Characterization of the identity and position ofTlx-1 expressing cells within the developing hindbrain.A, B, Colocalization ofTlx-1 mRNA and β-tubulin (A) or the 160 kDa (NF-M) neurofilament protein (B) in dissociated hindbrain cell cultures from stage 21–25 embryos. Thetop panels in A and B show phase-contrast photomicrographs of the bright-field images.Tlx-1-positive cell bodies are stainedblue. The same view under fluorescent illumination is shown in the bottom panels. Cells that express β-tubulin, an early neuronal marker, or the 160 kDa (NF-M) neurofilament protein, a marker of differentiated neurons, are stained red orgreen, respectively. Arrowheads mark examples of double-labeled cells. C, Confocal photomicrograph of a transverse section through a stage 27 hindbrain, showing that BrdU-labeled cells (green) in S-phase (fixed 30 min after BrdU injection) do not expressTlx-1 (blue). D,Parasagittal section through the hindbrain of an embryo injected with BrdU at the same stage as C but allowed to survive for 24 hr. Some Tlx-1-positive cells are also fluorescently labeled, indicating that they have become postmitotic within the last 24 hr. The ventricular surface is toward the top in bothC and D. E, Confocal photomicrograph of a flat-mounted stage 24 hindbrain showing the relative positions of Tlx-1-expressing cell bodies (monochrome dark label) and incoming sensory axons from the seventh/eighth ganglion anterogradely labeled with rhodamine–fluorescent dextran (red). Facial motor neurons whose cell bodies lie medial to the expression ofTlx-1 in r4 and r5 have also been retrogradely labeled.F, Confocal photomicrograph of a flat-mounted stage 27 hindbrain, viewed from the pial surface, showing the descending sensory tract of the fifth (trigeminal) ganglion labeled anterogradely with DiI. G, High-power view of F showing collateral axons projecting medially from the lateral longitudinal tract, which are unbranched and tipped with growth cones.H, Stage 19 hindbrain in which the descending tectobulbar and mesencephalic trigeminal neuron axons have been labeled by DiI injection into the caudal optic tectum. The tract widens and is displaced dorsally (laterally) as it crosses r1. Scale bars: A, B, 50 μm; all other panels, 100 μm.
