 for pax3 and zic1 expression. Defects of CNC lateral streams and trigeminal placode are shown by arrows and arrowhead respectively. (B) ISH for vgll3 and pax3 shows overlapping expression at the r2 level (blue arrow). (C) Stage 19 embryos injected with pax3GR/zic1GR mRNAs show a faint vgll3 and a strong snail2 expression after dexamethasone treatment (+dex). (D) cMO/LacZ mRNA control and quantification. Three independent experiments were performed (n=40). (*), injected side. All views are dorsal-anterior. A dotted line indicates the midline embryo. (E) ISH for vgll3 and zic1 shows overlapping expression at the r2 level (blue arrow). Zic1 expression in stage 21 embryo.