The 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/ExEx to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, Nkx6 plays a distinct role to promote axonogenesis; axon growth of Nkx6-positive 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 (Broihier, 2004).

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. 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. 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 (Broihier, 2004).

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. Drosophila MNs are classified by the location of the body wall muscles they innervate. MNs that innervate dorsal body wall muscles in Drosophila express the homeodomain (HD) transcription factor Even-skipped (Eve). Furthermore, genetic analyses indicate that Eve is a key determinant of the fate of dorsally projecting MNs (Landgraf, 1999). Eve engages in a cross-repressive interaction with the HD protein Hb9, a determinant of ventrally projecting MNs (Broihier, 2002; Broihier, 2004 and references therein).

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, 2002; Odden, 2002; Thor, 1997; Thor, 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 (Broihier, 2004 and references therein).

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. In vertebrates, the genetic hierarchy linking the three transcription factors appears more linear than in Drosophila, since Hb9 regulates Lhx3/4 and Isl1/2 expression. As in Drosophila, the vertebrate Eve homolog, Evx1, is expressed in a distinct population of neurons -- in this case, a subset of vertebrate interneurons (Broihier, 2004 and references therein).

In Drosophila and 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. 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, 2000; Cai, 1999). Nkx6.1/Nkx6.2 compound mutants exhibit a near complete loss of somatic MNs, demonstrating that Nkx6 proteins are essential for MN generation (Sander, 2000a; Vallstedt, 2001). Expression of Nkx6 proteins persists in postmitotic MNs, where they regulate proper nuclear migration and axon guidance in visceral MNs in the hindbrain (Müller, 2003; Pattyn, 2003; Broihier, 2004 and references therein).

To explore further the genetic networks behind neuronal diversification in Drosophila, the role of the Drosophila Nkx6 homolog in regulating distinct MN fates was investigated. Genetic interactions were characterized between Nkx6 and factors essential for neuronal fate acquisition. Evidence that Nkx6 collaborates with hb9 (exex – FlyBase) to regulate the fate of distinct neuronal populations. This analysis of hb9 Nkx6 double mutant embryos indicates that ventrally projecting MNs fail to develop properly in these embryos, while expression of eve, a key determinant of dorsally projecting MN identity, expands. In addition, Nkx6 promotes axonogenesis of Nkx6-positive neurons. Consistent with a direct regulatory role in this process, Nkx6 activates the expression of the neural adhesion molecule Fasciclin III in ventrally projecting motoneurons. These data suggest that Nkx6 is a primary transcriptional regulator of molecules essential for axon growth and guidance in a specific neuronal population (Broihier, 2004).

The findings that Nkx6 has roles in both the specification and differentiation of ventrally projecting MNs places Nkx6 in the regulatory circuit that specifies distinct postmitotic neuron fates in the Drosophila CNS. In the mouse, Nkx6 protein function in MN progenitors regulates Hb9 expression in postmitotic MNs (Arber, 1999; Sander, 2000a; Thaler, 1999; Vallstedt, 2001). Drosophila Nkx6 is expressed in neural precursors and postmitotic neurons while Hb9 expression is nearly exclusive to postmitotic neurons (Broihier, 2002; Odden, 2002). However, in contrast to the linear relationship of Nkx6.1/2 and Hb9 in vertebrates, Nkx6 and hb9 were found to act in parallel to specify neuronal fate in Drosophila. Nkx6 and hb9 act in concert both to repress expression of the dorsal MN determinant Eve and to promote expression of Lim3 and Islet in ventrally projecting RP MNs. It will be of interest to extend this genetic analysis to other groups of ventrally projecting MNs. It will be also be important to examine the directness of these genetic interactions. Both Nkx6 and hb9 contain conserved TN domains that in vertebrate HD proteins have been shown to interact with the Groucho co-repressor, suggesting that Nkx6 and hb9 function as transcriptional repressors (Broihier, 2002; Uhler, 2002). This raises the possibility that Nkx6 and hb9 bind to sequences in the eve enhancer and directly repress its transcription. In addition, Nkx6 and hb9 activate lim3/islet gene expression within ventrally projecting MNs, raising the possibility that they do so by repressing an unidentified repressor of ventrally projecting MN identity (Broihier, 2004).

eve represents an appealing candidate for the unidentified repressor in this model. Ectopic Eve expression in RP MNs in hb9 Nkx6 double mutants may repress Lim3 and Islet. Consistent with this, though it was not possible to unambiguously identify the ectopic Eve neurons in hb9 Nkx6 mutants, many of them are situated close to the midline, suggesting they may represent mis-specified RP MNs. Furthermore, pan-neuronal eve expression represses Lim3 and Islet expression in the RP MNs demonstrating that Eve can repress Lim3 and Islet (Landgraf, 1999). A direct test of this model will require resolving the identity of the ectopic Eve neurons in hb9 Nkx6 mutant embryos (Broihier, 2004).

While Nkx6 and hb9 play conserved roles in MN specification in Drosophila and in vertebrates, the genetic network within which they act differs. In vertebrates, Nkx6 is upstream of dHb9, while in Drosophila, Nkx6 and hb9 display a parallel requirement in MN generation. Why might the genetic relationship between Nkx6 and hb9 vary between Drosophila and vertebrates? It is proposed that the reason may relate to the different relationship between regional identity and neuronal subtype identity in Drosophila relative to vertebrates. In vertebrates, a given neuronal population is generated at a distinct dorsoventral position in the spinal cord in response to graded Sonic hedgehog levels. Thus, gene expression in neural precursors can simultaneously promote both precursor and neuronal subtype identity. In Drosophila, no obvious link ties regional identity to neuronal subtype. For example, Drosophila NBs arise within three dorsoventral columns. However, the dorsoventral position of neural precursors does not regulate postmitotic neuronal identity. NBs at all dorsoventral positions give rise to diverse populations of neurons, and neurons of given subtypes develop at many dorsoventral positions. For example, MNs are generated from NBs across the dorsoventral axis. Therefore in Drosophila embryos, gene expression in NBs does not directly promote neuronal subtype identity (Broihier, 2004).

A possible mechanism that might contribute to neuronal subtype identity in Drosophila is suggested by the temporal gene cascade in NBs (Kambadur, 1998; Brody, 2000; Isshiki, 2001). In this cascade, Hunchback (Hb) is expressed in the earliest-born one or two GMCs in a NB lineage followed by sequential expression of Kruppel, Pdm, and Castor in later-born GMCs. The majority of MNs arise from early-born GMCs, consistent with the idea that hb promotes MN identity. Thus while regional identity promotes postmitotic identity in vertebrates, temporal identity may play a similar role in Drosophila. However, many early-born GMCs do not produce MNs, indicating additional layers of complexity. Regional identity may interface with the temporal gene cascade to activate the proper combination of transcription factors to promote the MN fate in a subset of early-born GMCs. In this paradigm, MN specification occurs relatively late in development, suggesting that cells may need to rapidly activate and execute the genetic pathways leading to MN identity. As a result, near simultaneous activation of factors -- such a Nkx6 and hb9 -- that act in parallel to promote MN identity, might be required (Broihier, 2004).

While Nkx6 and hb9 exhibit parallel requirements in cell fate specification, Nkx6 plays a specific non-redundant role to promote axon growth and guidance in Nkx6-positive neurons. Nkx6 is, therefore, probably an element of the transcriptional code regulating the differential transcription of receptor and signal transduction molecules required to promote unique patterns of axon growth and guidance in distinct MN subsets. In support of this, Nkx6 activity is necessary for Fas3 expression in ventrally projecting RP MNs. Clearly, it will be necessary to elucidate the entire cassette of genes that Nkx6 activates to promote axonogenesis. In this regard, determining whether Nkx6 activates the same gene battery in all Nkx6-positive neurons or if Nkx6 regulation of such genes is cell-type-specific will be of interest. The singular requirement for Nkx6 in axon growth combined with the redundant functions of Nkx6 and hb9 in neuronal specification hints at the transcriptional complexity of neuronal specification and differentiation. It will be important to further distinguish the specification and differentiation functions of Nkx6, either by identifying additional interacting proteins, or by identifying protein domains within Nkx6 required specifically to promote either specification or differentiation. Precedence for the latter comes from the elucidation of distinct domains within the bHLH proteins Mash1 and Math1 required to promote neuronal differentiation and specification (Broihier, 2004).

Nkx6 is one of a number of transcription factors that have been implicated in controlling fundamental aspects of neuronal morphology. In Drosophila, several transcription factors have recently been shown to regulate dendritic morphogenesis. For example, different levels of the homeodomain protein Cut have been shown to regulate distinct dendritic branching patterns of peripheral nervous system (PNS) neurons, with higher Cut levels directing the development of more complex dendritic arbors. Interestingly, it was found that Nkx6 protein levels vary dramatically and reproducibly between CNS neurons. This raises the possibility that Nkx6 directs distinct patterns of axon outgrowth as a function of expression level, potentially adding another layer of complexity to Nkx6-transcriptional output (Broihier, 2004 and references therein),

This study also indicates that Nkx6 proteins have evolutionarily conserved functions in neuronal fate specification. In Drosophila, the role of Nkx6 in neuronal specification is uncovered in hb9 Nkx6 double mutants. The phenotype observed in Nkx6 mutants is a dramatic block to axon growth of Nkx6-positive neurons. Is it possible that this activity of Nkx6 in postmitotic neurons is conserved? Similar to Nkx6, vertebrate Nkx6.1 is expressed in postmitotic neurons, consistent with a functional role in neuronal differentiation. Interestingly, Müller shows that hindbrain visceral MNs in Nkx6.1 mutant mice display aberrant axon guidance and ectopically express the Ret and Unc5h3 receptors (Müller, 2003). Hence, Nkx6-class proteins appear to play conserved roles in regulating axon growth and guidance. It will be critical to determine whether the downstream factors they regulate are conserved as well (Broihier, 2004).

GENE STRUCTURE

cDNA clone length - 3041

Bases in 5' UTR - 855

Exons - 4

Bases in 3' UTR - 644

PROTEIN STRUCTURE

Amino Acids - 513

Structural Domains

Two overlapping Nk6 cDNAs were isolated from a Drosophila embryonic cDNA library whose combined insert length was 3041 bp. Multiple genomic clones, which map to polytene band 70E4-5, were also isolated and used to identify the intron/exon boundaries. Both low stringency hybridization of genomic Southern blots and BLAST searches of the Drosophila genome sequence databases failed to identify other Nkx6 homologs. The cDNA has an open reading frame of 1539 nucleotides. Alignment of Drosophila Nk6 with murine, rat and human Nkx6 sequences reveals considerable similarity within the homeodomain region, and in the amino and carboxy termini. The NK decapeptide domain is also conserved, suggesting that Nk6 may recruit the Groucho co-repressor as shown for vertebrate Nkx6 proteins. Nk6 does not possess the putative DNA binding interference domain present in the carboxy termini of Nkx6.1 and Nkx6.2 (Uhler, 2002).

An emerging feature of developmental genes is that many evolutionarily conserved genes are homologous not only in sequence, but often also have conserved expression patterns (for example, scarecrow and Nkx2.1), conserved regulation (e.g. targets of apterous homologs) and in some instances also conserved functions (vnd and Nkx2.2). Overall homology of the fly Nk6 gene is not prejudiced towards either individual vertebrate Nkx6 gene. The apparent absence of additional Nk6 homologs, ascertained both by sequence homology searches of the Drosophila genome and by hybridization analysis, suggests that Nkx6.1 and Nkx6.2 are each derived from an ancestral Nk(x)6 gene (Uhler, 2002).

date revised: 15 October 2004

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