Summary Drosophila homeodomain protein Nkx 6 coordinates motoneuron subtype identity and axonogenesis

The development of neuromuscular circuits depends critically on the specification of distinct motoneuron (MN) subtypes during development. Conserved transcriptional regulators help establish MN subtype identity (see Jurata et al., 2000; Thor and Thomas, 2002). The expression of unique combinations of transcription factors in distinct MN subtypes probably regulates the differential expression of cell-surface receptors that translate guidance cues to downstream effectors of cytoskeletal changes (e.g. Kania and Jessell, 2003). Such cytoskeletal rearrangements enable motor axons of different MN subtypes to make strikingly distinct guidance choices in a common environment of guidance cues. However, the manner in which distinct transcription factor profiles are translated into unique patterns of motor axon projections remains an outstanding question. In many model systems, MNs that extend axons along common trajectories express similar sets of transcriptional regulators – which in turn regulate key aspects of the differentiation of these MN subtypes (for a review, see Thor and Thomas, 2002). Drosophila MNs are classified by the location of the body wall muscles they innervate. MNs that innervate dorsal body wall muscles in Drosophilaexpress the homeodomain (HD) transcription factor Even-skipped (Eve) (Doe et al., 1988). Furthermore, genetic analyses indicate that Eve is a key determinant of the fate of dorsally projecting MNs (Landgraf et al., 1999). Eve engages in a cross-repressive interaction with the HD protein Hb9, a determinant of ventrally projecting MNs (Broihier and Skeath, 2002). Ventrally projecting MNs also express the HD transcription factors Lim3 and Islet. Functional analyses have demonstrated that these three HD factors are required for proper axon guidance of ventrally projecting MNs (Broihier and Skeath, 2002; Odden et al., 2002; Thor and Thomas, 1997; Thor et al., 1999). The genetic hierarchy governing the fate of ventrally projecting neurons has, however, remained elusive as Lim3, Islet, and Hb9 are expressed independently of each other. Lim3, Islet, and Hb9 are conserved regulators of MN cell fate whose vertebrate homologs – Lhx3/4, Islet 1/2, and Hb9 – play key roles in vertebrate MN specification (Arber et al., 1999; Sharma et al., 1998; Thaler et al., 1999; Tsuchida et al., 1994). In vertebrates, the genetic hierarchy linking the three transcription factors appears more linear than in Drosophila, as Hb9 regulates Lhx3/4 and Isl1/2 expression (Arber et al., 1999; Thaler et al., 1999). As in Drosophila, the vertebrate Eve homolog, Evx1, is expressed in a distinct population of neurons – in this case, a subset of vertebrate interneurons (MoranRivard et al., 2001). In Drosophilaand vertebrates, Hb9, Islet1/2, and Lhx3/4 are expressed almost exclusively by postmitotic neurons. In vertebrates, the expression of these factors in MNs depends on proper establishment of the MN progenitor domain by the coordinated action of upstream HD transcription factors (Briscoe et al., 2000). For example, the pair of Nkx-class HD proteins, Nkx6.1 and Nkx6.2 (Nkx6 proteins), have complementary expression patterns in MN and interneuron progenitors (Briscoe et al., 2000; Cai et al., 1999). Nkx6.1/Nkx6.2 compound mutants exhibit a near complete loss of somatic MNs, demonstrating that Nkx6 proteins are The regulatory networks acting in individual neurons to control their stereotyped differentiation, connectivity, and function are not well understood. Here, we demonstrate that homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9 to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6acts in parallel with hb9 to regulate motoneuron fate, we find that Nkx6plays a distinct role to promote axonogenesis, as axon growth of Nkx6positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity.