Segmental and neuronal architecture of the hindbrain of Krox-20 mouse mutants

The vertebrate hindbrain is transiently segmented during its early development with the formation of reiterated bulges, the rhombomeres (r). The Krox-20 gene, which encodes a zinc finger transcription factor, has been shown previously to be implicated in the maintenance of r3 and r5 (Schneider-Maunoury, S., Topilko, P., Seitanidou, T., Levi, G., Cohen-Tannoudji, M., Pournin, S., Babinet, C. and Charnay, P. (1993) Cell 75, 1199-1214; Swiatek, P. J. and Gridley, T. (1993) Genes Dev. 7, 2071-2084. However, it was not clear from these analyses how extensive the deletion of r3 and r5 was and whether the overall segmentation and internal architecture of the hindbrain was affected. We have now reinvestigated these issues by analysis of rhombomere boundaries, using both morphological and molecular markers, and of the fate of specific motor neuron populations, using retrograde and anterograde carbocyanine dye tracing. We conclude that r3 and r5 and their derivatives are completely eliminated in Krox-20(-/-) embryos while overall hindbrain segmentation is maintained. In addition, we show that the disappearance of these territories has important consequences for even-numbered rhombomeres as well, in particular on axonal navigation: (i) a population of r6 motoneurons, presumably normally fated to join the glossopharyngeal nerve, has its axons misrouted toward the facial exit point in r4; (ii) the trigeminal motor axons are also misrouted, presumably because of the proximity of the trigeminal and facial exit points. They fasciculate with facial axons outside the neural tube and enter the second branchial arch instead of the first arch. This navigational error could explain the disappearance, at around 17.5 dpc, of the trigeminal motor nucleus in Krox-20(-/-) embryos by inadequate supply of essential, possibly arch-specific survival factors.