The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Here, we provide genetic evidence that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs.
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