Fig. 6. In e12.5 Lmx1a/b DKO mutants, inner ear central projections from left and right ears interdigitate but do not fuse at the hindbrain dorsal midline.

Dorsal view of whole mount hindbrain (A-B’) or transverse sections (C-I) of e12.5 embryos. Selected afferents were labeled with dye (A-C, E-G) or anti-Neurofilament/anti-acetylated α-Tubulin immunohistochemistry (D, H, I). Cranial nerves are indicated by Roman numbers. (A, C) Selected ipsilateral afferents labeled with dye (cranial nerves VII/VIII are green, nerves dV/VI are red, and nerves IX/X/XII are lilac. In control embryos, beyond r1-derived cerebellum (CB) (A), vestibular inner ear (cranial nerve VIII) and trigeminal (dV) projections do not come close to the dorsal midline.

(B-G) Labeling of the trigeminal (lilac), inner ear (red) and facial/inner ear projection from the opposite side (green) in a Lmx1a/b DKO embryo. B’ shows higher magnification of B. Panels F and G show consecutive sections from the same embryo, Panel E shows a different embryo. All these nerves, after entering the brainstem, aberrantly extend across the roof plate (RP) that occupies the brainstem dorsal midline. Afferents from the two ears clearly interdigitate at the roof plate but do not fuse (B, B’ F, G). In the double mutant, afferents of the cranial nerve VII-derived solitary tract and trigeminal afferents cross the roof plate below the vestibular inner ear nerve VIII (F, G). In contrast to pathfinding errors of afferents that normally target the alar plate, facial branchial motor neurons (FBM) and inner ear efferents (IEE) were appropriately located in the basal plate of e12.5 Lmx1a/b DKO embryos (E).

(D, H, I) In Lmx1a/b DKO mutants, fiber bundles crossing the roof plate can also be visualized using anti-Neurofilament (red)/anti-acetylated α-Tubulin (green) immunohistochemistry.

Hoechst counterstaining is blue (H, I). Panels H and I show sections from two different embryos. No comparably located fibers were detected in control embryos (D). Note that because the IV^th ventricle does not properly form in Lmx1a/b DKO embryos, the double mutant overall brainstem morphology is somewhat similar to the spinal cord (E-I).

Scale bars: 1 mm (A), 100 μm (all other panels).