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Fig 6. Neural crest cell marker gene expression is decreased in pax9-depleted X. tropicalis embryos.
(A) Schematic depicting in situ hybridization experiments for neural crest markers. At the 2-cell stage, one cell was injected with a Pax9 MO and fluoro-ruby tracer dye. Embryos were grown to stage 18 before fixation and staining for Sox9, Twist, and Slug neural crest cell markers. (B) Reduced expression of Sox9, Twist, and Slug neural crest cell markers revealed by in situ hybridization in Xenopus embryos after Pax9 depletion. Control uninjected embryos are shown to the left. Embryos injected with the Pax9 MO and fluoro-ruby dye on the right side are shown in the middle. Embryos injected with the Pax9 MO and fluoro-ruby dye on the left side are shown to the right. (C) Reduced Slug expansion upon Pax9 depletion in X. tropicalis embryos. Ventral, Mid, and Dorsal Slug trajectories were quantified as indicated (μm, white lines) on both sides along the ventral dorsal axis in control uninjected embryos (left), embryos injected with 15 ng Pax9 MO at the one-cell stage (middle), and embryos injected with 20 ng Pax9 MO at the one-cell stage (right). N = 18. (D) Overexpression of human PAX9 mRNA alone in X. tropicalis embryos results in reduced Slug expression. At the 2-cell stage, human PAX9 mRNA was injected into 1 cell of the X. tropicalis embryos, with the other cell serving as an uninjected control (UIC). At stage 18, embryos were fixed and in situ hybridizations were performed to observe expression of the neural crest cell marker, Slug. (E) The injection of human PAX9 mRNA rescues Slug expression in Pax9-depleted X. tropicalis embryos, At the one-cell stage, embryos are injected with a MO targeting Pax9. At the 2-cell stage, one cell is injected with human PAX9 mRNA. Embryos were grown to stage 18 before in situ hybridizations were performed to monitor Slug expression. (F) Quantitation of Slug expression from rescue experiments shown in (E). Above: The side of the embryos that received both the PAX9 mRNA and MO (right side) had higher Slug expression than the side receiving only the MO (left side). Slug area manually segmented and measured with ImageJ. RNA injected side demonstrated increased expansion of slug. Below: The side of the embryos that received both the PAX9 mRNA and MO (left side) had higher Slug expression than the side receiving only the MO (right side). Slug area manually segmented and measured with ImageJ. RNA injected side demonstrated increased expansion of Slug. Student`s t-test; paired, used parametric test, ** p<0.01.
https://doi.org/10.1371/journal.pgen.1008967.g006 |
