The transcription factor genes Hoxa1 and Krox-20 have been shown to play im
portant roles in vertebrate hindbrain segmentation. In this report, we pres
ent evidence for novel functions of these genes which co-operate in specify
ing cellular identity in rhombomere (r) 3. Although Hoxal has not been obse
rved to be expressed rostrally to the prospective r3/r4 boundary, its inact
ivation results in (i) the appearance of patches of cells presenting an r2-
1ike molecular identity within r3, (ii) early neuronal differentiation in r
3, normally characteristic of even-numbered rhombomeres, and (iii) abnormal
navigation of r3 motor axons, similar to that observed in even-numbered rh
ombomeres. These phenotypic manifestations become more severe in the contex
t of the additional inactivation of one allele of the Krox-20 gene, demonst
rating that Hoxal and Krox-20 synergize in a dosage-dependent manner to spe
cify r3 identity and odd- versus even-numbered rhombomere characters. In ad
dition, these data suggest that the control of the development of r3 may no
t be autonomous but dependent on interactions with Hoxa1-expressing cells.