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Fig. 1. gli2 and Ptc1 expression patterns in Xenopus embryos. (A–G, J–K, M–O) Dorsal views of Xenopus laevis embryos, anterior side on the left. (L) Dorsal view, anterior side at the top. (J′, K′): Transversal sections, dorsal side at the top. (H–I) Lateral views, anterior side on the left. (K′) Anterior view. (A–B) gli2 is expressed in the dorsolateral region of the embryo at stages 12.5 and 13 (yellow arrowheads). (C–D) gli2 transcripts are detected in the NPB at early neurula stage (yellow arrowheads) and in the anterior NP (black arrow). No expression can be observed in the dorsal midline (red arrowhead) (E–G). From stage 17 to 19, gli2 becomes more prominent in the lateral and anterior NPB. (H–I) At tailbud stages, gli2 is expressed in the mandibular (mb) and hyoid (hy) NC streams, in the branchial arches (b.a.), in the otic (Ot) and optic (Op) vescicle and in the presomitic mesoderm (S). (J–K) Double ISH showing gli2 expression with two gene markers. (J–J') gli2 is expressed in an overlapping territory with the NC marker foxd3. (K–K′) The domain of gli2 expression extends laterally from the NP, as evidenced by the double ISH with sox2 in whole embryos. (K′) Anterior view showing that the expression of gli2 extends mores anteriorly than that of sox2. (L) At the late gastrula stage, Ptch1 is expressed in the dorsal side of the blastopore. (M–O) At neurula stages, Ptch1 can be detected in the NPB and in the neural folds. (O′) Expression of Ptch1 in the cell membranes of ectodermal cells. (P) Diagram representing the expression domains of the Ptch1 protein (green) and the genes gli2 (blue), foxd3 (light yellow) and sox2 (pink).
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Fig. 2. gli2 participates in the early induction of the NC. (A–E, H–M) Dorsal views of Xenopus laevis embryos at mid-neurula stage; anterior side is on the left. Arrowheads indicate the injected side, shown in turquoise and by GFP fluorescence in A–E. The expression of gene markers is shown in purple. (A–F) Analysis of gli2MO efficiency in vivo and in vitro. (A–E) in vivo assay. Merged fluorescence and clear field images of each embryo are shown as insets. (A) Normal GFP fluorescence was detected in the embryo coinjected with gli2GFP mRNA (2.6 ng/E) and CoMO (27 ng/E). (B–E) Embryos injected with gli2GFP and increasing concentrations of gli2MO (2.5 to 27 ng/E) evidenced a reduction in GFP fluorescence intensity. (F) in vitro analysis by Western blot shows a marked reduction in the expression of gli2GFP. α-tubulin expression was used as loading control. (G) Schematic representation of the gain- and loss-of-function experiments. (H–J) Analysis of gli2 requirement on NC induction. gli2MO-injected embryos show a reduced expression of the NC induction markers pax3, msx1 and snail1. (K–M) gli2 mRNA-injected embryos show an increase in the expression of the NPB markers pax3, msx1 and snail1. (N–O) Quantification of phenotypes shown in H–J and K–M, respectively. (P–R) The specific inhibition of Gli2 in NC explants incubated from stage 12.5 to 16 with GANT61 diminishes the expression of the NC marker foxd3.
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Fig. 3. gli2 requirement in NC specification. (B–G, I–N, P–U) Dorsal views of Xenopus laevis embryos at mid-neurula stage; anterior side is on the left. Arrowheads indicate the injected side (turquoise). The expression of the genes is shown in purple. (A) Graphic representation of the experiments. (B–D) gli2MO-injected embryos show reduced expression of the NC markers foxd3 (B), snail2 (C) and sox9 (D). (E–G) Increase in the expression of the NP marker sox2 and the epidermal marker xk81a after gli2MO injection. Figure G shows a double ISH of sox2 and xk81a, with brackets indicating the width of the NC domain. (I–N) The opposite effect is seen when gli2 is overexpressed in the NC territory. (H, O) Quantification of gli2MO and of gli2 mRNA effects on the expression of NC markers, respectively. (P–Q) Overexpression of gli2 with the construct α3000gli2 produced an increase in the expression of foxd3 and snail2. (R–S) Control of morpholino specificity. Coinjection of gli2MO morpholino along with a plasmid encoding a mutated gli2 at the morpholino binding site (CRgli2) rescued the expression of the NC markers foxd3 and snail2. (T) The injection of the α3000CRgli2 construct also rescued foxd3 expression. (U) The microinjection of CoMO did not alter foxd3 expression. (V) Quantification of the experiments shown in Fig. 3P–U.
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Fig. 4. gli2 requirement for the maintenance of NC specification. (B–E, G–J, M–N) Dorsal views of Xenopus laevis embryos at midneurula stage; anterior side is on the left. Arrowheads indicate the injected or implanted side. (A–E) GANT61 injection at stage 3 produces a decrease in the expression of foxd3 and snail2, with a concomitant increase in the expression of sox2 and xk81a. (F) Quantification of phenotypes in the injected embryos of figures B–E. (G–J) Injection of the GANT61 diluent (DMSO 0,03%) produced no changes in the expression of the gene markers analyzed. (K) Quantification of phenotypes. (L–O) Implanted GANT61-soaked beads from stage 12.5 to 16 produced a reduction in foxd3 expression, as opposed to the implantation of DMSO-soaked beads. (P–R) NC explants with mesoderm incubated in GANT61 from stage 14–18 lose foxd3 expression.
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Fig. 5. The activator form of gli2 is required for NC specification. (A) Schematic procedure of the experiments. (B–K, M–P, R–T) Dorsal views of Xenopus laevis embryos at mid-neurula stage, anterior side to the left. Arrowheads indicate the injected side, shown in turquoise. (B–C) gli2act increases the expression of foxd3 and snail2. (D-G) gli2act and α3000gli2act are able to rescue the expression of foxd3 and snail2 in the morphant embryo. (H–K) gli2rep represses the expression of foxd3 and snail2 and therefore is not able to rescue these expressions in the morphant embryo. (L) Quantification of effects shown in B–K. (M–P) The phosphorylation mutant gli2PSM increases the expression of NC markers and represses the activity of sox2 and xk81a. (R-T) The α3000gli2PSM construct also increases the expression of foxd3 and is able to rescue the effects of gli2MO. (Q, U) Quantification of the experiments shown in M–P and R–T, respectively.
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Fig. 6. gli2 acts through a transcriptional activation mechanism and affects the expression of several NC markers. (A–B) Gli-Luciferase reporter assay. (A) Schematic representation of the Luciferase assay experiment. (B) Transcriptional activity of Gli2 in NC explants. Intensities are shown as relative units of Luciferase normalized to the internal control Renilla Luciferase. An increase in Gli2 and Ihh function increases the expression of the Gli-dependent reporter construct. (C-D) RT-qPCR analysis of Gli2 function on the expression of NPB and NC markers. Each color coded bar represents the relative expression level of the markers for each condition (gain and loss-of-function of gli2). All the expression levels denote Ct values that were normalized first to the expression level of gadph, and then they were normalized to the expression of each gene in the uninjected wildtype condition, and are presented as fold change. (C) Schematic representation of the grafting experiment. At stage 15–16 total RNA was isolated and then RT-qPCR was performed. (D) Expression levels of the markers pax3, hairy2b, snail1, snail2, twist-b, e-cad and n-cad. Data are presented as mean ± s.d. of triplicates of two independent qPCR experiments. Reference: **, P < 0.05; ***, P < 0.001 (P-values correspond to Student's t-Test).
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Fig. 7. gli2 is involved in the apoptosis of NCC but not in their proliferation. (A–B) Dorsal views of Xenopus laevis embryos, anterior side to the top. White arrowheads indicate the injected side. Gain- and loss-of-function of gli2 did not alter the number of proliferating cells between the injected and the control side. (A′, A′, B′, B′) Insets showing the quantified area of each embryo. (C) Quantification of proliferating cells in the embryos treated with gli2 mRNA or gli2MO. (D) Schematic diagram showing the approach used to evaluate the apoptosis status of NCC. (E) Graph showing that the overexpression of gli2 prevents NCC from undergoing normal apoptosis. The inhibition of gli2 expression did not produce any significant changes in the apoptosis level of NCC. The activator form of Gli2 also decreased apoptosis in NC explants. Values are mean ± s.d. from two independent experiments. P ≤ 0.05 (Student's t-test).
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Fig. 8. gli2 acts in a cell-autonomous manner in NC migration. (B–E, G, J–K, M–N, Q–S) Lateral views of embryos with the anterior side on the left showing the expression of the NC migration markers sox10 and foxd3. Figures Q–S show merged images of fluorescence and bright field captures. (A, F, I, L, P) Schematic representation of the different assays. (B) Stage 20 embryos showing a reduction in the migration front in the injected side evidenced by the expression of foxd3. (C) In stage 22 embryos the difference in expression between the control and the injected side is notably higher, as evidenced by the marker sox10. (D–E) Coinjection of either CRgli2 or gli2act with gli2MO rescued NC migration as shown by foxd3 expression. (F–G) Injection of GANT61 produced a loss of gli2 function and a noticeable decrease in NC cell migration, as shown by the expression of foxd3. (H) Quantification of phenotypes shown in B–G. (I–N) Implantation of GANT61-soaked beads into NC at two different time frames (stages 14–22 and 17–22) produced a reduction in the front of migration of the NC as shown by sox10 expression. (O) Quantification of experiments shown in Figures I–N. (P–S) gli2 acts cell-autonomously in NC migration. Grafting of uninjected NC explant into an uninjected wild type embryo produced no alteration in the front of migration of the NC. The injection of gli2MO in the transplanted explant altered the normal pattern of NC migration. Grafting of a wild type NC explant into a gli2MO injected host produced no changes in normal NC migration.
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Fig. 9. gli2 is required for the proper formation of NC derivatives. (A, H, K) Schematic representation of the experiments performed. (B) Diagram indicating the different components of the craniofacial cartilages. (C–F, I–M) Craniofacial cartilages preparation of stage-45 tadpoles. Alcian blue staining, ventral views of embryos, anterior side is at the top. Injected side is indicated by arrowhead. (C) GANT61 injection at stage 3 produced a reduction in craniofacial cartilages, with a more prominent phenotype in the ceratobranchial cartilage. (D) The gli2MO-injected side of embryos (arrowhead) presents a marked reduction in Meckel's, ceratohyal and ceratobranchial cartilages. (E) Rescue of cartilage morphology after coinjection of CRgli2 along with gli2MO. (F) CoMO-injected embryos showed no effect on the treated side. (G) Quantification of phenotypes shown in C–F. (H–N) Grafting of GANT61-soaked beads at two different time frames (st. 12.5 to 45 and 17 to 45) produced a consistent reduction in craniofacial cartilages. (O–Q) Lateral view of a gli2MO injected embryo. On the injected side there is a reduction in the number of melanocytes in the zone of the future gut. Quantification is shown in Fig. 9Q. (R–T) tbx2 expression shows a reduction in RB neurons and cranial ganglia primordia on the gli2MO injected side. Pr, profoundal placode; L, lens placode; Ll, lateral line placode; Tp, trigeminal placode; RB, Rohon-Beard sensory neurons.
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Fig. 10. gli2 epistatic relationships with ihh, gli3, ednra and eg5. (A) Schematic representation of the experiments performed. (B–Q) Dorsal views of Xenopus laevis embryos at mid-neurula stage; anterior side is on the left. Arrowheads indicate the injected side, shown in turquoise. The images show combinatorial experiments performed to evaluate the epistatic relationship between gli2 and different genes involved in the gene regulatory network that induces the NC. They also show the effect of individual genes on the NC. (B–E) gli2 is downstream of ihh during NC induction. (F–I) gli2 and gli3 rescue each other's deficiency during NC induction. (J–Q) Both genes ednra and eg5 act upstream of the gli2 transcription factor during NC induction and specification. (R) Quantification of the phenotypes shown in B–Q. (S) Hierarchy diagram plotting the epistatic relationships between the genes analyzed.
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Figure S1. Graphic representation of gli2 constructs used in the functional studies. (A) pCS2+MT-gli2-HIS: construct encoding the full-length Xenopus gli2 construct. (B) ï¡3000_pCS2+MT-gli2: construct encoding gli2 full-length, which only is expressed in the NC. (C) pCS2+MT-CRgli2: construct encoding the full-length gli2 protein in which the morpholino binding sequence is altered so that gli2MO can no longer bind to the protein. (D) ï¡3000_pCS2+MT-CRgli2: CRgli2 construct, specifically designed to be expressed in the NC. (E) pCS2+MT-gli2act: Activator form of gli2, without the repressor region. (F) ï¡3000_pCS2+MT-gli2act: gli2act construct with expression only in the NC. (G) pCS2+MT-gli2rep: Repressor form of gli2, without the activator region. (H) pCS2+MT-gli2PSM-HIS: Construct with PKA-sites mutated. (I) ï¡3000_pCS2+MT-CRgli2PSM: gli2PSM expressed only in NCC. (J) pCS2+GFP-gli2: Short region of gli2 fused to the GFP protein. MT: myc tag; MBS: Morpholino binding site; MBS: Codon replacement in the Morpholino binding site; N-Rep: N-terminal repressor domain (1044 bp); ZF: Zinc fingers domain; PKA1-6: Conserved PKA (protein kinase A) phosphorylation sites 1 to 6; C-Act: C-terminal activator domain (3519bp).
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