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. 2017 Dec 4;6:e28415. doi: 10.7554/eLife.28415

Figure 6. Expression and functional perturbations of selected genes enriched in Trailblazers.

(A–M) Typical cranial neural crest cell migratory streams at HHSt13 adjacent to rhombomere 4 (r4) with HNK-1-positive cells color coded by the number of PKP2 (Plakophilin 2; A), Desmin (B), KAZALD1 (Kazal Type Serine Peptidase Inhibitor Domain1; C), Tescalcin (D), Troponin I1 (E), Annexin1 (F), Glypican 3 (G), BDNF (Brain Derived Neurotrophic Factor; H), Nexilin (I), BAMBI (BMP and Activin Membrane Bound Inhibitor; J), Desmoplakin (K), TFPI2 (Tissue Factor Pathway Inhibitor 2; L), and CDH5 (Cadherin 5; M). RNAscope spots detected per cell. (A’–M’) Single cells outlined with expression were selected from each neural crest cell migratory stream and (A”–M”) the number of RNAscope spots per cell volume for a cell with high expression and adjacent neighboring cells is shown in the bar graphs. (N) Schematic representation of morpholino and electroporation procedure. (O) Morpholino transfected migrating cranial neural crest migration at HHST15 (n = 16 control MO embryos, n = 20 EDN1 MO embryos, n = 20 PKP2 MO embryos, n = 20 MMP2 MO embryos, n = 7 GATA5 MO embryos, n = 5 WNT5B MO embryos, n = 10 GPC3 MO embryos, n = 7 ANXA1 MO embryos, n = 22 LUM MO embryos). (P) Correlation plot of distance migrated and expression in trailblazers. Circle size correlates with statistical significance: PKP2 MO, p=0.017, EDN1 MO, p=0.0004, MMP2 MO, p=0.0032, GATA5 MO, p=0.022, WNT5B MO, p=0.0001 (Q) Box plot of the distance migrated of morpholino transfected neural crest cells as a percentage of the distance from the neural tube to the tip of the branchial arch. HH, Hamburger and Hamilton (1951); St, stage; OV, otic vesicle; MO, morpholino. Bar = 20 um (A–G). Bar = 15 um (H–M). Bar = 50 um (O).

Figure 6—source data 1. Primer sequences used to test activity of splice blocking morpholinos.
The three morpholinos tested as well as the gene Ensemble IDs and primer sequences used to test morpholino activity are shown. Primers were also designed and used for sequence upstream of the region targeted by morpholino for controls.
elife-28415-fig6-data1.xlsx (369.3KB, xlsx)
DOI: 10.7554/eLife.28415.020

Figure 6.

Figure 6—figure supplement 1. Trailblazer genes chosen for perturbation experiments.

Figure 6—figure supplement 1.

Genes were chosen for perturbation experiments based on percentage of trailblazers expression, level of expression, significance and relevance to neural crest cell migration.
Figure 6—figure supplement 2. Testing of splice blocking morpholinos.

Figure 6—figure supplement 2.

LMH chicken cell line was transfected with control or experimental morpholinos. RT-PCR primers flanking the region affected by each morpholino were then used to demonstrate perturbation of translation. (A) Transfection with PKP2 MO lead to an excision of exon 3 as determined using primers designed within exons 2 and 4 (*). (B) Transfection with EDN1 MO lead to an excision of some of the gene as well as larger fragments, probably due to inclusion of intron 3 and/or frameshift effects. (C) Transfection with ANXA1 MO lead to an excision of exon 4 as determined using primers designed within exons 3 and 5 (*).
Figure 6—figure supplement 3. Analysis of stream neural crest migration after morpholino transfections in a subset of neural crest.

Figure 6—figure supplement 3.

(A) Example images on which analysis was performed showing morpholino (green) and HNK-1 (purple). (B) Box plot of the distance migrated of HNK1 positive neural crest cells as a percentage of the distance from the neural tube to the tip of the branchial arch after morpholino transfection. HNK1 distance measurements were made on the same embryos as analyzed in Figure 6Q. n = 16 control MO embryos, n = 20 EDN1 MO embryos, n = 20 PKP2 MO embryos, n = 20 MMP2 MO embryos. MMP2 MO, p=0.028, GATA5 MO, p=0.02, WNT5B MO, p=0.05, LUM MO, p=0.02.