Tg(isl1:GFP)^rw0 reporter expression reveals a facial motor neuron migration defect in olig2 MO-injected embryos as compared to wild-type and control MO-injected embryos. Panels are z-stack images showing dorsal views of whole embryos with anterior to the left. A–C: Wild-type embryos. D–F:olig2 MO-injected embryos. G–I: Control MO-injected embryos. Arrows indicate posteriorly migrated facial motor neurons. Bar indicates facial motor neurons along their caudal migration. Cranial motor neurons are labeled “n.” Otic vesicles are outlined. Scale bar = 30 μm.

Facial motor neurons fail to completely exit r4 and r5 to form clusters in r6 and r7. Panels show a single z-plane image of dorsally mounted whole embryos with anterior to the left. Tg(isl1:GFP)^rw0 reporter expression, amplified using an anti-GFP antibody and an Alexa 488 secondary antibody, is revealed as green and RNA expression patterns are blue. A,B:hoxb1a expression marks r4. C–F:egr2b expression labels r3 and r5. G–L:hoxb4a expression begins at the r6/r7 boundary and extends posteriorly. Arrows indicate region where facial motor neurons normally migrate. Approximate location of the otic vesicles is outlined with red ovals. Scale bar = 32 μm.

olig2 MO does not disrupt lateral line migration. All panels show dorsal views of whole embryos, anterior to the left. Elavl labeling is shown in red and Tg(isl1:GFP)^rw0 reporter expression is green. A–C: 28-hpf embryos. D–F: 33-hpf embryos. G–I: 50-hpf embryos. A–F show projections of z-stack images in both red and green channels. The red channel in G–I is a single z-plane, focused on the lateral line, whereas the green channel is a z-stack image projection of the green channel. Circles mark the posterior lateral line primordia. Arrows indicate lagging facial motor neurons in olig2 MO-injected embryos. Scale bar = 30 μm.

Normal facial motor neuron migration revealed by time-lapse imaging. Panels show frames captured from a time-lapse imaging sequence obtained from a control Tg(isl1:GFP)^rw0 embryo. Z-stack projection images are shown from dorsal orientation with anterior to the left. Developmental time is indicated in the upper right-hand corner of each panel. Cranial motor neurons are labeled “n.” Arrows indicate axons of facial motor neurons (nVII). Arrowheads indicate cluster of nVII neurons in r7.

Facial motor neuron migration revealed by time-lapse imaging. Panels show frames captured from a time-lapse imaging sequence obtained from an olig2 MO-injected Tg(isl1:GFP)^rw0 embryo. Z-stack projection images are shown from dorsal orientation with anterior to the left. Developmental time is indicated in the upper right-hand corner of each panel. Cranial motor neurons are labeled “n.” Arrows indicate axons of facial motor neurons (nVII). Asterisks indicate a small number of cells migrating caudally.

olig2 and sdf1a expression in wild-type, mafba^-/- and olig2 MO-injected embryos. All images show dorsal views with anterior to the left. A,B:olig2 expression in wild-type (A) and mafba^-/- (B) embryos at 24 hpf. Arrow in B indicates loss of r5/r6 olig2 expression in mafba^-/- in mutant embryos. Otic vesicles are outlined in red. C,D:sdf1a (blue) and egr2b (red) expression in wild-type (C) and mafba^-/- (D) embryos at 18 hpf. Absence of r5 egr2b expression identifies mafba mutants. E,F:sdf1a expression in wild-type (E) and mafba^-/- (F) embryos at 24 hpf. Mutant embryos lack r6 expression (arrow). G–J:sdf1a expression in control (G,I) and olig2 (H,J) MO-injected embryos (H, J).

Distribution of branchiomotor neurons marked by Tg(isl1:gfp)rw0 reporter expression at 3 dpf. Dorsa| whole mount with anterior to the left. A: Wild-type embryo. B: olig2 MO-injected embryo. Cranial motor neurons labeled “n.”

Expression of isl RNA and protein in wild-type, control MO-injected, and olig2 MO-injected embryos. Dorsal whole mount with anterior to the left. A–C: isl1 RNA expression in 24-hpf embryos. D–F: Isl protein expression detected by immunocytochemistry in 24-hpf embryos. G–I: isl1 RNA expression in 33-hpf embryos. J–L: Isl protein expression detected by immunocytochemistry in 33-hpf embryos. M–O: isl1 RNA expression in 50-hpf embryos. P–R: Isl protein expression detected by immunocytochemistry in 50-hpf embryos. Otic vesicles are outlined.

Tg(isl1:gfp)rw0 reporter expression reveals that the facial motor neuron migration defect in olig2 MO-injected embryos is not affected by co-injection of p53 MO. Panels are images showing dorsal views of whole embryos with anterior to the left. A–C: Wild-type embryos. D–F: olig2 MO-injected embryos. G–I: olig2 MO-injected embryos co-injected with p53 MO. Arrows indicate posteriorly migrated facial motor neurons. Bar indicates facial motor neurons along their caudal migration. Cranial motor neurons are labeled “n.”

olig2 is not expressed in r4 and olig2 loss of function has no effect on hoxb1a or prickle1b expression. A: Wild-type expression at 22 hpf of olig2 in r5 and r6 in blue and egr2b in r3 and r5 in red. B: qPCR of hoxb1a expression in wild-type, control MO-injected, and olig2 MO-injected embryos at 24 and 33 hpf. C: qPCR of prickel1b expression in wild type, control MO-injected, and olig2 MO-injected embryos at 24 hpf. Error bars represent standard deviation. P value calculated by Student′s t-test is listed above each bar and compared to wild-type embryos.

Glossopharyngeal motor neuron formation does not require olig2 function. Tg(olig2:EGFP) embryos labeled with anti-Alcama antibody (red) in wild-type and olig2 MO-injected embryos. Lateral sections, dorsal to the top, anterior to the left. A,B: Wild-type embryos. C,D: olig2 MO-injected embryos. Glossopharyngeal motor neurons indicated by arrows. Rhombomeres labeled “r.”