Fig. 1. Loss-of-function of hoxb1a alters the disposition of r4-derived branchiomotor neurons. The disposition of the BM neurons is revealed by expression of islet1, either in live islet-GFP transgenic embryos (A-D,G-J) or by islet1 in situ hybridization (E,F). Uninjected (wild-type) control embryos are shown in left-hand panels and embryos injected with MOb1a at 1 mg/ml in right-hand panels. The BM neurons of the following cranial nerves are indicated: III, occulomotor; IV, trochlear; V, trigeminal; VII, facial; IX, glossopharyngeal; X – vagal. Rhombomeres (r) are numbered. (A-H) Embryos are dorsal-side uppermost with anterior towards the left. (A-D) islet-GFP transgenic embryos at 24 hours of development. (A,B) Merged bright-field and fluorescent images. o, otic vesicle; n, notochord. (C,D) Fluorescent images alone. In wild-type embryos (A,C), the Vth (trigeminal) nerve cell bodies lie in r2 (Va cluster; see E-J) and r3 (Vp cluster; see E-J); the VIIth (facial) nerve cell bodies migrate posteriorly, close to the floorplate, from r4 and r5, to ultimately reach r6 and r7. In MOb1a-injected embryos (B,D), the VIIth nerve cell bodies do not migrate, and instead lie in laterally positioned clusters similar to Vth nerve cell bodies. In both control and injected embryos, axons can be seen exiting the hindbrain at the r4 level and projecting towards the second pharyngeal arch. (E,F) islet1 in situ hybridization at 30 hours reveals the same neuronal disposition. Several islet1 expression sites additional to those in the GFP line can also be seen. These include the laterally located cranial ganglia, as well as the r6 and r7 located glossopharyngeal (IXth) nerve cell bodies. In the absence of r6/7-located VIIth nerve neurons, the IXth nerve neurons are revealed after MOb1a injection (F). These neurons express islet1 mRNA but are not labeled by the islet-GFP transgene (compare F with D). (G,H) Merged confocal images of 40 hour embryos. In wild-type specimens (G), Vth and VIIth nerve neurons have now reached their final locations. In MOb1a-injected embryos (H), the r4-derived neurons remain at the r4 level (VII) and show a similar mediolateral localization to r2-derived Vth nerve neurons. (I,J) Confocal analysis of 48 hour larvae in lateral view; anterior towards the left. In wild-type larvae (I), the VIIth nerve neurons are localized significantly posterior to the Vth nerve neurons (red labels); Vth nerve neurons project axons out of r2 to innervate the first pharyngeal arch; VIIth nerve neurons project axons anteriorly to exit the hindbrain in r4 and innervate the second arch; the Xth (vagal) nerve neurons innervate arches 4 through 7 (axons indicated by red arrows). The red arrowhead indicates VIIIth nerve/octavolateralis efferent (OLe) axons projecting into the otic region. In MOb1a-injected larvae (J), the projections into the pharyngeal arches are indistinguishable from normal (red arrows). However, the r4-derived cell bodies (VII) continue to be localized in r4, immediately posterior to Vth nerve cell bodies, and the VIIIth/OLe axon tract is absent (red asterisk). The BM neuron axons followed the same general pathways into the arches in all specimens analyzed, but we observed occasional individual stray axons that were not fasciculated with the main bundles in both wild-type and injected embryos.