Fig. 2

Validation of adam19b as critical for epibranchial sensory axon pathfinding. (A) adam19b start morpholino (MO1; 4 ng) injected into Tg(p2rx3.2:gfp) embryos. This image shows the disruption of the projections of sVII and sIX (dotted arrows) and the malformed plexus (white arrow), recapitulating the phenotype of sl19. sV appears unaffected. A 5 bp-mismatched MO gave no phenotype (for image of control fish, see Figure 1A). (B) Phenotype from CRISPR targeting of adam19b exon 2. White arrow shows malformed plexus and dotted arrows show disrupted axon projections of the sVII and sIX, again phenocopying sl19 (for image of control fish, see Figure 1A). Injection of gRNAs targeting other genes (e.g., drg11, golden) did not produce these phenotypes). sVII, sIX and sX are the projections of the facial (gVII), glossopharyngeal (gIX) and vagus (gX) ganglia, respectively. (C) Confocal image of 4 dpf Tg(3.2:nsfB-mcherry/cfos:gfp.sill) wild-type larvae, showing the anterior lateral line ganglia (allg) and the posterior lateral line ganglia [pllg; green fluorescent protein (gfp) channel: cfos:gfp.sill), which project to the lateral line plexus (white arrowhead) in the hindbrain. (D) Confocal image of 4 dpf Tg(3.2:nsfB-mcherry/cfos:gfp.sill] adam19b CRISPR/Cas9 mutants demonstrating the lateral line projections to the hindbrain are not affected by the adam19b CRISPR indels (gfp channel: cfos:gfp.sill) *neuromasts. (E) Confocal image of banchiomotor nerves in 4 dpf wild-type Tg(3.2:gfp/hsp70:nsfB-mcherry.crest) larvae, showing the segmental organization of the motor neurons (mV, mVII and mX; red channel: hsp70:nsfB-mcherry.crest). (F) Confocal image of branchiomotor nerves in Tg(3.2:gfp/hsp70:nsfB-mcherry.crest). adam19b CRISPR/Cas9 mutants show that the segmental organization of the motor nuclei (mV, mVII and mX) and their projections appear normal (red channel: hsp70:nsfB-mcherry.crest). (G,H) The hindbrain organization is not affected in sl19 mutants. Anti-acetylated tubulin immunostaining of 4 dpf wild-type (G) and sl19 mutant larvae (H) shows no disruption to the cerebellum (Cb), the optic tectum (ot) or the commissural neurons (cn) in the mutants. For panels (C–F), cognate images in the channel showing epibranchial afferents are shown in Supplementary Figures S5, S6. All images are oriented anterior to the left and dorsal at top. Scale bars = 100 μm.