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FIGURE 1. 16p12.1-affected genes are expressed in migrating neural crest cells during embryonic development. (A) Lateral view of whole-mount in situ hybridization at stage 25 for twist, a NCC-enriched transcription factor. Anterior of embryo is to left. Arrows indicate the pharyngeal arches (PAs). (B–E) In situ hybridization at stage 25–28 for polr3e, mosmo, uqcrc2, and cdr2 demonstrate expression in NCCs that occupy the PAs (n = 10 per probe). Scalebar = 300 μm.
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FIGURE 2. Knockdown of Polr3e, Mosmo, and Uqcrc2 impact cartilage morphology. (A–E) Ventral view of stage 42 embryos following single 16p12.1-associated gene KD, stained with Alcian blue to label cartilage elements. Anterior of embryo is at the top. In the control, the red arrow points to ceratohyal cartilage, while the red bar spans the first branchial arch. (F–G) Measurements of the average ceratohyal area and width of the first branchial arch. The data was normalized to the control MO condition. Partial depletion of Polr3e, Mosmo, and Uqcrc2 significantly reduced the size of both of these cartilage elements compared to controls, while depletion of Cdr2 had no effect on cartilage size. Significance determined using a student’s unpaired t-test. (Embryos quantified: Control = 48, Polr3e KD = 32, Mosmo KD = 51, Uqcrc2 KD = 34, Cdr2 KD = 39). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 300 μm.
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FIGURE 3. Knockdown of Polr3e and Mosmo affect CNC migration in vivo. (A–C, A′–C) (A–E, A′–E′) Lateral views of stage 25 embryos following whole-mount in situ hybridization against twist. Each column of panels are lateral views of two sides of the same embryo. Embryos were unilaterally injected to KD each individual 16p12.1-affected gene in half of the embryo and the other half was left uninjected. The left panels (A–E) represent the uninjected side and the right panels (A′–E′) represent the injected side. (F–Q) Measurements were taken for the total area of the three PAs (posterior PA, anterior PA, and hyoid PA), the length of each individual arch, and the NCC migration distance, as measured from the dorsal-most tip of each arch to the neural tube, by taking ratios of the injected side versus the uninjected side. Significance was determined using an unpaired students t-test with these ratios. (F–H) Partial depletion of both Polr3e and Mosmo significantly reduced the total area of the three PAs, while partial depletion of Uqcrc2 slightly increased the total area of the three PAs. (J–K) Polr3e KD significantly reduced the length of the posterior PA but had no effect on the length of the anterior PA or hyoid PA. However, partial depletion of this gene significantly reduced the total NCC migration distances for all three PAs. (L–M) Mosmo KD significantly reduced the length of the anterior PA and hyoid PA but had no effect on the length of the posterior PA. Partial depletion of this gene also significantly reduced the total NCC migration distance for all three PAs. (N–O) Uqcrc2 KD slightly increased the length of the anterior and hyoid PA but had no effect on the length of the posterior PA, nor did it affect the total NCC migration distance. (I, P–Q) Cdr2 KD had no effect on the total area of the three PAs, nor did it affect the total NCC migration distance. However, partial depletion of this gene slightly increased the length of the hyoid PA but did not affect the posterior or anterior PAs. (Embryos quantified: Control for Polr3e = 35, Control for Mosmo = 48, Control for Uqcrc2 = 48, Control for Cdr2 = 45, Polr3e KD = 41, Mosmo KD = 75, Uqcrc2 KD = 68, Cdr2 KD = 45). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 300 μm.
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FIGURE 4. Manipulation of Polr3e, Uqcrc2, and Cdr2 impacts NCC migration speeds in vitro. Dissected NCC explants from control, Polr3e KD, Mosmo KD, Uqcrc2 KD, or Cdr2 KD embryos were plated on fibronectin-coated coverslips, allowed to adhere and begin migration, and imaged for 6 h using 20x phase microscopy. (A–E) Representative images of explants at initial timepoint (0 min). (A′–E′) Representative images of explants after 6 h migration (360 min). (F) Representative tracks generated by FiJi Trackmate plug-in overlaid on panel A’. (G–J) Mean track speeds of Polr3e KD, Mosmo KD, Uqcrc2 KD, and Cdr2 KD explants compared to their controls. Partial depletion of Polr3e and Uqcrc2 significantly reduced mean NCC speed, while depletion of Cdr2 increased mean NCC speed. Partial depletion of Mosmo had no effect on mean NCC speed. (Explants quantified: 6-7 explants from control and KD embryos were plated for each experiment. Three separate experiments were performed for each depletion.) ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 200 μm.
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FIGURE 5. Manipulation of 16p12.1-affected genes does not impact NCC proliferation in vitro. Dissected NCC explants from control, Polr3e KD, Mosmo KD, Uqcrc2 KD, and Cdr2 KD embryos were plated on fibronectin-coated coverslips, allowed to adhere and begin migration for 4 h before being fixed in 4% PFA and immunostained with PH3 antibody as a marker for NCC proliferation, phalloidin to label the actin cytoskeleton, and Hoechst to label nuclei. (A,A′) Representative image of control NCC explant immunostained with PH3, phalloidin, and Hoechst. White arrows denote cells positively labeled for PH3. (B–E) Quantification of the number of positively PH3-labeled cells versus the total number of cells per NCC explant for Polr3e KD, Mosmo KD, Uqcrc2 KD, and Cdr2 KD compared to controls. Partial depletion of each individual 16p12.1-affected did not have a significant impact on NCC proliferation in vitro. (Explants quantified: 6-7 explants from control and KD embryos were plated for each experiment. Three separate experiments were performed for each depletion). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 300 μm.
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FIGURE 6. Manipulation of Polr3e and Mosmo affects NCC specification In situ hybridization utilized (A,C,E,G,I) antisense mRNA probe against sox9 and (B,D,F,H,J) antisense mRNA probe against twist. Each column of panels are dorsal views of two sides of the same embryo. Embryos were unilaterally injected to KD each individual 16p12.1-affected gene in half of the embryo and the other half was left uninjected. The left side represents the uninjected side and the right side, indicated with an asterisk (*), represents the injected side. Anterior of embryo is at the top. (K–L) Measurements were taken for the total area of the expression pattern for either sox9 (K) or twist (L) using the polygon tool in ImageJ by taking ratios of the injected side versus the uninjected side. Significance was determined using an unpaired students t-test with these ratios. Partial depletion of either Mosmo or Polr3e significantly reduced the total area of expression for both sox9 and twist, while partial depletion of either Uqcrc2 or Cdr2 did not significantly affect the total area of expression for either NCC specification marker. (Embryos quantified with sox9 probe: Control for Polr3e = 60, Control for Mosmo = 66, Control for Uqcrc2 = 25, Control for Cdr2 = 36, Polr3e KD = 55, Mosmo KD = 69, Uqcrc2 KD = 27, Cdr2 KD = 59. Embryos quantified with twist probe: Control for Polr3e = 20, Control for Mosmo = 33, Control for Uqcrc2 = 23, Control for Cdr2 = 46, Polr3e KD = 25, Mosmo KD = 41, Uqcrc2 KD = 44, Cdr2 KD = 50). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 300 μm.
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FIGURE 7. Summary table of 16p12.1-affected gene craniofacial, cartilage, and NCC phenotypes. Partial depletion of 16p12.1-affected genes demonstrates numerous impacts on craniofacial, cartilage, and CNC development. Tissues are denoted as affected (checked box) if phenotypes were significantly different from control (p < 0.05); see individual figures for data distribution and statistics.
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Figure S1. Validation of 16p12.1 morpholinos in X. laevis. (A-D) Electrophoretic gels show altered RNA expression of 16p12.1 gene homologs due to splice-site morpholino (MO) knockdown with increasing concentrations in X. laevis embryos. RNA was extracted from 10 two-day old whole embryos for each RT-PCR reaction. At least two replicates (uninjected and multiple MO concentrations) were performed for each MO, and band intensities were compared with expression of ODC1 controls taken from the same cDNA samples and run on gels processed in parallel. Splice site MOs were validated and densitometry was performed to quantify the effectiveness of alternative splicing in our previous manuscript, Pizzo et al., 2021.
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Figure S2. Craniofacial defects caused by 16p12.1-associated gene KD are rescued by co- injection of exogenous mRNA co-expression. (A) Row 1: Representative frontal view images of st. 42 tadpoles injected with control MO or MOs for 16p12.1 homologs show defects in craniofacial features, including a decrease in facial width compared to the control. Row 2: Representative frontal view images of st. 42 tadpoles show facial width defects were rescued with co-injection and overexpression of mRNA for each 16p12.1 gene homolog, respectively. (B) Boxplot representing the quantification and comparison of facial width measurements with knockdown and overexpression of mRNA of individual 16p12.1 gene homologs normalized to controls. Significance determined using a student’s unpaired t-test. (Embryos quantified: Control = 65, Polr3e KD = 31, Polr3e KD+OE = 24, Mosmo KD = 33, Mosmo KD+OE = 42, Uqcrc2 KD = 44, Uqcrc2 KD+OE = 32, Cdr2 KD = 30). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. Scalebar = 500μm.
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Figure S3: Expression patterns for 16p12.1-affected genes across early development. In situ hybridization utilized (A-D) antisense mRNA probe to polr3e, (E-H) antisense mRNA probe to mosmo, (I-L) antisense mRNA probe to uqcrc2, and (M-P) antisense mRNA probe to cdr2. Anterior to the left. Lateral and dorsal view images of embryos shown at stage 20 (A-B, E-F, I- J, M-N), lateral view at stage 35 (C, G, K, O), and lateral view at stage 40 (D, H, L, P) (n = 10 per probe) Scalebar = 300μm.
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Figure S4: In situ hybridization probes generated against sense strands of 16p12.1-affected gene mRNAs. In situ hybridization utilized (A) sense mRNA probe against polr3e, (B) sense mRNA probe against mosmo, (C) sense mRNA probe against uqcrc2, and (D) sense mRNA probe against cdr2, shown at stage 25. (n = 10 per probe). Scalebar = 300μm.
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Figure S5: Representative image of stage 28 X. laevis embryo labeled for twist, a transcription factor that is critical for NCC specification and enriched in the pharyngeal arches. Measurements of the length (orange line) and area (red dotted line) of each individual pharyngeal arch can be done using ImageJ. cg, cement gland; pa, pharyngeal arch. Scale bar = 500μm.
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