Morpholinos for splice modificatio

Morpholinos for splice modification

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Supplementary Material

DEV076075 Supplementary Material

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  • Supplemental Figure S1 -

    Fig. S1. Characterization of the Tg(pax2a:GFP)e1 PPA domain. (A-A′′) Confocal projection of a Tg(pax2a:GFP)e1 embryo at 12 hpf immunostained for Pax2a. Significant overlap was seen between transgenic GFP and endogenous Pax2a; 97% of α-Pax2+ cells were GFP+ (n=380 cells from five embryos) and 87% of GFP+ cells were α-Pax2+ (n=420 cells from five embryos). (B-B′′) Confocal projection of a Tg(pax2a:GFP)e1 embryo at 24 hpf co-stained with the Pax2 antibody. Few cells in the EB placodes are labeled with the transgene at this stage. Ectopic expression of transgene is seen in the acoustic and anterior lateral line ganglion (gVIII/gAll) and rhombomeres 3 and 5 (R3 and R5). (C) Confocal projection of a Tg(pax2a:GFP)e1 transgenic embryo at 12 hpf. (D) Total number of cells of the PPA, in addition to number of cell diameters along the A-P (red marker) and M-L (yellow marker) axes, were counted in 11 embryos (1186 cells total from 11 embryos) to determine variability in dimensions of this domain. Counts reveal a consistent average (Ave) with a small standard error of mean (SEM) for both total number of cells and cells across the A-P and M-L axes in the PPA of 12 hpf embryos. ov, otic vesicle; gVIII/gAll, acoustic/anterior lateral line ganglia; A-P, anterior-posterior; M-L, medial-lateral. Scale bars: 50 µm.

  • Supplemental Figure S2 -

    Fig. S2. Fate of Pax2a-expressing cells in the otic vesicle. (A) Small groups of cells labeled within the PPA at 12 hpf. (B) At 24 hpf, the location of labeled cells within the otic vesicle was recorded using polar coordinates transposed over the otic vesicle. Cells were cataloged by proximity to one of 12 markings (30° radial sectors, corresponding to hour marks on an analog clock). (C) Radial charts of the proportion of cells that ended up closest to each of the sectors for regions 1-12 of the Pax2a domain (A). Each concentric line represents 10% of all labeling instances. (D) Simplified fate map of Pax2a cells within the otic vesicle. The Pax2a domain is divided into four columns from anterior to posterior (colored grid) and position of cells from each column in the otic vesicle is mapped to a radial chart. Similar to the individual maps for regions 1-12 of the Pax2a domain (A), cells from the anterior portion of the Pax2a domain are strongly biased towards the anterolateral region of the otic vesicle. Cells from more posterior regions are more equally distributed around the otic vesicle. (E) Fate map of the Pax2a domain along the mediolateral axis. The pax2a domain is divided into three rows from medial to lateral. The anterolateral bias obscures the pattern, but cells from the medial, intermediate or lateral regions of the Pax2a domain tend to remain in these positions as they are incorporated into the otic vesicle.

  • Supplemental Figure S3 -

    Fig. S3. Differential levels of pax2a/8 are observed in the PPA and mature placodes. (A-C) In situ hybridization of pax2a reveals differential pax2a expression levels at both 12 (B) and 24 (C) hpf. (D-F) In situ hybridization for pax8 reveals presence of this transcript in the PPA at 11 (D) and 12 (E) hpf; pax8 is also expressed in the EB placodes and in distinct foci in the otic vesicle at 24 hpf (F). Note the differential expression levels of pax8 at 12 and 24 hpf with low level pax8 in the EB placode. Arrowheads indicate low expression; arrows indicate high pax expression. Scale bars: 50 µm.

  • Supplemental Figure S4 -

    Fig. S4. Injection of hsp70-dTomato-pax2a plasmid induces robust Pax2a expression following heat-shock. (A-A′′) Embryos injected with a heat-shock-inducible construct expressing both Pax2a and the dTomato reporter were heat-shocked at 12hpf. These embryos display mosaic Pax2a expression 3 hours post-heat-shock. Arrows indicate cells with ectopic Pax2a expression. (B) Quantification of induced Pax2a levels relative to endogenous Pax2a. Note that on average ectopic Pax2a levels are 2.9-fold higher than the endogenous levels (n=42 cells from five embryos; Student’s t-test, ***P<<0.001). Error bars represent s.e.m. (C) Distribution of Pax2a fluorescence intensity comparing endogenous Pax2a with ectopically induced Pax2a. Note that overall intensity levels of the exogenous Pax2a fall within a high endogenous spectrum (n=198 cells from five embryos). Scale bar: 50 µm.

  • Supplemental Figure S5 -

    Fig. S5. Combined activity of Pax2a and Pax8 is necessary for formation of the EB ganglia. (A,B) Embryos derived from a pax2ab593/+ incross immunolabeled with anti-Elavl3/4 to mark differentiated EB neurons in control (A) and embryos injected with 1 ng of pax8-MO (B). Note near-complete loss of Elavl3/4+ neurons in the EB ganglia of the pax2a mutant that also received pax8-MO (B). (C-F) Confocal projection from 50 hpf Tg(phox2b:EGFP)w37 embryos. Control embryos (C,E) were compared with embryos injected with either 0.25 ng of pax8-MO (D) or 3.0 ng of pax2a-MO (F). (G,H) Quantification of phox2b+ neurons at 50 hpf in the facial, glossopharyngeal and most anterior small vagal ganglia in embryos injected with 0.25 ng of pax8-MO (G) and 3.0 ng pax2a-MO (H) versus controls (n≥34 cells from five embryos per condition). Note the lack of significant difference in ganglion sizes when MOs are singly injected at suboptimal concentrations. Error bars represent s.e.m. (I) RT-PCR of pax2a splice variant products from embryos that were injected with increasing amounts of pax2a MO. The amount of an RT-PCR product corresponding to an aberrant splice variant (arrow) increased with respect to injected pax2a-MO in a dose-dependent manner. Scale bars: 25 µm.

  • Supplemental Figure S6 -

    Fig. S6. Injection of suboptimal amounts of pax2a MO results in a reduction in Pax2a protein levels in cells of the PPA at 12 hpf. (A-B′) Control embryos and embryos injected with 3 ng of pax2a-MO were immunolabeled with anti-Pax2 antibody at 12 hpf. Suboptimal MO levels were defined by titration experiments. Note that 3 ng of pax2a MO results in an overall reduction, but not complete absence, of Pax2a protein in the PPA (A′ vs B′). (C) Distribution of mean gray values of Pax2a levels in control and pax2a-MO injected embryos (n≥393 cells from seven embryos per condition). Note the significant decrease (χ2-test, P<<0.001) in the number of cells with high Pax2a expression in embryos injected with pax2a-MO. Scale bar: 50 µm.

  • Supplemental Figure S7 -

    Fig. S7. Modulating Fgf signaling affects size of the otic and EB placodes. (A-C) Representative confocal projections of embryos immunolabeled with anti-Pax2 antibody at 24 hpf including: control (A), constitutively active Fgfr1 Tg(hsp70:ca-fgfr1)pd3 transgene (B) and dominant-negative Fgfr1 Tg(hsp70:dnfgfr1-EGFP)pd1 transgene (C). Heat-shock induction for both constructs was carried out at 10 hpf. Note an increase in the number of Pax2+ cells in the facial placode (arrow in B) and concurrent reduction in the G+V placodes and dysmorphia in the otic vesicle following Fgf upregulation. By contrast, inhibition of Fgf results in loss of the facial placode with a concomitant reduction in the otic vesicle and G+V placodes (C). (D) Quantification of Pax2+ cells in the otic vesicle and EB placodes in control embryos (no heat-shock) and Tg(hsp70:ca-fgfr1)pd3 embryos heat-shocked at various temperatures (36.9, 37.5 and 38°C). Note the 3.4-fold increase in the number of Pax2+ cells in the facial placode following heat-shock at 38°C, with concomitant decrease of the G+V placodes and otic vesicle (Student’s t-test, **P<0.0075). (E) Quantification of Pax2+ cells in the otic vesicle and EB placodes in control embryos (no heat-shock) and Tg(hsp70:dnfgfr1-EGFP)pd1 embryos following heat-shock at various temperatures (35, 36.3 and 37.2°C). Note the ∼95% reduction in the facial placode following 35°C heat-shock (Student’s t-test, **P<0.002), and complete loss of this placode at higher inductive temperatures. Whereas the G+V placodes are unaffected at lower inductive conditions, there is a 68% reduction (Student’s t-test **P<0.004) and a complete loss of this domain following heat-shock at 36.3°C and 37.2°C, respectively. By contrast, the number of Pax2a+ cells in the otic vesicle is only reduced following heat-shock at the 37.2°C (46% reduction; Student’s t-test **P<0.001). (F) Percentage of Pax2a+ cells undergoing mitosis was measured by immunolabeling with a pH3 antibody at 18 hpf. There is no significant change in proliferation following 10 hpf heat-shock of Tg(hsp70:ca-fgfr1)pd3 embryos, compared with uninduced controls (n=12 embryos per condition). (G) Percentage of Pax2a+ cells undergoing apoptosis as measured by immunolabeling with Caspase-3 antibody at 18 hpf. There is a significant increase of cell death in the otic placode following 10 hpf heat-shock of Tg(hsp70:ca-fgfr1)pd3 embryos (n=12 embryos per condition). (H-M) Pax2a expression at 12, 15 and 18 hpf in control embryos (H-J) or Tg(hsp70:ca-fgfr1)pd3 transgenic embryos following heat-shock induction at 10 hpf (K-M). Note additional Pax2a+ cells lateral to the PPA at 15 hpf (L, arrowheads); and expansion and fusion of Pax2a+ cells in the pronephros (pn) with the PPA. By 18 hpf a striking expansion of the facial placode can be observed (arrowheads), concurrent with a dysmorphic otic vesicle (filled arrow) expansion and mislocalization of the pronephros (M, open arrow). (N) Distribution of Pax2a fluorescence intensity in PPA cells at 12 hpf in control, Tg(hsp70:dnfgfr1-EGFP)pd1 and Tg(hsp70:ca-fgfr1)pd3 following 10 hpf heat- shock. Note that the overall distribution of Pax2a intensity levels is unchanged regardless of Fgf levels (n≥504 cells from eight embryos per condition). Scale bars: 50 µm.

  • Supplemental Figure S8 -

    Fig. S8. Number of Dlx3b+ cells in the otic vesicle is altered when Fgf and Wnt pathways are modulated. Confocal projections of 18 hpf embryos co-labeled with anti-Pax2a and anti-Dlx3b to assess expression in the developing otic vesicle. (A-A′′) Control embryos reveal that at 18 hpf Pax2a and Dlx3b exhibit extensive colocalization; however, a number of lateral otic cells that either express very low Pax2a levels or do not express Pax2a at all are Dlx3b+. (B-B′′) Embryos treated with 2.5 µM BIO starting at 11 hpf show a concomitant increase in both Pax2a and Dlx3b+ cells. (C-C′′) 10 hpf heat-shock of Tg(hsp70:ca-fgfr1)pd3 embryos caused an increase in number of Dlx3b+ cells but also yielded a high level of cell disorganization. (D-D′′) Pax2a expression in the otic cells is completely lost following inhibition of Wnt signaling by 10 hpf heat-shock of Tg(hsp70:tcfΔC-EGFP). Otic cells retained Dlx3b expression; however, the overall number of otic cells is reduced versus control. (E) Quantification of Dlx3b+ cells in the otic vesicle at 18 hpf following various treatments. This analysis revealed a 60% increase in Dlx3b+ cells in the otic vesicle after BIO treatment (Student’s t-test ***P<<0.001) a 49% increase in Dlx3b+ cells after Tg(hsp70:ca-fgfr1)pd3 induction (Student’s t-test **P<0.006), and a 25% reduction in the number of Dlx3b+ cells after induction of Tg(hsp70:tcfΔC-EGFP) (Student’s t-test ***P<<0.001; n=6 embryos per condition). (F) Quantification of Pax2a+ cells in the otic vesicle at 18 hpf following the above treatments. The analysis revealed a 51% increase in Pax2a+ cells in the otic vesicle after BIO treatment (Student’s t-test ***P<<0.001), no significant change in Pax2a+ cells in Tg(hsp70:ca-fgfr1)pd3 induced embryos, and a 61% reduction in Pax2a+ cells after Tg(hsp70:tcfΔC-EGFP) induction (Student’s t-test ***P<<0.001; n=6 embryos per condition). Error bars represent s.e.m. Scale bar: 50 µm.

  • Supplemental Figure S9 -

    Fig. S9. Overactivation of Wnt signaling severely reduces or blocks EB ganglia formation. Confocal projection images of the otic vesicle and EB ganglia at 50 hpf. Tg(phox2b:EGFP)w37 embryos were treated with 2.5 and 5.0 µM BIO beginning 11 hpf, then washed out at 24 hpf. Embryos were allowed to develop until 50 hpf, at which time they were assessed for EB ganglion formation by EGFP expression and for otic vesicle formation by DAPI labeling. (A-C) Whereas DMSO-treated control embryos had normal development of the otic vesicle and EB ganglia, approximately half of 2.5 µM BIO-treated embryos had severely reduced EB ganglia and the remaining half had no EBs. (D) BIO (5.0 µM)-treated embryos resulted in a complete loss of EBs. Scale bar: 25 µm.

  • Supplemental Figure S10 -

    Fig. S10. BIO regulates Pax2a levels and promotes otic cell commitment through the Wnt pathway. (A,B) Tg(hsp70:tcfΔC-EGFP) embryos in which transgene was not activated were treated at 11 hpf with either DMSO (A) or 2.5µM BIO (B). In embryos treated with BIO we saw supernumerary Pax2+ cells in the otic vesicle and increased Pax2a levels. (C,D) Tg(hsp70:tcfΔC-EGFP) embryos that were heat-shocked at 10 hpf, yielded the same phenotype in both DMSO- (C) and 2.5µM BIO- (D) treated embryos, indicating that BIO is modulating Pax2 levels and otic fate via canonical Wnt (n=5 embryos per condition). Scale bar: 50 µm.

  • Supplemental Figure S11 -

    Fig. S11. Global Wnt inhibition by Dkk1 expression attenuates Pax2a levels. (A-D) Control and Tg(hsp70:dkk1-GFP)w32 embryos were heat-shocked at 10 hpf and assayed for Pax2a expression at 12 and 24 hpf. Heat maps generated from control (A′) and transgenic embryos (B′) reveal reduced Pax2a levels in the PPA at 12 hpf following heat-shock. Note that upregulation of Dkk1 caused an increase in facial placode size with a concurrent reduction in the size of the otic vesicle. (E) Mean gray values of Pax2a levels in the otic vesicle at 24 hpf revealed a 2.6-fold reduction in the Dkk1-induced embryos (Student’s t-test, ***P<<0.001). (F) Control and Tg(hsp70:dkk1-GFP)w32 embryos were heat-shocked at 10 hpf, collected at 24 hpf and analyzed for Pax2a+ cell number in the otic vesicle and EB placodes. This analysis revealed a 48% increase in the number of cells that segregate to the facial placode (Student’s t-test, ***P<0.001), with a concomitant 47% reduction in the number of cells in the otic placode (Student’s t-test ***P<0.001). There was no significant difference in the number of cells in the glossopharyngeal/vagal placode in these experiments. Scale bar: 50 µm.

  • Supplemental Figure S12 -

    Fig. S12. Modulation of Wnt signaling at early somitogenesis does not affect expression of fgf3 and fgf8 in the hindbrain. fgf3 and fgf8 expression were assayed by in situ hybridization at 13 hpf after modulating Wnt signaling at 10 hpf. (A-H) Neither fgf3 nor fgf8 expression is grossly altered when Wnt signaling is blocked by induction of Tg(hsp70:tcfΔC-EGFP) transgene or overactivated by BIO treatment, compared with uninduced Tg(hsp70:tcfΔC-EGFP) embryos or DMSO-treated controls. Scale bar: 50 µm.

  • Movie 1 -

    Movie 1. Time-lapse of wild-type Tg(pax2a:GFP)e1 embryo during placode segregation and formation. Time-lapse of confocal projection images were acquired at 8-minute intervals over ∼7.3 hours. First frame begins at 11 hpf with the PPA circled in yellow. Otic assembly commences ∼17 hpf. Acoustic ganglion emanates from the forming otic vesicle during this time. Transgene is missing crucial regulator elements resulting in loss of expression in EB placodes and ectopic expression of GFP in rhombomeres 3 and 5. Note that the otic vesicle originates primarily from the posterior region of the PPE and the gVIII/gAll from the anterior region. Last frame highlights otic vesicle (OV), facial placode (F), glossopharyngeal/vagal placode (G+V), acoustic/All ganglia (gVIII/gAll) and rhombomeres 3 and 5 (R3, R5). Scale bar: 50 µm.

  • Movie 2 -

    Movie 2. Extensive cell movements are rare in PPA cells. Time-lapse of a Tg(pax2a:GFP)e1 transgenic embryo injected with pa-tagrfp mRNA. One-cell-stage embryos were microinjected with 250 pg of pa-tagrfp mRNA and a small number of cells in the anterior and middle regions of the PPA were photo-activated at 12 hpf. Confocal z-stacks were acquired at 10-minute intervals over ∼9.5 hours. Time-lapse movie, consisting of z-projections, begins at 12.5 hpf with the PPA (green cells) circled in yellow. Note two clusters of photo-activated cells (red). The majority of photo-activated cells do not undergo extensive cell movements; however, at t=290 min one of the labeled anterior cells (arrow) undergoes extensive posterior movement from the presumptive acoustic and anterior lateral line ganglia (gVIII/gAll) region towards the forming otic placode. Last frame highlights the gVIII/gAll ganglia and the otic vesicle (OV). Scale bar: 50 µm.

  • Movie 3 -

    Movie 3. Small number of PPA cells move laterally and contribute to the EB placodes. Time-lapse of a Tg(pax2a:GFP)e1 transgenic injected with 250 pg pa-tagrfp mRNA; a small subset of cells in the anterior and middle regions of the PPA were photo-activated at 12 hpf (red cells). Time-lapse of confocal z-stacks acquired at 10 minute intervals over ∼7.2 hours beginning at 13 hpf. PPA (green cells) is circled in yellow. The most labeled cells exhibit little movement and maintain positions close to initial locations. Note a lateral cell in the middle of the PPA which did not integrate into the otic vesicle, acquired increased motility and migrated laterally to the G+V placode (yellow arrow). The acoustic/All ganglia (gVIII/gAll), glossopharyngeal/vagal placode (G+V), rhombomere 3 (R3) and the otic vesicle (OV) are marked on the last time frame. Scale bar: 50 µm.

  • Movie 4 -

    Movie 4. Transient expression of dTomato:HSE:EGFP transgene generates mosaic distribution of dTomato+ cells in the PPA. Time-lapse of a Tg(pax2a:GFP)e1 transgenic was injected with 25 pg of dTomato:HSE:EGFP DNA construct at 1-cell stage. The transgene was induced by heat-shock at 10 hpf and confocal z-stacks were acquired at 10 minute intervals over ∼7 hours starting at 13 hpf. PPA (green cells) circled in yellow. Note a number of dTomato+ cells in the PPA (red). A small number of these cells undergo cell death during placode formation. Also note neural crest dTomato+ cells that migrate to the acoustic/All ganglia (gVIII/gAll) region (yellow arrows). Last frame highlights the acoustic/All ganglia (gVIII/gAll) and the otic vesicle (OV). Scale bar: 50 µm.

  • Movie 5 -

    Movie 5. High levels of Pax2a induce formation of placodal precursors from the non-neural ectoderm and their subsequent incorporation into the otic placode. One-cell-stage Tg(pax2a:GFP)e1 transgenic embryos were injected with 25 pg of dTomato:HSE:Pax2a DNA construct and heat-shocked at 10 hpf. Time-lapse of confocal z-stacks acquired at 10 minute intervals over ∼10.2 hours beginning at 12 hpf (PPA circled in yellow). Note mosaic expression of dTomato+ cells in red. Time-lapse reveals ectopic Pax2a-expressing cells appearing in the lateral non-neural ectoderm and migrating medially towards the forming otic placode (yellow and white arrows). Some of these cells are able to incorporate into the otic vesicle (white arrow), while a number of ectopic Pax2a cells that cannot incorporate undergo cell death (fragmented cells; green arrows). Last frame highlights the acoustic/All ganglia (gVIII/gAll), rhombomere 3 (R3) and the otic vesicle (OV). Scale bar: 50 µm.

  • Movie 6 -

    Movie 6. Time-lapse of a Tg(hsp70:ca-fgfr1)pd3 and Tg(pax2a:GFP)e1 transgenic cross during placode segregation and formation. Time-lapse of confocal projection images acquired at 8-minute intervals over ∼12 hours. Embryos were heat-shocked at 10 hpf to induce transgene expression. First frame begins at 12 hpf with the PPA circled in yellow. By 14 hpf, continuous ectopic induction of Pax2a is seen (yellow arrows) in the lateral non-neural ectoderm which continues until after otic vesicle assembly (∼18 hpf). Note axial extension of the otic vesicle with concurrent failure of invagination. Last frame highlights otic vesicle (OV), glossopharyngeal/vagal placode (G+V), potential acoustic/All ganglia (gVIII/gAll ?), and rhombomere 5 (R5). Scale bar: 50 µm.

  • Movie 7 -

    Movie 7. Anterior PPA cells contribute to the otic vesicle following over activation of Wnt signaling. Time-lapse of a Tg(pax2a:GFP)e1 transgenic embryo treated with 2.5 µM BIO continuously between 11 and 24 hpf (end of time lapse). Confocal z-stacks were acquired at 10-minute intervals over ∼11.7 hours starting at 12 hpf. PPA is circled in yellow. By t=180 minutes, anterior cells of the prospective gVIII/gAll (marked by yellow arrows) begin to move posteriorly toward the forming otic vesicle. Last frame highlights the reduced acoustic/All ganglia (gVIII/gAll) and the enlarged otic vesicle (OV). There is a level of autofluorescence owing to the presence of BIO inhibitor. Scale bar: 50 µm.